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The role of adipocytes in acute lymphoblastic leukemia cell migration and survival against daunorubicin
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The role of adipocytes in acute lymphoblastic leukemia cell migration and survival against daunorubicin
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Content
The role of adipocytes in acute lymphoblastic leukemia cell migration and survival
against daunorubicin
by
Susan (Xia) Sheng
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(INTEGRATIVE BIOLOGY OF DISEASE)
December 2015
Copyright 2015 Susan (Xia) Sheng
ii
Table of Contents
Dedication………………………………………………………………………………………………………………………………………………iv
Acknowledgements…………………………………………………………………………………………………………………………………v
List of Tables…………………………………………………………………………………………………………………………………………..vi
List of Figures…………………………………………………………………………………………………………………………………………vii
Abbreviations…………………………………………………………………………………………………………………………………………ix
Chapter 1: Introduction…………………………………………………………………………………………………………………………..1
1.1 Obesity and cancer……………………………………………………………………………………………………………………..1
1.2 Acute lymphoblastic leukemia…………………………………………………………………………………………………….1
1.3 Adipocytes in the tumor microenvironment……………………………………………………………………………….4
1.4 SDF-1α and ALL migration……………………………………………………………………….………………………………….6
1.5 Adipose tissue alters chemotherapy pharmacokinetics………………………….…………………………………..6
1.6 Oxidative stress……………………………………………………………………….………………………………………………….7
1.7 Significance and potential therapeutic targets……………………….…………………………………………………..8
Chapter 2: Adipose tissue attracts acute lymphoblastic leukemia cells.………………………………………………10
2.1 Abstract………………………………………………………………………………….…………………………………………………10
2.2 Introduction…………………………………………………………………………….……………………………………………….10
2.3 Results……………………………………………..…………………………………….…………………………………………………10
2.4 Discussion……………………………………………..…………………………………….……………………………………………17
Chapter 3: Optimization of an in vitro model to study the interaction between ALL cells and
adipocytes………………………………………………………………………………………………………………………………………….…20
3.1 Abstract………………………………………………………………………………….…………………………………………………20
3.2 Introduction…………………………………………………………………………….……………………………………………….20
3.3 Results……………………………………………..…………………………………….…………………………………………………21
3.4 Discussion……………………………………………..…………………………………….……………………………………………26
Chapter 4: Adipocytes decrease ALL intracellular daunorubicin without changing surface MDR-1
expression………………………………………………………………………………….…………………………………………………………28
4.1 Abstract………………………………………………………………………………….…………………………………………………28
4.2 Introduction…………………………………………………………………………….……………………………………………….28
4.3 Results……………………………………………..…………………………………….…………………………………………………29
4.4 Discussion……………………………………………..…………………………………….……………………………………………32
iii
Chapter 5: Adipocytes protect ALL cells from chemotherapy by reducing oxidative stress………………...34
5.1 Abstract………………………………………………………………………………….…………………………………………………34
5.2 Introduction…………………………………………………………………………….……………………………………………….34
5.3 Results……………………………………………..…………………………………….…………………………………………………35
5.4 Discussion……………………………………………..…………………………………….……………………………………………43
Chapter 6: Concluding remarks & future directions………………………………………………………………………..……46
6.1 Summary and general discussion………………………………………………………………………………………………46
6.2 Limitations of the study…………………………………………………………………………………………………………….47
6.3 Future research…………………………………………………………………………………………………………………………49
Chapter 7: Materials and methods……………………………………………………………………………………………………….51
7.1 Experimental animals……………………………………………………………………………………………………………….51
7.2 Tissue harvesting and flow cytometry………………………………………………………………………………………51
7.3 Cell culture……………………………………………………………………………………………………………………………….51
7.4 Adipocyte differentiation…………………………………………………………………………………………………………52
7.5 Migration assays………………………………………………………………………………………………………………………53
7.6 Oil Red O staining and lipid quantification……………………………………………………………………………….53
7.7 Confocal microscopy………………………………………………………………………………………………………………..53
7.8 PCR analysis……………………………………………………………………………………………………………………………..53
7.9 Western blots…………………………………………………………………………………………………………………….......54
7.10 ELISA.............................................................................................................................................55
7.11 Insulin inhibition of lipolysis.........................................................................................................55
7.12 Oxidative stress measurement.....................................................................................................55
7.13 Intracellular GSH measurement...................................................................................................55
7.14 Statistical analysis........................................................................................................................56
References..................................................................................................................................................57
iv
Dedication
I dedicate this dissertation to my mother and father, Yun Liu ( 刘云) and Yang Sheng ( 盛扬), without
whom I would not have accomplished any of this. Thank you for supporting me with everything that I
choose to do, be it backpacking around China at the age of 15, or embarking on a journey to pursue a
PhD. Thank you for moving to the United States for me, and for providing me with the opportunity to a
great educational experience. Thank you for your constant encouragement and for always being my
number one fans. Dad, thank you for writing a book on the adventures of me and my pets. It provided
endless entertainment when I was feeling blue and lots of valuable life lessons. Mom, thank you for
loving all of our pets and taking such great care of them, especially when they are sick. I love you both,
so, so much!
v
Acknowledgements
First and foremost, I would like to thank my Principle Investigator Dr. Steven D Mittelman for persuading
me to pursue a PhD in biomedical research. If it weren’t for him, I would have never considered
becoming a research scientist. Besides science, Steve is constantly teaching me about not being too
literal and being more light-hearted with his sense of humor, even during his grand rounds.
Thanks to Dr. James Behan, who held me to the highest standards during my training, He is the reason
why I am meticulous, border lining anal retentive with my experimental techniques. Seriously, I don’t
know anyone else who must have all the Eppendorf tubes facing one direction before they can all be
labeled. Jim taught me to have a system in everything I do, so mistakes can be avoided.
I would also like to thank Anna Arutyunyan and Ara Moses, for teaching me all about cell culture, RNA
extractions, and Western blots. And I would like to thank Ehsan Ehsanipour, for encouraging me to
design mini projects and experiments to test my hypotheses, even before I started graduate school. I
would never forget the long nights we spent optimizing the HPLC protocol for amino acid analysis. I
learned valuable lessons on troubleshooting and what it means to take responsibility. That was also
when I realized that being a good scientist really meant being a remarkable learner, one that knows how
to assemble information and put it into practice.
Last but not least, I must thank my comrade, Jonathan Tucci, who in his own words “learned to
compensate” while working with me for the past three years. Being the only two PhD students in the lab,
we formed an alliance taking on multiple research projects. Thank you for being a good partner! I will
never forget all the pranks, songs, funny cat videos, sandwiches, and fish tacos. You really made my PhD
process much, much more enjoyable!
vi
List of Tables
Table 1.1 Retrospective studies examining the association between overweight/obesity and ALL
outcome ………………………..............……………………………………………………………………………….........2
Table 3.1
O
P
2
(mmHg) at the cell layer with different ambient oxygen percentages and media volumes
in a 24-well plate……………………………………………………………………………………………………...……...24
Table 5.1 Top 10 most upregulated genes in adipocytes treated with LCM……………………………………...38
Table 7.1 Primer list...............................................................................................................................54
vii
List of Figures
Figure 1.1 Bone marrow biopsy of ALL patient pre- (left) and post- (right) induction
treatment
5
Figure 2.1 Number of mice with detectable ALL cells by flow cytometry in various
tissues
11
Figure 2.2 Flow cytometry quantification of ALL cells detected from various tissues 12
Figure 2.3 Weight of visceral, omental, epidydimal, and perirenal fat pads from obese
and lean mice
12
Figure 2.4 Migration of ALL cells to mouse tissue explants 13
Figure 2.5 Migration of murine preB ALL cells to adipocytes and ACM 14
Figure 2.6 Human ALL cells migrate to ACM and SDF-1α 15
Figure 2.7 Quantification of secreted SDF-1α in FCM and ACM by ELISA 15
Figure 2.8 SDF-1 α concentrations in plasma and secreted by various tissues from
mice
15
Figure 2.9 AMD3100 blocks migration of ALL cells 16
Figure 2.10 Protein expression of SDF-1α receptor, CXCR4, in human ALL cells 17
Figure 2.11 Protection of ALL cells by fat explants against daunorubicin and vincristine 17
Figure 3.1 Lipid accumulation in adipocytes differentiated with three different
volumes
21
Figure 3.2 Lipid accumulation in mature 3T3-L1 adipocytes is directly proportional to
media height
22
Figure 3.3 Gene expressions of adipocyte-specific markers in relation to media height
during differentiation
23
Figure 3.4 Adipokine secretion from adipocytes differentiated with three volumes 23
Figure 3.5 Lipid accumulation in 3T3-L1 cells differentiated under different oxygen
contents
24
Figure 3.6 Insulin sensitivity in 3T3-L1 cells differentiated under different volumes 25
Figure 3.7 Insulin signaling in 3T3-L1 cells differentiated under different volumes 25
Figure 4.1 Adipocytes protect ALL cells from DNR 29
Figure 4.2 Quantification of intracellular DNR and surface MDR-1 expression in ALL
cells
30
Figure 4.3 DNR efflux from BV173 cells co-cultured with adipocytes 31
Figure 4.4 Adipocytes absorb DNR in a time-dependent manner 32
Figure 4.5 BV173 intracellular DNR and daunorubicinol measurements by HPLC 32
Figure 5.1 GCLC and GCLM gene expressions are suppressed in ALL cells in the
presence of adipocytes despite DNR treatment
36
Figure 5.2 Adipocytes alleviate DNR-induced ROS in ALL cells 36
viii
Figure 5.3 Adipocytes prevent DNR-induced cleaved caspase 3 expressions in ALL cells 37
Figure 5.4 Adipocytes protect ALL cells from oxidative stress 37
Figure 5.5 ALL cells induced oxidative stress in adipocytes 38
Figure 5.6 Nrf2 is translocated into the nucleus in 3T3-L1 adipocytes upon LCM
treatment
39
Figure 5.7 Nrf2 downstream genes are upregulated in 3T3-L1 adipocytes co-cultured
with ALL
39
Figure 5.8 ALL cells stimulate adipocytes to secrete survival factors 40
Figure 5.9 Oxidative stress in adipocytes leads to secretion of survival factors 41
Figure 5.10 Glutathione synthesis is partially involved in adipocyte protection of ALL
cells
42
Figure 5.11 3T3-L1 intracellular GSH measurements 42
Figure 5.12 Exogenous antioxidants cause ALL DNR resistance 43
Figure 5.13 Exogenous antioxidants increase ALL viability and decreases ROS despite
DNR treatment
43
Figure 6.1 Schematic depicting the interaction between adipocytes and ALL cells 49
ix
Abbreviations
A Adipocytes
ABC ATP-binding cassette
ACM Adipocyte-conditioned media
ADM Adrenomedullin
AHC Adipogenic hormone cocktail
ALCM Adipocyte-leukemia cell conditioned media
ALL Acute lymphoblastic leukemia
AML Acute myelogenous leukemia
APC Allophycocyanin
AUR Auranofin
BCA Bicinchoninic assay
Bcl-2 B-cell lymphoma 2
BSO Buthionine sulfoximine
CCG Children's cancer group
C/EBP CCAAT-enhancer-binding protein
CEIR Chronic energy-intake restriction
CLL Chronic lymphoblastic leukemia
CML Chronic myelogenous leukemia
CXCL12 C-X-C motif chemokine 12
CXCR4 C-X-C motif receptor 4
DCFH-DA Dichloro-dihydro-fluorescein diacetate
DIO Diet-induced obesity
DNR Daunorubicin
EC Effective concentration
EFS Event-free survival
F Fibroblast
FACS Fluorescence-activated cell sorting
FCM fibroblast-conditioned media
FFA Free fatty acid
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GCLC Glutamate-cysteine ligase, catalytic subunit
GCLM Glutamate-cysteine ligase, modifying subunit
GFP Green fluorescence protein
GSH Glutathione
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HO-1
HPLC
Heme-oxygenase 1
High pressure liquid chromatography
HR Hazard ratio
IBMX 3-isobutyl-1-methylxanthine
IGF-1 Insulin-like growth factor 1
IRS-1 Insulin receptor substrate 1
x
Keap-1
LCM
Kelch-like ECH-associated protein 1
Leukemia conditioned media
MCP-1 Monocyte chemotactic protein 1
M-CSF Macrophage colony-stimulating factor
MDR-1 Multidrug resistance protein 1
MFI Median fluorescence intensity
MSC mesenchymal stem cell
Mt-2 Metallothionine 2
NAC N-acetylcysteine
NEFA Non-esterified fatty acid
NF No feeder
NK Natural killer
NKT Natural killer T cells
Nrf2 Nuclear factor (erythroid 2-) related factor 2
Pgp P-glycoprotein
PPARγ Peroxisome proliferator-activated receptor gamma
qPCR Quantitative polymerase chain reaction
RANTES Regulated on activation, normal T cell expressed and secreted
ROS Reactive oxygen species
SDF-1α Stromal-derived factor 1 alpha
TXN Thioredoxin
1
Chapter 1
1
:
Introduction
Cancer is complicated. You just won’t believe how vastly, hugely, mind-boggling complicated it is. I mean,
you may think algebra is complicated, but that’s just peanuts to cancer.
- Stolen from Douglas Adams, The Hitchhiker’s Guide to the Galaxy
1.1 Obesity and cancer
Obesity is a serious health problem in both adults and children, as it is associated with a variety of health
conditions, including type II diabetes (2), cardiovascular diseases, hypertension, osteoarthritis, and
cancer (3). Data from the National Health and Nutrition Examination Survey of 2009 to 2010 revealed
that more than 35% of adults and almost 17% of youth were obese
2
in the United States (4). Another
one third of adults and one sixth of children are overweight, meaning that overall most adults and about
a third of children have unhealthy weight. The estimated annual medical cost of obesity in the U.S. was
$209.7 billion, or $2,741 for each obese person (5).
Obesity is strongly associated with increased cancer incidence and mortality. A large prospective study
published in 2003 by Eugenia Calle and colleagues was one of the first to show that increased BMI is
associated with increased mortality from many types of cancer (6). The study found an overall 52%
increased mortality in men with BMI greater than or equal to 35, and an 88% increase in women of the
same BMI category. While much of this increase in mortality can be attributed to increased cancer
incidence, obesity has also been shown to associate with a poorer prognosis in a variety of cancers.
Obesity can be defined as having excess fat mass. Healthy men and women are composed of 8-19% and
21-33% fat, respectively (7). Morbidly obese individuals can have well over 40% body fat. By volume,
adipocytes are the major cell type found in adipose tissue. However, preadipocytes, fibroblasts,
endothelial cells, immune cells, adipose stem cells and adipose tissue macrophages are also found in
adipose tissue (8–11). Particularly in obese individuals, these other cell types can account for more than
half of the number of cells in adipose tissue.
1.2 Childhood leukemias
Hematologic malignancies represent a substantial cancer burden in the United States. Approximately
55,000 people die per year from leukemia, lymphoma, and myeloma (Howlader
http://seer.cancer.gov/csr/1975_2011/). Leukemia is the most common cancer in children and teens,
accounting for almost 1 out of 3 cancers. About 3 out of 4 leukemias among children and teens are
acute lymphoblastic leukemia (ALL). Most of the remaining cases are acute myelogenous leukemia
(AML).
1
This chapter is a modified version of the published “mini review article”, The role of adipose tissue and obesity in
causing treatment resistance of acute lymphoblastic leukemia, of which I was first author(1).
2
Obesity is defined by the Center for Disease Control as a body mass index (BMI) ≥30 kg/m
2
in adults or ≥95
th
percentile in children. Overweight is considered a BMI ≥25 but less than 30 kg/m
2
in adults, and ≥85
th
but less than
95
th
percentile in children.
2
1948 marked the beginning of chemotherapy for ALL, when Farber et al. described “temporary
remissions” induced by aminopterin, a folic acid antagonist, in five children (12). Since then, the five-
year event-free survival rate has increased to more than 85% and five-year overall survival rate to more
than 90% (13–17).
Several studies have found an increased risk of developing hematological malignancies in obese adults
(18–21). In a meta-analysis of cohort studies, Larsson and Wolk found that excess body weight is
associated with an increased risk of developing all four major subtypes of hematologic malignancies
(ALL, AML, chronic lymphocytic leukemia [CLL], and chronic myelogenous leukemia [CML]) (22). They
estimated that each 5 kg/m
2
of increased BMI is associated with a 13% increase risk of developing
leukemia. Others have shown that obesity increases the risk of developing Hodgkin’s lymphoma, non-
Hodgkin’s lymphoma, and multiple myeloma (23–25).
The association between obesity and leukemia prognosis has also been examined, with some detecting
an effect of obesity to worsen prognosis, and others not (Table 1.1). Two of the studies acknowledged
that their failure to detect an association between BMI and ALL outcome may have been due to small
sample size (26,27). Interestingly, the risk estimates of overall survival and event-free survival from both
studies showed a trend of worsened outcome in the overweight/obese patients. The largest study was
done by the Children’s Cancer Group (CCG), and included over 5000 children. This study found that
obesity was associated with a significantly increased risk of relapse, particularly in children over 10 years
of age (considered high risk) (28). This latter caveat is consistent with the findings of a relatively small
study of 377 patients from the UKALL X treatment trial, which included only standard risk patients, and
concluded that overweight or obesity at diagnosis was unlikely to impair prognosis (29). The CCG
findings were also confirmed later in a cohort of 181 ALL children (mainly <10 years old) treated with
Berlin-Frankfurt-Munich protocols. The retrospective analysis clearly indicated that overweight/obesity
is an independent predictor of relapse risk in the intermediate- and high-risk groups(30). Thus, it
appears that obesity can impair ALL outcomes at least in high risk patients. A recent study on a cohort of
198 B-precursor ALL patients ranging from 1 to 19 years of age reported an association between obesity
and end-induction minimal residual disease(31).
Study Population
(age)
Cooperative
Group/Regimen
# Results Conclusion
Baillargeon
(26)
Predominantly
Hispanic B-
precursor ALL
patients (2-9
yo)
South Texas Pediatric
Minority Based
Community Clinical
Oncology
Program/Pediatric
Oncology Group
legacy protocols
241 HR of obesity on
EFS = 1.09 [0.54-
2.20]
“no association
between obesity
and survival”
Predominantly
Hispanic B-
precursor ALL
South Texas Pediatric
Minority Based
Community Clinical
81 HR of obesity on
EFS = 1.48 [0.73-
3.01]
3
patients (10-18
yo)
Oncology
Program/Pediatric
Oncology Group
legacy protocols
Hijiya (27) Predominantly
White ALL
St Jude Total Therapy
Studies/Total XII,
XIIIA, XIIIB and XIV
621 5 year EFS: obese
= 72.7±5.9%,
normal weight =
78.7±2.1%, p-
0.722
“No association
between BMI and
outcome or
toxicity in children
with ALL”
Subset ≥10 yo St Jude Total Therapy
Studies/Total XII,
XIIIA, XIIIB and XIV
185 P=0.103 for
effect of obesity
on EFS
“relatively small
number of
patients and the
use of different
treatments
compared to
those of the CCG
study”
Aldhafiri
(29)
UK national
cohort
excluding high
risk and low
risk (2-15 yo)
UKALL X treatment
trial
337 Relapse rate in
overweight/obes
e = 36.2%,
healthy weight =
36.6%
“No evidence that
being
overweight/obese
at diagnosis
impairs prognosis
in childhood ALL in
the UK”
Butturini
(28)
Predominantly
White newly
diagnosed ALL
CCG1881 CCG1922
CCG1891 CCG1882
CCG1901
4260 HR of obesity on
events 1.26
[1.03-1.55],
p=0.02
“In this study,
obesity seems to
be one of the
main
determinants of
relapse
in…patients
diagnosed with
ALL after their 10
th
birthday…”
Predominantly
White newly
diagnosed ALL
(≥10 yo)
CCG1882 CCG1901 1003 HR of obesity on
events 1.48
[1.07-2.03],
p=0.01
Verification
cohort (≥10 yo)
CCG1961 1160 Obese and > or =
10 yrs old: 1.42
[1.03-1.96]
p=0.032
Gelelete
(30)
Children with
ALL (mainly
IPPMG/UFRJ/Berlin-
Frankfurt-Munich
181 HR of overweight
and obese on EFS
“…overweight or
obesity at
4
<10 yo) protocols 1.92 [1.42-2.6]
p=0.031
diagnosis was an
independent
prognostic factor
to the 5-years EFS
in children with
ALL…”
Orgel (31) Predominantly
Hispanic B-
precursor ALL
patients
(between 1
and 19 yo)
Children’s Oncology
Group induction
regimens
198 p=0.012 “Obesity and
overweight were
associated with
poorer EFS
irrespective of
end-induction
MRD.”
Table 1.1 Retrospective studies examining the association between overweight/obesity and ALL
outcome (yo = years old; EFS = event free survival; HR = hazard ratio; MRD = minimal residual disease).
First three studies showed no association between overweight/obesity and ALL outcomes. Latter three
studies showed obesity to be a determinant of ALL outcomes.
1.3 Adipocytes in the tumor microenvironment
There is now increased understanding that the cancer microenvironment plays an important role in
spread, metastasis, and treatment response. This microenvironment consists of cancer cells, normal
cells and the intracellular matrix and signals surrounding them. In solid tumors, cancer cells interact with
several types of host cells including fibroblasts, macrophages, lymphocytes, endothelial cells and
adipocytes. Through a complex set of interactions, which are not completely understood, these host
cells are recruited to transform the local environment into a hospitable niche, improving access to
nutrients, protecting from immune surveillance, and providing growth factors and survival signals (32).
Together, these changes allow the cancer to survive, proliferate, and metastasize (33). Given their
mobile nature and propensity to travel throughout the body, leukemia cells are exposed to several
microenvironments, including the bone marrow, spleen, lymphatic system, the intravascular
environment, and various other extramedullary tissues. Understanding the roles of these
microenvironments is important for further improving treatment outcome.
5
Figure 1.1 Bone marrow biopsy of ALL patient pre- (left) and post- (right) induction treatment(1).
Adipose tissue and adipocytes have been shown to play an important role in supporting progression of
several types of cancer. Bone marrow, a major site of metastasis for solid tumors and an important
microenvironment for hematological malignancies, is also rich in adipocytes. In fact, after induction
chemotherapy for acute lymphoblastic leukemia (ALL), adipocytes can represent the primary cellular
component of bone marrow (Figure 1.1)(1). Given the effects of obesity on cancer prognosis, we and
others have investigated whether these fat cells may contribute to treatment resistance in leukemias
and other cancers.
Traditionally, adipose tissue has been recognized as primarily a site for fuel storage. However, since the
discovery of leptin by Zhang et al. in 1994, it is now well-established that adipose tissue is an active
endocrine organ (34). There are now more than 50 identified adipokines (i.e. cytokines secreted
primarily by adipose tissue) (35). These adipokines regulate appetite, energy expenditure, immune
function, growth, and metabolism of other tissues(36). Many adipokines (such as leptin) and other
cytokines secreted by adipose tissue (such as IGF-1), have been linked to cancer pathogenesis(37–42).
Numerous immune cells are found in normal adipose tissue, including T and B lymphocytes, NK cells,
NKT cells, mast cells, macrophages and neutrophils. Adipose tissue inflammation likely contributes to
many of the negative sequalae of obesity, including diabetes, heart disease, and possibly cancer (43).
Obesity is associated with an increased number of lymphocytes in adipose tissue in mice (12). These
lymphocytes are believed to interact with adipocytes and adipose tissue macrophages, and may play a
role in obesity-induced insulin resistance and diabetes (44).
Several cancers occur in close proximity to adipose tissue. Breast, colon, pancreas, ovary, uterus, and
liver are all surrounded by and/or infiltrated by adipose tissue. Extension of these cancer types outside
of their originating organ often takes them into direct contact with adipose tissue. Furthermore,
adipocytes are found in the bone marrow, a common site for solid tumor metastasis (45), and an
important microenvironment for many hematologic malignancies (46). Bone marrow adiposity is not
only affected by obesity (47), but has recently been shown to be influenced by ALL treatment (48). Lopez
et al. isolated mesenchymal stem cells (MSC) from bone marrow aspirates of ALL patients at various
time points: diagnosis, during therapy, and after therapy. ALL-MSC from treated patients showed an
6
increased adipogenic differentiation potential, including a higher expression of adipogenic genes (CEBP
and PPARγ), compared to healthy MSC(48).
1.4 SDF-1α and ALL migration
Stromal derived factor 1 alpha (SDF-1α), also known as CXCL12, is a chemoattractant for cells of
lymphoid origin, including hematopoietic cells, mature lymphocytes, and leukemia cells (49–51). It acts
by binding to the surface receptor CXCR4, causing multiple intracellular changes including actin
cytoskeletal reorganization and activation of integrins and adhesion molecules (52). SDF-1α has since
been found to be expressed in other cell types, such as fibroblasts, adipocytes, and endothelial cells(53–
55).
Given the fact that lymphocytes infiltrate adipose tissue, it is not surprising that adipocytes could attract
pre-B leukemia cells in the same manner via the SDF-1α/CXCR4 axis. We found that ALL cells were
present in adipose tissue of mice which developed progressive leukemia despite vincristine treatment
(56). In addition, syngeneic ALL cells implanted into mice by a retro-orbital injection infiltrated adipose
tissue within ten days, to a similar degree (quantified as percentage of ALL cells in the tissue digest) as
other more classic sites for ALL, such as spleen and liver (57). ALL migration towards adipocytes is
mediated by adipocyte secretion of SDF-1α (or CXCL12). While obesity was not associated with
increased serum levels of SDF-1α (measured by ELISA), obese mice had a significantly higher burden of
leukemia cells in visceral fat compared to control mice (57). Adipocytes have been shown by others to
facilitate leukemia bone marrow engraftment via secretion of SDF-1α and leptin (58).
In Chapter 2, I will describe our findings on how adipocytes facilitate ALL migration into the adipose
tissue using the SDF-1α/CXCR4 axis. We also show that adipose tissue explants protect ALL cells from
chemotherapy treatment, such as daunorubicin and vincristine.
1.5 Adipose tissue alters chemotherapy pharmacokinetics
Obesity influences many aspects of drug pharmacokinetics (PK). For lipid-soluble drugs, obesity
increases the volume of distribution by accumulation of drug in the excess adipose tissue (59–61).
Obesity is also associated with increased alpha 1-acid glycoproteins, which could increase the binding of
basic drugs in plasma (60). Hepatic and renal drug clearance could be altered in obesity due to increased
activity of cytochrome P450 2E1 and increased glomerular filtration and tubular secretion (62,63). This
in turn could alter the PK of water-soluble drugs, since they can be readily excreted from the kidneys
(64). Since the overall exposure to a drug depends on both the volume of distribution and the clearance,
obesity could be associated with both toxicity and impaired efficacy of different medications. While
there have been no studies of which we are aware examining the effects of weight status on PK in
children with leukemia, a retrospective study by Hijiya et al reported no difference in mean systemic
clearance and intracellular levels of thioguanine nucleotides and methotrexate polyglutamates among
four BMI groups (27).
Vincristine, a potent anti-microtubule agent, is a key drug used in childhood ALL combination
chemotherapy, as well as treatment of many other cancers. In pediatric and adult leukemia patients,
vincristine is dosed proportional to body surface area, and this dose is generally “capped” at 1.33 square
7
meters or 2 milligrams. We found that DIO mice implanted with syngeneic leukemia cells exhibited
poorer survival compared to control mice, despite vincristine treatment being dosed proportional to
body weight (56). However, there have been few PK studies examining the impact of obesity on
chemotherapy treatments. Given that vincristine is a lipophilic agent, it may be sequestered in adipose
tissue and thus have altered tissue distribution in obese individuals. We explored this idea using
injections of tritiated vincristine in obese and control mice (65). Upon a single intravenous injection of
vincristine proportional to body weight, blood and tissue levels of the drug were measured at different
time points up to 24 hours. Over the initial 24 hours, blood vincristine concentrations were higher in the
obese mice than the control, while tissue concentrations were comparable in spleen, liver, brown fat
and bone marrow. However, by 3 hours, there was significantly higher vincristine in the white adipose
tissue of obese mice than that of control mice. PK modeling showed that overall exposure of ALL cells to
vincristine is impaired by obesity, and this may be exacerbated when the drug is dosed per body surface
area and is capped.
In 2012, the American Society of Clinical Oncology (ASCO) published recommendations for appropriate
cytotoxic chemotherapy dosing for obese adult cancer patients (66). The ASCO assembled a panel of
experts to conduct a systematic review of studies published between 1996 and 2010. They reported that
up to 40% of obese patients received reduced chemotherapy doses that were not based on actual body
weight. Meanwhile, they found no evidence that short- or long-term toxicity was increased among
obese patients receiving full weight-based doses. The panel thus recommends that “full weight-based
chemotherapy doses be used in the treatment of the obese patient with cancer, particularly when the
goal of treatment is cure.”
We have previously shown that adipocytes and adipose tissue explants could protect ALL cells from
daunorubicin (DNR), an anthracycline routinely given to leukemia patients(56,57). However, the
mechanism of protection is largely unknown. In Chapter 4, I present that DNR is taken up by adipocytes,
and that ALL cells accumulate less DNR intracellularly when co-cultured with adipocytes. The alterations
in DNR partitioning could, in part, explain the protective effects of adipose tissue explants against DNR,
as described in Chapter 2. We also provide evidence that adipocytes convert DNR to its less cytotoxic
metabolite, daunorubicinol.
1.6 Oxidative stress and ALL
Oxidative stress is induced by an increase in intracellular reactive oxygen species (ROS), which are
normal byproducts generated by many cellular processes. The amount of intracellular ROS is tightly
controlled by antioxidants, which are products of the oxidative stress response pathways. ROS act as
signaling molecules to promote proliferation and survival(67–69). However, when ROS levels increase,
they could cause protein and DNA oxidation and exert oxidative stress on cells. In normal cells, sustained
high levels of ROS could lead to cell senescence or death. Because of their high proliferation and
metabolic rates, cancer cells are under greater oxidative stress compared to normal cells, and therefore
have an active oxidative stress response pathway. However, despite having an enhanced antioxidant
system, cancer cells maintain higher ROS levels than normal cells(70). In fact, many chemotherapeutics
induce high levels of oxidative stress, targeting the sensitivity of cancer cells to further accumulation of
8
ROS(69,71). Anthracyclines, which are routinely given to relapsed ALL patients, are one of such
chemotherapy(71).
One of the key pathways in regulating oxidative stress is the nuclear factor (erythroid-derived 2)-like 2
(Nrf2)-mediated oxidative stress response pathway. Under basal conditions, Nrf2 is sequestered in the
cytoplasm, bound to Kelch-like ECH-associated protein 1 (Keap1), leading to its ubiquitination and
subsequent degradation. Under oxidative stress conditions, Keap1 is oxidized and can no longer bind to
Nrf2, leading to the stabilization of Nrf2 and translocation into the nucleus. Nrf2 activates genes such as
heme oxygenase-1 (HO-1) and the two subunits of glutamate cysteine synthetase, modifier (GCLM) and
catalyzing (GCLC), which control the rate-limiting step of glutathione synthesis(72–74). Nrf2 has been
shown to be induced by oncogenes to promote cancer cell growth and control of ROS(75). Specifically,
Nrf2 expression in AML is linked to chemoresistance(72).
Besides induction of tumorigenesis, oxidative stress also plays an important role in adipocyte
differentiation, where preadipocytes acquire the ability to deposit lipids in lipid droplets(76). The
mitochondria are where free fatty acids are converted to triglycerides, and are a major source of ROS.
Numerous reports have been made on the importance of regulation of ROS in adipogenesis and
adipocyte function(77–80). During differentiation, expressions of manganese superoxide dismutase,
Cu/Zn superoxide dismutase, and catalase are induced to help balance intracellular ROS(81). In fact, it
has been reported that over-expression of Nrf2 by knocking down Keap1 enhances adipogenesis in 3T3-
L1 cells(82).
With conditioned media experiments, we found that ALL cells increase oxidative stress in adipocytes via
soluble factors, and this oxidative stress response in adipocytes may partially explain the drug resistance
of ALL cells that are in this microenvironment. We found adipocytes exposed to either ALL cells or ALL
conditioned media had increased ROS and that ALL cells co-cultured together with adipocytes had lower
intracellular ROS despite daunorubicin treatment. The protection from adipocytes against daunorubicin
was partially reversed when the adipocytes were pretreated with buthionine sulfoximine (BSO), which is
an irreversible inhibitor of γ-glutamylcysteine synthetase. Interestingly, a recent publication
demonstrated that combined inhibition of glutathione (GSH) and thioredoxin (TXN) antioxidant
pathways leads to a synergistic cancer cell death in vitro and in vivo (83). There is evidence that
increasing the already elevated oxidative stress in CLL cells leads to preferential killing of the CLL cells
and sparing normal lymphocytes (84). In Chapter 5, the interactions between adipocytes and ALL cells
mediated by oxidative stress will be discussed in detail.
1.7 Significance and potential therapeutic targets
With the prevalence of obesity and the mounting evidence of increased adiposity as a risk factor for
cancer incidence and mortality, it is important to understand the adipocyte-cancer interaction and
expand traditional therapies to address obesity. More than two decades ago, Shields et al. showed that
chronic energy-intake restriction (CEIR) has profound effects to delay lymphoma development in AKR
mice (85). As a more practical strategy than CEIR, short-term fasting has been demonstrated to provide
differential stress resistance to chemotherapy, where normal cells are protected while cancerous cells
9
remain sensitive. There is evidence that fasting is not only safe, but also effective in reducing common
side effects associated with chemotherapy (86).
There are a number of clinically available metabolic agents targeting adipocyte differentiation, insulin
resistance and inflammation that have been shown to have anti-cancer effects. One of such agents is
metformin, which is clinically used to treat type 2 diabetes mellitus. While metformin has not been
tested in leukemia patients, analysis of a retrospective cohort of 363 women diagnosed with
endometrial cancer and diabetes mellitus analysis demonstrated that metformin use was associated
with prolonged recurrence-free survival time, despite the fact that metformin users were statistically
heavier (87). There are studies that speculate the anti-tumorigenic effects of metformin to be due to
increasing insulin sensitivity, inhibiting liver gluconeogenesis, reducing hyperglycemia and insulin levels,
and activating AMP-activated protein kinase(88,89).
Since adipocytes may be providing cancer cells with free fatty acids (FFA) as a fuel source, inhibition of
lipolysis and FFA efflux from adipocytes or blocking of cancer cell FFA oxidation could represent
therapeutic targets. Inhibition of FFA oxidation, such as with etomoxir, has been shown to inhibit
proliferation and induce apoptosis in a variety of hematological malignancies, including myeloma cell
(90), AML (91), CLL (92) and mantle cell lymphoma (93). Since cancer cells, such as ovarian cancer,
increase their FFA oxidation rate when co-cultured with adipocytes (94), targeting this pathway may be
particularly beneficial in obese patients. Besides provision of fuel, adipocyte-derived FFAs have been
shown to mediate chemotherapy resistance in CLL cells(95,96).
Over the past two decades, there has been ground-breaking research in the field of adipocyte biology,
leading to a reassessment of the role of adipose tissue in man. There has also been mounting
epidemiological evidence of the important effect of obesity on cancer incidence and mortality.
Adipocytes play an active role in the tumor microenvironment in many types of cancer, including those
that have strong associations with obesity. With obesity rates reaching unprecedented levels worldwide
and the alarming rise in childhood obesity, teasing apart the mechanisms linking obesity and cancer
represents a crucial step in the quest to improve overall cancer survival. In the present thesis, I
hypothesize that adipocytes play an important role in ALL treatment by 1) attracting ALL cells into
adipose tissue; 2) altering chemotherapy distribution and metabolism in the microenvironment; and 3)
alleviating chemotherapy-induced oxidative stress, leading to survival of ALL cells despite chemotherapy
treatment.
10
Chapter 2
3
:
Adipose tissue attracts acute lymphoblastic leukemia cells
2.1 Abstract
Obesity is associated with an increased risk of acute lymphoblastic leukemia (ALL) relapse. Using mouse
and cell co-culture models, we investigated whether adipose tissue attracts ALL to a protective
microenvironment. Syngeneically implanted ALL cells migrated into adipose tissue within ten days. In
vitro, murine ALL cells migrated towards adipose tissue explants and 3T3-L1 adipocytes. Human and
mouse ALL cells migrated toward adipocyte conditioned media, which was mediated by SDF-1 . In
addition, adipose tissue explants protected ALL cells against daunorubicin and vincristine. Our findings
suggest that ALL migration into adipose tissue could contribute to drug resistance and potentially
relapse.
2.2 Introduction
ALL is the most common type of cancer in children. With aggressive combination chemotherapy, the overall
cure rate is about 80% in children(97) but only 50% in adults(98). Leukemia relapse continues to be a
problem, and is thought to be due to drug resistance(99). While many studies of drug resistance have
focused on acquired gene mutations in leukemia cells, in some studies the leukemia microenvironment has
also been shown to play a major role in de novo chemotherapy resistance in ALL(100). It is thought that
leukemia cells home to the bone marrow, which acts as a niche that protects ALL cells from drug-induced
death(100,101). Since bone marrow is comprised of many different cell types (e.g. mesenchymal stem cells,
osteoblasts, endothelial cells, hematopoietic cells, adipocytes), it is not clear which marrow cells are
responsible for ALL homing and induction of chemotherapy resistance.
There is growing evidence that adipocytes may interact with cancer cells to promote invasion, proliferation,
and/or drug resistance(56,102,103). Adipocytes secrete numerous factors which have roles in cancer cell
proliferation, migration, and metastasis, such as insulin-like growth factor 1, leptin, platelet-derived growth
factor, matrix metalloproteinase 11, interleukin 6 and stromal cell-derived factor 1 (SDF-1α)(104 –106).
Some of these factors may contribute to the strong associations observed between obesity and cancer
mortality (6) including studies which show that obesity is associated with increased risk of relapse in
ALL(28). However, the precise mechanisms whereby adipocytes may contribute to ALL relapse remain
unknown.
Previously, we observed the presence of transplanted ALL cells in the fat depots of obese mice by
fluorescence microscopy after vincristine treatment(56). However, since these mice had developed a
substantial leukemia burden, and we did not look for ALL cells in other organs, it was not known whether
the leukemia cells actively and preferentially migrated into the adipose tissue. Furthermore, the
mechanism(s) regulating leukemia cell migration into adipose tissue are not known. In the present study,
we report that adipocyte secretion of SDF-1α induces ALL cells to migrate into adipose tissue, further
demonstrating the importance of adipocytes in the leukemia cell microenvironment.
2.3 Results
3
This chapter is a modified version of the published research article Adipose tissue attracts and protects acute
lymphoblastic leukemia cells from chemotherapy, of which I was a co-first author(57).
11
2.3.1 ALL cells migrate into adipose tissue.
To investigate whether leukemia cells actively migrate into adipose tissue in vivo, we transplanted 20
week-old obese C57Bl/6 mice syngeneically with 10,000 GFP positive 8093 ALL cells. We collected
tissues at an early time point, ten days after implantation, to measure homing to different locations. The
presence of 8093 cells in these tissues was analyzed by FACS. At this early time point, there were few
circulating ALL cells, with all 6 obese mice (HF = high fat) and 4 of 6 control mice (LC = lean chow) having
detectible leukemia, though at <0.1% of events. Half the mice from each group had detectible leukemia
in the bone marrow and 2 obese and 3 control mice had detectible leukemia in the spleen, all at <11% of
events (Fig. 2.1).
Figure 2.1 Number of mice with detectable ALL cells by flow cytometry in various tissues. The detection
limit is set for each tissue using samples from a non-implanted mouse (HF = high fat; LC = lean chow).
At this time point, all mice also had detectible leukemia in their visceral fat pads, and most of other fat
pads as well. Leukemia burden of each tissue site was calculated as a percentage of GFP+ cells, using the
total number of isolated viable single cells from the tissues as a denominator. For example, a fraction of
fat was digested and the burden was calculated by dividing the number of GFP+ cells by the total live
stromal vascular fraction quantified by flow cytometry. When leukemia cells were present in marrow,
spleen and liver, they were present in large numbers (>0.5% of events on average), while the burdens in
kidney, lung, and brain were lower (Fig. 2.2). Leukemia burden was also high in both visceral and
perirenal fat depots. There was a significantly higher burden of leukemia cells in visceral fat from obese
compared to control mice. The percentage of GFP+ cells was calculated from each tissue site by
digesting a portion of the tissue and dividing the number of GFP+ cells by the total number of live cells
analyzed using flow cytometry. Although diet group had no effect on leukemia burden in any other
tissue, obese mice had significantly more fat in all four depots compared to lean mice (Fig. 2.3).
Therefore the absolute number of leukemia cells in these depots would be expected to be higher in the
obese mice.
12
B lo o d
M a rro w
S p le e n
L iv e r
K id n e y
M u s c le
L u n g
B ra in
S u b Q
V is c e ra l
E p id id y m a l
P e rire n a l
O m e n ta l
0
1
2
3
4
5
2 0
*
N .D
% o f G F P c e lls
Figure 2.2 Flow cytometry quantification of ALL cells detected from various tissues in Figure 2.1 in obese
(black bar) and lean (white bar) mice (refer to Figure 2.1 for number of mice per tissue site with
detectable ALL cells). Percentages are calculated by dividing the amount of GFP+ cells detected by flow
cytometry by the total number of live cells from each tissue site. In the case of adipose tissue, the
denominator was live cells in the stromovascular fraction (i.e. the centrifuged pellet from the digested
adipose tissue). No ALL cells were detected in muscle (N.D.). * indicates p<0.05 compared to the
corresponding tissue of the lean mice.
NT Visc OM PR Epi SQ Musc
0
10
20
30
**
**
*
Tissue
% migration
V is O m E p i P R
0
5 0 0
1 0 0 0
1 5 0 0
***
***
***
***
m g
Figure 2.3 Weight of visceral (Vis), omental (Om), epidydimal (Epi), and perirenal (PR) fat pads from
obese (black bar) and lean (white bar) mice. *** indicates p<0.001 compared to the corresponding
tissue of the lean mice.
The leukemia burden in the adipose tissue apparently increased over time; in two additional mice, we
found that adipose tissue leukemia burden was substantially higher when mice were sacrificed 21 days
after leukemia transplantation (not shown). Therefore, leukemia cells appear to migrate into adipose
tissue early after transplantation, when the leukemia burden is still relatively low. We have previously
reported that ALL cells isolated from adipose tissue from mice after vincristine chemotherapy treatment
are viable, and able to proliferate in vitro(56). However, we did not test whether these cells were
actively proliferating within the adipose tissue depots in vivo, and so whether ALL cell expansion occurs
within adipose tissue remains to be tested.
13
To test whether adipose tissue attracts leukemia cells, we performed ex vivo migration assays with
mouse adipose tissue explants. Murine preB ALL cells migrated through TransWells toward various fat
depots (3-15% within 90 minutes; n=3, Fig. 2.4). Consistent with the in vivo findings, very few cells
migrated toward muscle or control media.
NT Visc OM PR Epi SQ Musc
0
10
20
30
**
**
*
Tissue
% migration
V is O m E p i P R
0
5 0 0
1 0 0 0
1 5 0 0
***
***
***
***
m g
Figure 2.4 Migration of ALL cells to mouse tissue explants after 90 minutes (NT = no tissue control).
Percentage was calculated as number of cells in bottom chamber divided by total number of cells in
both chambers. * p<0.05, ** p<0.01, compared to NT.
2.3.2 Leukemia cells exhibit chemotaxis towards adipocytes.
Adipose tissue is comprised of multiple cell types, including adipocytes, endothelial cells, stromal cells,
and immune cells. Since adipocytes occupy the most volume in adipose tissue, we next tested whether
the chemotaxis of leukemia cells was specifically toward adipocytes. Murine preB ALL cells migrated
toward differentiated 3T3-L1 and OP9 adipocytes, and to media conditioned by these adipocytes (Fig.
2.5 A). Undifferentiated pre-adipocytes also caused significant migration of leukemia cells; for 3T3-L1
cells, migration toward pre-adipocytes and preadipocyte-conditioned media was less than toward
adipocytes or ACM. However, migration toward OP-9 adipocytes and preadipocytes (and respective
conditioned media) was similar. Interestingly, when both chambers contained adipocyte conditioned
media (ACM, Fig. 2.5 B), an intermediate number of 8093 cells migrated through the TransWell, implying
that ACM increases both directional and non-directional leukemia cell motility. Migration toward ACM
was relatively rapid, with about half of the total migration taking place within the first 60 minutes of
incubation (Fig. 2.5 C).
14
Figure 2.5 Migration of murine preB ALL cells to adipocytes and ACM. (A) Migration of 8093 murine ALL
cells towards FCM, ACM, pre-adipocyte feeder layer (Fibro), and adipocyte feeder layer (Adipo) of 3T3-
L1 and OP9 cells. Percentage of migrated cells is calculated as before. Control condition was performed
with RPMI1640 medium in both chambers. (B) ACM causes both directional and non-directional
movement of 8093 cells (C = RPMI; A = ACM). (C) Time course of 8093 ALL migration towards ACM. *
p<0.05, ** p<0.01, compared to control conditions, unless otherwise indicated.
2.3.3 SDF-1 is the adipocyte-derived chemoattractant responsible for leukemia cell migration.
Several adipocyte-derived molecules function as chemoattractants to immune cells. To identify which
molecule(s) were responsible for the adipocyte-mediated chemoattraction, we tested various
adipocyte-secreted molecules for their ability to stimulate 8093-ALL cell migration. In a screening of 6
different recombinant molecules (SDF-1 , leptin, adiponectin, resistin, MCP-1 and RANTES, all are
known to be secreted by adipocytes), we found only SDF-1 to significantly stimulate migration of 8093-
ALL cells in our experimental conditions (data not shown; doses tested 10-180 ng/mL). Murine
recombinant SDF-1 induced migration of all 3 human ALL cell lines we tested, to a similar degree as
ACM (Fig. 2.6 left). In addition, three out of five primary human leukemia cells, which were isolated from
ALL patients and grown in vitro with murine OP9 fibroblasts as a feeder layer as described
previously(107,108), showed a significantly increased migration toward SDF-1 (TXL2, ICN13, US.7, BLQ1
and UCSF02, Fig. 2.6 right).
15
Figure 2.6 Human ALL cells migrate to ACM and SDF-1α. Left: commercial human ALL cell lines; right:
primary human ALL cell strains. Migration was quantified after 3 hours. * p<0.05, ** p<0.01 compared to
control conditions of each cell line.
We next tested whether adipocytes secrete SDF-1 . Media conditioned for 48 hours by 3T3-L1 and OP9
preadipocytes and adipocytes were measured using an SDF-1 ELISA kit. In both cell lines, adipocytes
secreted significantly more SDF-1 than their corresponding pre-adipocytes (Fig. 2.7). We also
measured SDF-1 secretion by tissue explants from control and obese mice. There was no significant
effect of diet group on SDF-1 secretion in any of the tissues (Fig. 2.8). There was no detectable amount
of SDF-1 in complete media.
Figure 2.7 Quantification of secreted SDF-1α in pre-adipocyte conditioned media (FCM) and adipocyte
conditioned media (ACM) by ELISA. Complete media without any conditioning with feeder layers were
included as a negative control, and their SDF-1α levels were below the limit of detection of the assay. **
p<0.01 compared to FCM of the same cell line. ** p<0.01 compared to its FCM.
Figure 2.8 SDF-1 α concentrations in plasma and secreted by various tissues from control (LC) and obese
(HF) mice measured by ELISA. Plasma was collected from mice by cardiac puncture. Tissue and fat
depots were collected and rinsed with cold PBS. Then equal amounts of each tissue type were plated in
16
1mL of complete media for 48 hours. SDF-1α measurements were then normalized for each tissue by
dividing by the actual milligram of tissue. Bone marrow (BM) samples were collected by flushing the
bone marrow from femurs of the mice and plated in 1 mL of complete media for 48 hours. The media
were then collected and filtered. SDF-1α concentrations were reported as pg/mL since the weight of the
bone marrow samples were not measured. No differences between control and obese mice were
detected.
To confirm that adipocyte-derived SDF-1α was responsible for the ALL chemotaxis, we used AMD3100, a
specific inhibitor of CXCR4. AMD3100 inhibited 8093 leukemia cell migration toward SDF1α and 3T3-L1
ACM (Fig. 2.9 left) in a dose dependent manner. At the highest dose tested (0.8 µg/mL), AMD3100
completely blocked ALL migration toward 3T3-L1 ACM. Interestingly, AMD3100 also blocked migration
of ALL toward OP-9 conditioned media, though at higher doses, possibly due to the higher concentration
of SDF-1α (Fig. 2.9 right). These results demonstrate that SDF-1 α is the functional chemoattractant for
ALL cells in ACM. The difference in AMD3100 activities against ACM and SDF-1α could be due to the fact
that the SDF-1α was recombinant, while ACM contained endogenous SDF-1α. In fact, the ELISA of SDF-
1α in ACM and FCM was measured to be between 200-700 pg/mL (Fig. 2.7), while 20 ng/mL of the
recombinant protein was used to achieve a similar degree of migration.
0 0 .8 4
0
2 5
5 0
7 5
1 0 0
A M D 3 1 0 0 (u g /m L )
**
**
*** ***
% m ig ra tio n
S D F
0 0 .0 0 6 0 .0 3 0 .1 6 0 .8
0
2 0
4 0
6 0
*
**
**
***
*
*
A M D 3 1 0 0 (u g /m L )
% m ig ra tio n
F C M
A C M
Figure 2.9 AMD3100 blocks migration of ALL cells. Left: inhibition of murine ALL cells towards 20 ng/mL
SDF-1α or 3T3-L1 ACM after pre-incubation of cells with the indicated amounts of AMD3100. Right:
migration of murine ALL cells towards OP9 FCM and ACM after pre-incubation of cells with the indicated
amounts of AMD3100. All groups were pre-incubated with indicated concentrations of AMD3100 at 4 C
for 30 minutes. * p<0.05, ** p<0.01, ***p<0.001 compared to corresponding conditions without
AMD3100.
We also verified that the ALL cells which migrated toward ACM express the SDF-1 receptor, CXCR4,
with western blots (Fig. 2.10). β-actin was used as loading control for each sample.
17
7 6
5 2
3 8
S D 1 B V 1 7 3 R S 4 ;1 1 8 0 9 3 K 5 6 2
C X C R 4
-a c tin
k D a
Figure 2.10 Protein expression of SDF-1α receptor, CXCR4, in ALL cells. β-actin was included as a loading
control.
2.3.4 Adipose tissue protects leukemia cells from chemotherapy in vitro.
Since we previously found that adipocytes in vitro protect ALL cells against various chemotherapeutic
agents, we tested the effects of weight-matched tissue explants on drug resistance of 8093-ALL cells. As
shown in Fig. 2.11, subcutaneous and visceral fat pads from obese and control mice both protected
8093-ALL cells from daunorubicin. Both fat pads also protected ALL from vincristine, though this did not
reach statistical significance for the obese mice. Muscle protected ALL cells in some conditions, while
lung and kidney showed no significant protection from either drug.
N o T is s u e
S u b Q
V is c e ra l
M u s c le
L u n g
K id n e y
0
5 0 ,0 0 0
1 0 0 ,0 0 0
1 5 0 ,0 0 0
2 0 0 ,0 0 0
2 5 0 ,0 0 0
*
**
**
L C
H F
***
**
N o T is s u e
S u b Q
V is c e ra l
M u s c le
L u n g
K id n e y
0
2 0 ,0 0 0
4 0 ,0 0 0
6 0 ,0 0 0
p = 0 .0 7
*
***
*
D a u n o ru b ic in
V in c ris tin e
C e lls /w e ll
C e lls /w e ll
Figure 2.11 Protection of ALL cells by fat explants against daunorubicin and vincristine. Weight-matched
tissue explants (subcutaneous fat, visceral fat, muscle, lungs, and kidneys) from control and obese mice
(n=6 each) were excised after cardiac perfusion. The doses of daunorubicin (19nM) and vincristine (3nM)
were EC
99
doses in the control conditions without tissue explants. ALL cells were quantified using trypan
blue exclusion after 48 hours of drug treatment. * p<0.05, ** p<0.01, ***p<0.001 compared to no tissue
controls.
2.4 Discussion
The tumor microenvironment plays an important role in cancer cell survival and growth. Primary
leukemia cells do not survive and proliferate well in vitro unless they are cultured with stromal
cells(109,110). It is thought that the bone marrow is the most important microenvironment for pre-B
18
ALL cells in vivo. Since ALL cells are malignantly transformed immature B-cells, it would not be surprising
that they share some of the immune-surveillance functions of more mature lymphocytes, such as
sensitivity to SDF-1α, and migration throughout various tissues of the body. Recent studies have
elaborated a complex trafficking of immune cells in adipose tissue, especially in the obese
state(111,112). In accordance, we previously found transplanted leukemia cells in mouse adipose tissue
after chemotherapy(56). In the present study, we have demonstrated that adipose tissue is actively
infiltrated by ALL cells, even when the leukemia burden is similar to that seen in patients with minimal
residual disease [0.01% in blood,(113)]. Our current results show that leukemia cells migrate into
adipose tissue, probably under the regulation of SDF-1α (stromal cell-derived factor-1α) secreted by
adipocytes.
Immune cell migration is regulated by chemokines(114,115), including SDF-1α. SDF-1α, also known as
CXCL12, is a chemoattractant for cells of lymphoid origin, including hematopoietic cells, mature
lymphocytes, and leukemia cells(50,51,116). It acts by binding to the surface receptor CXCR4, causing
multiple intracellular changes including actin cytoskeletal reorganization and activation of integrins and
adhesion molecules(52). While it was first identified in bone marrow stromal cells, SDF-1α has since
been found to be expressed in other cell types, such as fibroblasts, adipocytes, and endothelial
cells(53,54,117). Many of these cells reside in adipose tissue, and indeed we found adipose tissue to be
a significant producer of SDF-1α ex vivo.
Numerous immune cells are found in normal adipose tissue, including T and B lymphocytes(118,119).
Although obesity clearly increases the number of T cells found in adipose tissue in mice(44,111,120), its
effect on B cells is less clear(44,120). These lymphocytes are believed to interact with adipose tissue
macrophages, and may play a role in obesity-induced insulin resistance and diabetes(111,121). Whether
adipocytes attract non-malignant lymphocytes via SDF-1α secretion has not been determined.
Although adipocytes express and secrete SDF-1α, serum levels are actually lower in obese
humans(122,123). Since SDF-1α is produced in multiple tissues of the body, it is not clear whether
decreased serum levels reflect a decrease in production from adipose tissue or other tissues, or perhaps
even an increase in SDF-1α clearance from the blood. In any case, as SDF-1α is a chemokine, its activity
depends on local gradients in capillary beds of tissue. Since obese patients can have several fold higher
amounts of adipose tissue than lean patients, circulating leukemia cells in obese patients are likely
exposed to more adipose tissue capillary beds, where they can encounter a local SDF-1α gradient and
have the opportunity to migrate into adipose tissue. Indeed, this hypothesis is consistent with our
finding that adipose tissue from control and obese leukemic mice have a similar ALL percentage of
adipose tissue in most depots (Fig. 2.2), while the increased amount of adipose tissue (Fig. 2.3) likely
reflects a higher overall number of leukemia cells in adipose tissue of obese mice.
Interestingly, adipocyte expression of SDF-1α decreases during differentiation(111), though our data
show that secretion is higher from both 3T3-L1 and OP-9 adipocytes than from preadipocytes (Fig. 2.7).
However, ALL migration was similar toward the conditioned media of differentiated and
undifferentiated OP-9 cells. Migration of ALL toward tissue explants was also not directly proportional to
SDF-1α concentrations measured in their respective conditioned media. Since AMD3100 was able to
completely abolish ALL migration toward towards OP9 conditioned media, it is likely that this migration
is dependent upon SDF-1α. However, there may be other signals which can modulate this SDF-1α
dependent migration.
While it has been shown that SDF-1α regulates bone marrow trafficking of leukemia cells(124), this is
the first study to demonstrate that pre-B acute lymphoblastic leukemia cells migrate toward adipose
19
tissue. Although this occurred in mice, it is not known whether adipose infiltration by leukemia cells is
common in humans. There is some evidence from the literature that this can occur with lymphoid
malignancies. For example, Maitra et al. reported on 9 patients with precursor B-cell lymphoblastic
leukemia with no evidence of marrow or blood involvement, and found that at least one of those
patients had blast cells within subcutaneous fat(125). In addition, malignant lymphocytes have been
described as “rimming” adipocytes in subcutaneous tissue(126). However, to our knowledge there has
been no systematic study examining the frequency of leukemia cells in adipose tissue, the true
prevalence of this phenomenon is not known.
One of the limitations of this study was that not all mice had detectable amounts of ALL cells in bone
marrow and spleen after retro-orbital implantation of ALL cells. Thus, this mouse model is not an
optimal model of childhood ALL. We have some preliminary data indicating that the retro-orbital route
is not as effective as tail vein for ALL engraftment. Further studies with more representative models,
such as tail vein injection of ALL cells in mice, could be used to help confirm the finding of ALL infiltration
into adipose tissue.
Since we have previously demonstrated that adipocytes protect leukemia cells from chemotherapy (56)
and absorb chemotherapeutic agents(65), it is possible that leukemia cells which migrate into adipose
tissue gain a survival advantage due to this microenvironment. Leukemia cells in this environment could
remain in a dormant state, or could receive survival signals which allow them to resist chemotherapy,
which could contribute to the increased relapse rate observed in children and adults who are obese at
the time they are diagnosed with leukemia(28). In fact, it is possible that adipocyte secretion of SDF-1α
could contribute to the links between obesity and poor outcome from other cancers which express
CXCR4, such as colon(127), breast(128), and prostate(129). Strategies which block leukemia cell
migration into adipose tissue may improve outcomes in both lean and obese patients. Indeed, AMD3100
is currently being investigated as an adjunct to chemotherapy for ALL, based on the theory that this
could mobilize leukemia stem cells from the bone marrow niche to the bloodstream, where they are
more susceptible to chemotherapy(130–132). Our findings suggest that this and other CXCR4
antagonists might also block leukemia cells infiltration to adipose tissue, which could be an additional
benefit of treatment, particularly in patients with excess adipose tissue.
20
Chapter 3
4
:
Optimization of an in vitro model to study the interaction between ALL cells and
adipocytes
3.1 Abstract
3T3-L1 cells have been widely used as a model for adipogenesis. However, despite its extensive usage,
differentiation of this cell line has been reported to be inconsistent with low efficiency. We investigated
the effect of media height during adipocyte differentiation on lipid accumulation and adipokine
secretion in mature adipocytes. Three cell lines (3T3-L1, OP9, and ChubS7) were used to test the
influence of media volume on adipogenesis. Total lipid content and lipid droplet size and number were
quantified. Adipocyte related gene expressions were quantified during the course of differentiation.
Secretion of leptin and adiponectin from mature adipocytes were measured using Enzyme-Linked
Immunosorbent Assays. The influence of oxygen partial pressure on adipogenesis was investigated using
three oxygen percentages: 5%, 21%, and 30%. Insulin sensitivity was measured by insulin inhibition of
isoproterenol-induced lipolysis and phosphorylation of Insulin Receptor Substrate-1 (IRS-1). We found
that a lower media height during adipogenesis increased total lipid accumulation, non-esterified fatty
acid release and leptin and adiponectin secretion in mature adipocytes. Insulin sensitivity was not
affected by media height during differentiation. In conclusion, media height during adipogenesis was
inversely correlated with lipid content in mature adipocytes in vitro. To achieve a high lipid content and
greater adipokine secretion, it is best to use a low media volume during differentiation.
3.2 Introduction
Biologically, adipogenesis is a process that replenishes adipocytes in the adipose tissue, which is not only
a key depot for energy storage but is also involved in the dynamic regulation of metabolism. The direct
precursors to adipocytes are pre-adipocytes, which, under the correct environmental cues, differentiate
into adipocytes. Under normal conditions, approximately 10% of adipocytes turn over in human adipose
tissue each year, maintaining the same number of adipocytes(134). Understanding the regulations of
adipogenesis is of major relevance to a variety of diseases. Adipogenesis and adipocyte physiology have
been widely studied in vitro using two cell culture models: 3T3-L1 and OP9. 3T3-L1 cells were originally
isolated from clones of Swiss 3T3 fibroblasts(135). OP9 cells were established from the calvaria of
newborn mice genetically deficient in functional macrophage colony-stimulating factor (M-
CSF)(136).Both cell lines can be induced to differentiate into adipocytes following a method utilizing an
adipogenic hormone cocktail (AHC), first described in 1980 by Reed et al.(137). The most commonly
used protocol involves treating the confluent cell monolayer with AHC containing
isobutylmethylxanthine (IBMX, a cyclic adenosine monophosphate inducer, which accelerates the
initiation of adipogenesis by inducing c/EBPβ and PPARδ), dexamethasone, and insulin for 48 hours,
then removing IBMX and dexamethasone while further treating with insulin for 48 hours. During
4
This chapter is a modified version of the published research article Adipocyte differentiation is affected by media
height above the cell layer, of which I was first author(133).
21
adipogenesis, markers such as c/EBPβ, ap2, and PPARγ are induced, accompanied by upregulation of
fatty acid binding and synthesis. There is also an increase in mRNA and protein levels of adipocytes, such
as adiponectin and leptin(138). Both 3T3-L1 and OP9 cells differentiate into mature adipocytes 7 days
post AHC treatment. However, there have been reports on low differentiation yields and variations in
differentiation efficiency(139–141). ChubS7 is an immortalized human pre-adipocyte cell line.
We have observed inconsistency in the degree of lipid accumulation (Oil Red O staining and visualization
of percent of differentiated cells) using this protocol in both 3T3-L1 and OP9 cells, particularly when cells
are differentiated in different tissue culture plates and flasks. One potential difference between tissue
culture containers is the variation in media height above the cell layer, when media volume is not
adjusted accordingly. This could be due to change in oxygen content at the cell layer, since oxygen
content decreases rapidly with increased medium height(142). Since hypoxia is known to inhibit
adipogenesis(143), we hypothesized that one of the contributors to poor yield of adipocyte
differentiation is the height of the media above the cell layer. The present study was designed to
determine the impact of media height on adipocyte differentiation using three adipocyte cell lines: 3T3-
L1, OP9, and ChubS7.
3.3 Results
3.3.1 Lipid accumulation in mature adipocytes is directly proportional to media height
To study the direct effect of media height on adipogenesis, 3T3-L1, OP9, and ChubS7 cells were
differentiated into mature adipocytes in 24-well plates using three media volumes: 1500, 650, and
325µL, corresponding to media heights of 0.75, 0.325, and 0.1625cm. As shown in figure 3.1, there was
a significant increase in total lipid content (measured by Oil Red O) associated with decreased media
volume (p<0.01). These three cell lines differed intrinsically in their differentiation capacity, where 3T3-
L1 cells were the most sensitive to volume changes, with the greatest relative increase in lipid content
with a decrease in media volume.
Figure 3.1 Lipid accumulation in adipocytes differentiated with three different volumes. Lipid content
were measured in three pre-adipocyte cell lines after differentiation in 24-well plates using three media
volumes (1500, 650, and 325 µL). Microscopy pictures were taken by a blinded observer in the middle
region of each well to avoid edge effects. Analysis was done treating media volume as a continuous
predictor. P-values indicate significant changes in total lipid content with decreased media volume.
22
A reason why a lower volume leads to greater lipid accumulation could be the concentration of secreted
molecules participating in autocrine signaling. We developed a tilting flask model in which 3T3L1 cells
were differentiated in the same media, though at different depths. Therefore, in this model, cells would
be exposed to similar concentrations of solutes in media at all locations, but different media heights at
different regions. The flask was arbitrarily divided into five equal regions, where region 1 had the
greatest height of media (~12 mm) and region 5 had the least (~2 mm). As in the 24 well plates, media
height was inversely associated with lipid accumulation (p<0.001) (Figure 1B). This effect was due to
both an increase in the mean droplet size (p<0.001) and total droplet number (p<0.001) with decreasing
media height (Figure 3.2A). Figure 3.2B shows 3T3-L1 mature adipocytes differentiated in three media
volumes stained with Oil Red O (red, for lipid droplets) and hematoxylin (blue, for nuclei) at x200
magnification. A greater amount of lipid is accumulated in the lower volume of differentiation, with
larger lipid droplet size as well.
Figure 3.2 Lipid accumulation in mature 3T3-L1 adipocytes is directly proportional to media height. (A)
3T3-L1 cells differentiated in a tilting flask accumulate lipids differently at different depths. (B)
Representative pictures of mature 3T3-L1 adipocytes differentiated using three volumes (1500, 650, and
325 µL). Cells stained with oil red O (lipid droplet) and hematoxylin (nuclei), shown at x200 magnification.
Volume and region, in the tilting flask experiment, were treated as a continuous variable. The log
10
transformation was utilized to lessen the effects of outliers due to the highly skewed distribution of lipid
content. P-values indicate significant changes from flask region 1 to 5.
3.3.2 Lower media height increases adipocyte related gene expressions and leptin secretion
23
To investigate the effect of media volume on adipocyte differentiation on a molecular level, we
measured adiponectin, leptin, and PPARγ expression by qPCR in 3T3-L1 cells differentiated with 5, 3.2,
and 1.6mL in a 6-well plate. These volumes yielded media heights comparable to those we used in the
24-well plates. Adiponectin and leptin expression were not different between the 1.6 and 3.2mL media
volumes, but were both lowest in the highest media volume (Figure 3.3 A B). PPARγ expression was
highest in the lowest media volume compared to other two volumes (Figure 3.3 C).
Figure 3.3 Gene expressions of adipocyte-specific markers in relation to media height during
differentiation. Three media heights were used to differentiate 3T3-L1 adipocytes. 5, 3.2 and 1.6 mL, in
6-well plates. The log
10
transformation was used on the data and analyzed separately for each gene.
*p<0.05, †p<0.001
Similar to gene expression patterns, both adiponectin and leptin secretion increased with decreasing
volume of media during differentiation (Figure 3.4). Interestingly, there were no detectable amounts of
leptin in OP9 conditioned media, though it is possible that levels were below the limit of detection for
our assay (0.2ng/mL; data not shown).
Figure 3.4 Adipokine secretion from adipocytes differentiated with three volumes. (A) Leptin and (B)
Adiponectin secretion over 48 hours were measured by ELISA. After differentiation, adipocytes were
switched to the same amount of complete media for each well and incubated for 48 hours. Media were
then collected, filtered with 0.22µm syringe filters, and stored at -80 C until ELISA analysis. Data was
log
10
transformed and analyzed separately for each cell line and cytokine. *p<0.05, **p<0.01, †p<0.001
3.3.3 Hypoxia inhibits adipogenesis in 3T3-L1 cells
To investigate whether differences in adipogenesis were caused by differing oxygen concentrations at
the cell layers, we differentiated 3T3-L1 preadipocytes in 5%, 21%, and 30% O
2
. Two differentiation
24
volumes were used in each oxygen percentage in 24-well plates: 1500µL and 325µL.The partial
pressures of oxygen at the cell layers were calculated for each condition (see formula in methods, Table
3.1). Three sets of hypoxia experiments and two sets of hyperoxia experiments were performed, each
including a simultaneous normoxia control.
5% 21% 30%
1500µL 0 115.41 183.81
650µL 18.85 140.45 208.85
325µL 28.57 150.02 218.57
Table 3.1
O
P
2
(mmHg) at the cell layer with different ambient oxygen percentages and media volumes in
a 24-well plate.
In all three oxygen percentages, the lower media volume was associated with a significant increase in
total lipid content (p<0.001), lipid droplet size (p<0.001), and droplet number (p<0.001) (Figure 3.5).
Hypoxia significantly inhibited lipid accumulation in mature 3T3-L1 cells (p<0.001). Lipid droplet number
(grouping both volumes of differentiation) increased significantly with hyperoxia (p<0.05, Figure 3.5C),
though there was no significant effect on overall lipid content (p=0.16, Figure 3.5A). Interestingly, in the
325µL condition, lipid droplet number decreased while the droplet diameter increased in 30% oxygen.
As a result, the difference in total lipid content between 21% and 30% were not statistically significant.
The increased lipid droplet diameter may be more reflective of adipocytes in vivo, since white adipose
tissue cells in vivo are unilocular. However, a true unilocular adipocyte cannot be readily differentiated
in a 2D culture system due to physical constraints.
Figure 3.5 Lipid accumulation in 3T3-L1 cells differentiated under different oxygen contents. (A) Lipid
content, (B) droplet diameter, and (C) droplet count in 3T3-L1 adipocytes differentiation in two different
media volumes (1500 and 325µL) and three oxygen contents (5, 21, and 30%). *p<0.05; †p<0.001
3.3.4 Insulin sensitivity is similar in 3T3-L1 adipocytes differentiated with different volumes
To investigate insulin sensitivity in 3T3-L1 adipocytes caused by a difference in differentiation volume,
we performed lipolysis assays and Western blotting of IRS-1 and phosphoIRS-1. In normal culture
conditions, adipocytes show very low levels of lipolysis. Therefore, isoproterenol, a beta-adrenergic
agonist, was used to stimulate lipolysis, simulating physiological FFA mobilization. Non-esterified fatty
acid (NEFA) release in media was significantly greater from adipocytes differentiated with the lower
volume following treatment with 1µM isoproterenol (Figure 3.6A). Upon addition of insulin, NEFA
25
release was significantly suppressed, by just under 50%, from both conditions. However, the absolute
amount of suppression was approximately 3 times greater in adipocytes differentiated in 325 µL (Figure
3.6B). Interestingly, both basal and stimulated phosphorylation of IRS-1 Y895 was lower in adipocytes
differentiated in the lower volume. However, insulin stimulation of pIRS-1 measured as fold-change over
basal phosphorylation was similar between volumes (Figure 3.7). GAPDH was used as a loading control.
It appears that less protein was loaded in the 1.6 mL conditions; however, the ratios between
phosphorylated IRS-1 and total IRS-1 indicate similar response to insulin stimulation (Figure 3.7 right).
-0.4
-0.3
-0.2
-0.1
0.0
**
NEFA (mEq/L)
0.00
0.25
0.50
0.75
1.00
1500 L
325 L
Insulin (100nM)
Isoproterenol (1 M)
-
+
+
+
**
**
**
*
NEFA (mEq/L)
A B
Figure 3.6 Insulin sensitivity in 3T3-L1 cells differentiated under different volumes. The experiments
were conducted in adipocytes differentiated in 24-well plates with two volumes (1500 and 325 µL).
When treating the adipocytes with isoproterenol and insulin, the volumes were the same for all the
conditions. Basal lipolysis, without insulin and isoproterenol, was measured and was below the
detection limit of the assay. (A) Adipocyte NEFA secretion plus 1µM isoproterenol with and without
insulin (100nM). (B) Suppression of NEFA release by 100nM insulin in the two volumes of differentiation.
*p<0.05, **p<0.01 comparing 325 µL to 1500 µL.
0 10 100
5 1.6 5 1.6 5 1.6
[Ins] (nM)
Volume (mL)
GAPDH
pIRS-1 Y895
IRS-1
0 10 100
0
2
4
6
5 mL
1.6 mL
Ins (nM)
Ratio of pIRS-1 over IRS-1
(Arbitrary Unit)
Figure 3.7 Insulin signaling in 3T3-L1 cells differentiated under different volumes. The experiments were
conducted in adipocytes differentiated in 6-well plates with two volumes (5 and 1.6 mL). Left:
representative western blot of phosphoIRS-1 Y895, total IRS-1 and GAPDH of adipocytes stimulated with
different concentrations of insulin. (B) Densitometric quantification of n=3 blots.
26
4.3 Discussion
Since the establishment of 3T3-L1 as a model of adipocytes in vitro, protocols have been established to
differentiate 3T3-L1 cells into adipocytes. The most widely used protocols now are modified versions of
the differentiation cocktail method first described by Reed et al. in 1980, in which they described using
3.5mL medium in a 6-cm dish(144), which we calculate would yield a media height of 0.124cm. However,
in an earlier publication in 1976, the same group described using 16mL medium in a 10-cm dish to
differentiate the 3T3-L1 cells(145), which is 0.204cm of media. Articles since then utilizing similar
protocols to induce adipogenesis have reported results of differentiation to be inconsistent(139). We
have also noticed inconsistency in the degree of differentiation (Oil Red O staining and visualization of
percent of differentiated cells) in 3T3-L1 cells, which prompted us to investigate the possible causes of
this problem.
In this study, we have shown that adipocyte differentiation is very sensitive to media volume. Lower
media volume during adipogenesis results in 1) a higher amount of lipid accumulation, due to increase in
both lipid droplet size and number; 2) up-regulation of genes involved in adipogenesis; and 3) higher
adiponectin secretion in mature 3T3-L1 and OP9 adipocytes, and 4) higher leptin secretion in mature
3T3-L1 adipocytes. We further show that adipocyte lipid accumulation is sensitive to oxygen, where
hypoxia inhibits lipid accumulation. Interestingly, we have also noticed using a low media volume during
differentiation, 3T3-L1 cells were able to retain their differentiation efficiency with increased passage
number (data not shown). Our data clearly demonstrated the importance of keeping the media height
consistent during differentiation, especially when using cell culture dishes and plates of various surface
areas.
The tilting flask experiment showed a clear linear correlation between total lipid accumulation and
media height above the cell layer in mature adipocytes. Since the cells in the flask were exposed to the
same media, the effect is not likely to be caused by differences in concentrations of secreted molecules
by the differentiating cells. Since media height is directly correlated with the partial pressure of oxygen
at the cell layer, we investigated into the effects of oxygen on adipogenesis.
Hypoxia is known to inhibit certain types of differentiation in stem cells(146–148). Our findings that
higher media volume and lower oxygen percentage inhibit 3T3-L1 adipogenesis may be related to this.
However, it is interesting that the differentiation of mesenchymal stem cells and preadipocytes into
adipocytes often occurs in relatively hypoxic environments, such as the bone marrow(149) and adipose
tissue(150), which could have oxygen level of 15.2mmHg (2%) in obese mice(151). Further investigation
is needed to determine the molecular mechanism linking oxygen tension and lipid accumulation.
In vivo, adipocytes are unilocular, while they accumulate multiple smaller lipid droplets in vitro. This is
most likely due to physical constraints of the flat growth surface of the monolayer of cells. We have
performed preliminary experiments of differentiating adipocytes in 3D, and obtained unilocular
adipocytes that resemble those seen in vivo. Therefore, mechanical pressure from the environment
could also influence lipid accumulating in adipocytes. We have not to date tested whether oxygen
concentration has additional effects on adipocyte differentiation in these 3D cultures. To test whether
27
the pressure caused by the differentiation media above the cell layer was a variable, we differentiated
adipocytes using “ceiling culture” (152,153). The system uses the buoyant property of adipocytes by
allowing pre-adipocytes to adhere to the bottom of a culture flask and then filling the flask completely
with media and flipping the flask upside down during the time course of differentiation. We did observe
a trend of increased lipid content in the flask that was upside down compared to the control flask which
was also filled completely but right side up (n=1). However, because the lipid content in both conditions
was not nearly as much as cells differentiated normally in plates, we did not pursue this line of
experimentation.
Although adipocytes differentiated in a lower volume exhibited amplified lipid accumulation, increased
adipogenic gene expression and greater basal lipolysis, these effects did not coincide with altered IRS-1
phosphorylation at tyrosine 895. Because phosphorylation of IRS-1 at various serine and tyrosine
residues determines its activity, further experiments are needed to confirm the effect of differentiation
volume on IRS-1 signaling. In any case, researchers measuring insulin signaling or sensitivity in
adipocytes should pay close attention to media volume during differentiation.
In conclusion, our study demonstrated that a small change in media volume during adipogenesis could
lead to dramatic changes in the lipid content and differentiation of mature adipocytes. A lower media
volume (media height of 0.16cm) leads to more complete adipogenesis in 3T3-L1, OP9, and ChubS7 pre-
adipocyte cell lines. This finding may be valuable to researchers using pre-adipocyte cell lines for
obtaining better, more consistent results in adipogenesis.
28
Chapter 4:
Adipocytes decrease ALL intracellular daunorubicin without changing surface
MDR-1 expression
4.1 Abstract
Daunorubicin (DNR) is an anthracycline widely used in childhood cancer treatments, including ALL and
AML. We have previously shown that murine adipocytes and adipose tissue explants protect murine ALL
cells from DNR treatment. In this Chapter, we examine the accumulation and metabolism of DNR in ALL
cells alone versus in co-culture with adipocytes. First, we showed that murine (3T3-L1) and human
(ChubS7) adipocyte are both capable of protecting human ALL cell lines, BV173, RS4;11, and Nalm6,
from DNR treatment. We then found the presence of adipocytes significantly lowered ALL intracellular
DNR, without altering surface multi-drug resistance protein 1 (MDR-1) expressions. Using confocal
fluorescence microscopy and high pressure liquid chromatography (HPLC), we detected and quantified
DNR accumulation in adipocytes. We also quantified the DNR metabolite, daunorubicinol, in adipocyte
lysate. In conclusion, adipocyte protection of ALL cells against DNR may be explained, in part, by the
shift in distribution of the drug and metabolism by adipocytes.
4.2 Introduction
Daunorubicin (DNR) is an anthracycline widely used in childhood cancer treatments, including ALL and
AML. It is a topoisomerase II inhibitor, and it intercalates in between DNA base pairs, causing DNA
double strand breaks and accumulation of intracellular ROS. Anthracyclines increase cellular oxidative
stress by altering mitochondrial bioenergetics. They stimulate oxygen radical generation in the
mitochondria by interfering with the electron transport chain(154). DNR distributes widely into tissues,
but is primarily metabolized in the liver to daunorubicinol, which has drastically decreased cytotoxic
effects compared to DNR(155,156). The efficacy of the drug is dependent on its delivery into target cells,
and there has been many studies on delivery of daunorubicin using liposomes or nanoparticles(157,158).
We have previously reported the protective effect of adipocytes on ALL cells against DNR. However, the
molecular mechanisms of the protection remain unknown. We have shown that diet-induced obesity in
mice alters the pharmacokinetics of vincristine. Therefore, we decided to explore whether adipocytes
might change DNR accumulation in a co-culture model of adipocytes and ALL cells.
Energy-dependent efflux of chemotherapy drugs is mostly mediated by transmembrane transporters in
the ATP-binding cassette (ABC) proteins superfamily. One member of the superfamily, P-glycoprotein
(Pgp, encoded by the gene abcb1, also known as MDR-1), is the most widely studied. Many studies have
shown that increased Pgp expression is correlated with drug resistance in cancer treatment(159–161).
We have previously performed preliminary experiments to show that ALL cells express a variety of ABC
transporter genes, however, exposure to DNR did not cause upregulation in any of them. We developed
a DNR resistant cell line using BV173 ALL cells using the pulse and rescue method, and the only ABC
29
transporter that was upregulated was Pgp. Therefore, we focus the bulk of our attention on this
important drug efflux protein.
4.3 Results
4.3.1 Adipocytes protect ALL cells from DNR
Using a murine diet-induced obesity (DIO) model of leukemia, we showed that DIO mice developed
progressive leukemia despite vincristine treatment(56). When we measured plasma vincristine
concentrations, there was no difference between the DIO and control groups. We recapitulated the in
vivo phenomenon in vitro and showed that adipocytes protect murine leukemia cells against four
chemotherapy drugs, including daunorubicin, in direct co-culture. We also found adipocytes offered
significant protection against vincristine to 8093 cells growing in TransWells. Here, to extend this finding
to other ALL cell lines and DNR treatment, we co-cultured 3T3-L1 and ChubS7 adipocytes each with 3
human leukemia cell lines. Both types of adipocytes significantly protected ALL cells from DNR
compared to no feeder control (Fig. 4.1). For RS4;11 and Nalm6, adipocytes protected significantly more
than pre-adipocytes. Adipocytes protected more than pre-adipocytes in BV173 cells in general, although
statistical significance was not reached (p=0.057 for 3T3-L1 and p=0.054 for ChubS7).
B V 1 7 3 R S 4 ;1 1 N a lm 6
0
1
2
2 .5
5 .0
7 .5
N F
F
A
*
*
V ia b le C e lls (x 1 0
5
)
*
p = 0 .0 5 7
*
*
B V 1 7 3 R S 4 ;1 1 N a lm 6
0
1
2
3
4
5
**
***
**
**
**
p = 0 .0 5 4
3 T 3 -L 1 C h u b S 7
Figure 4.1 Adipocytes protect ALL cells from DNR. 3T3-L1 and ChubS7 pre-adipocytes (F, lined bar) and
adipocytes (A, black bar) co-cultured with ALL cells in TransWells with DNR treatment (BV173 100nM,
RS4;11 100nM, and Nalm6 200nM). Cell counts were performed using trypan blue exclusion after 72
hours. Approximately 200,000 to 250,000 cells were plated initially. The doses of DNR were 100nM for
BV173 and RS4;11 and 200nM for Nalm6, which are EC
90
doses (as seen in the No Feeder (NF) conditions,
gray bar). * p<0.05, ** p<0.01, *** p<0.001 compared to NF, or F compared to A, as indicated by the
brackets.
4.3.2 ALL cells accumulate less DNR with no change in MDR-1 surface expression in the presence of
adipocytes
To address whether adipocyte protection against daunorubicin was via decreasing leukemia cell
intracellular DNR, we next measured intracellular DNR by flow cytometry. We treated BV173 with 60nM
30
DNR and SEM with 200nM DNR for 24 and 48 hours alone or in co-culture with 3T3-L1 pre-adipocytes or
adipocytes. At each time point, the cells were collected and labeled with MDR-1 surface antibody
conjugated to APC. Flow cytometry analysis was then performed to detect intracellular DNR (PE channel)
and surface MDR-1 expression (APC channel). Cells that were not treated with DNR and without labeling
with MDR-1 antibody served as negative controls for DNR and MDR-1, respectively. DNR-resistant BV173
cells made by long term culture in progressively increased DNR concentrations were used as positive
control for MDR-1. We found a significant decrease in intracellular DNR in both BV173 and SEM co-
cultured with adipocytes at both time points (Fig. 4.2A). Interestingly, co-culturing with pre-adipocytes
did not decrease ALL intracellular DNR, indicating that this is may be an effect specific to adipocytes.
However, the presence of feeder cells did not preferentially alter MDR-1 expression in ALL cells. There
was an overall increase in MDR-1 with DNR treatment in BV173 but not in SEM (Fig. 4.2B). Therefore,
the decreased intracellular DNR and increased survival of BV173 when co-cultured with adipocytes is
independent of its MDR-1 expression. Further experiments using Western blots to detect cell membrane
MDR-1 expression on these ALL cells would be needed to better quantify the protein expressions.
B V 1 7 3
N F A N F A
0
2
4
6
8
2 4 h r 4 8 h r
**
*
D N R M F I
S E M
N F A N F A
0
6
1 2
1 8
2 4 h r 4 8 h r
** **
**
**
B V 1 7 3
N F A N F A
0 .0
0 .5
1 .0
1 .5
2 4 h r 4 8 h r
* **
M D R -1 M F I
S E M
N F A N F A
0 .0
0 .2
0 .4
0 .6
0 .8
2 4 h r 4 8 h r
D N R M D R -1
Figure 4.2 Quantification of (A) intracellular DNR and (B) surface MDR-1 expression in BV173 and SEM
over no feeder (N), pre-adipocyte (F), and adipocytes (A) after 24 or 48 hours of DNR treatment. Flow
cytometry was used to record the median intensity of DNR (PE channel) and MDR-1 (APC channel).
Single color controls were included to compensate for interference between the two channels and
establish the background fluorescence. Median fluorescence intensity (MFI) was calculated by
subtracting the background fluorescence from each readout, and then dividing by the background
fluorescence. Therefore, the control conditions have an MFI of 0. * p<0.05, ** p<0.01 compared as a
group at 24 hr and 48 hr to control without DNR treatment.
DNR efflux experiments were done with BV173 cells by pre-loading the cells with 17µM DNR for 1 hour.
Then the cells were washed with ice cold PBS and plated into fresh media. Flow cytometry measurement
of intracellular DNR was performed to establish starting point (100%). Samples were then taken at each
time point from 30 minutes up to 4 hours for flow cytometry analysis on intracellular DNR. There were
no significant differences of drug efflux in cells plated with adipocytes compared to control conditions,
which was consistent with the no change in surface MDR-1 expressions.
31
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
0
2 0
4 0
6 0
8 0
1 0 0
C o m ple te
A d ip o
T im e (m in .)
%
Figure 4.3 DNR efflux from BV173 cells co-cultured with adipocytes. Flow cytometry quantification of
intracellular DNR in BV173 cells after pre-loading with 17µM DNR for 1 hour. Pre-loaded cells were then
placed in either complete media (Complete) or in TransWells in co-culture with adipocytes (Adipo).
Samples were then taken at 20, 40, 60, 120, and 240 minutes. Percentage of intracellular DNR was
calculated by dividing by the intracellular DNR median fluorescence at time=0 min. immediately after
pre-loading.
4.3.3 Adipocytes take up DNR in a time-dependent manner
To test the possibility that adipocytes might protect leukemia cells by sequestering DNR and then
metabolizing it to daunorubicinol, we treated adipocytes with 100nM DNR for 4, 24, and 48 hours.
Media were collected and filtered through 0.22 µm syringe filters and frozen in -80 degrees Celsius until
they were ready for use. Support of cell growth was tested by plating BV173 cells in these media,
including a freshly prepared 100nM condition as control (time 0). Cell counts were performed using
Trypan blue exclusion. There was a significant increase in cell proliferation in DNR media incubated for
as short as 4 hours, and trending towards more cell growth with longer incubation time (Figure 4.4A).
This indicates that adipocytes are depleting DNR from media in a time-dependent manner. Interestingly,
there was no significant difference between media incubated for 4 hours versus 24 hours, which could
indicate a rapid initial uptake that tapers off until another detoxification mechanism takes place.
Therefore, to test whether adipocytes metabolize DNR, we increased its dose from 100nM to 250, 500,
and 1000nM and treated adipocytes for 48 hours. Then, both media and cell lysates were collected for
BV173 proliferation assays. As shown in Figure 4.4B, even the adipocyte lysate from the 1000nM DNR
media did not cause cell death (dotted line indicates number of cells initially plated). This implies that
adipocytes could be sequestering DNR and then metabolizing it to its less toxic form, daunorubicinol.
Figure 4.4C shows BV173 growth in DNR media conditioned with adipocytes for 48 hours compared to in
regular media of those same doses of DNR. Conditioning over adipocytes significantly decreases DNR
toxicity (at 100nM DNR). We have performed some preliminary HPLC measurements on intracellular
DNR and daunorubicinol in BV173 cells with and without co-culturing with 3T3-L1 adipocytes. We found
a trend of decreased DNR and increased daunorubicinol with 3T3-L1 adipocyte co-culture (Figure 4.5).
32
0 1 0 2 0 3 0 4 0 5 0
0
1
2
3
4
5
T im e (h r)
c e lls/w e ll (x1 0
5
)
*
*
p = 0 .0 6
0 2 5 0 5 0 0 1 0 0 0
0
2
4
6
8
D N R (n M )
c e lls/w e ll (x1 0
5
)
0 1 0 0 2 5 0 5 0 0 1 0 0 0
0 .0
0 .6
1 .2
1 .8
3 .0
4 .5
6 .0
D N R (n M )
c e lls/w e ll (x1 0
5
)
A C M w / D N R
D N R c o n tro l
* *
A B C
Figure 4.4 Adipocytes absorb DNR in a time-dependent manner. BV173 cell growth was quantified with
trypan blue after 72 hours in (A) media containing 100nM DNR that were conditioned with adipocytes
for 4, 24, and 48 hours; (B) lysates of adipocytes that were treated with 250, 500, and 1000nM DNR for
48 hours; (C) media containing various doses of DNR that have been conditioned with adipocytes for 48
hours (grey bars), compared to media of the same DNR doses that were not conditioned (black bars). *
p<0.05, ** p<0.01.
B V 1 7 3 B V 1 7 3 + A d ip o
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
B V 1 7 3 In tra c e llu la r D N R
p g /m L
B V 1 7 3 B V 1 7 3 + A d ip o
0
2
4
6
B V 1 7 3 In tra c e llu la r D a u n o ru b ic in o l
p g /m L
p = 0 .0 5
p = 0 .0 8
Figure 4.5 BV173 intracellular DNR and daunorubicinol measurements by HPLC. BV173 cells were
treated with 100nM DNR for 16 hours with and without co-culturing with 3T3-L1 adipocytes. n=5.
4.4 Discussion
We have previously shown that adipocytes sequester the lipophilic vincristine in a co-culture system
with leukemia cells(162). However, the concentrations of vincristine in media and in leukemia cells were
unaltered. DNR is amphiphilic, due to its tetracenequinone ring structure and the sugar moiety. It enters
cells by passive diffusion, which largely depends on the interaction between DNR and the cell membrane.
Interestingly, confocal images of adipocytes treated with different concentrations of DNR revealed that
DNR preferentially accumulate in the cytosol. When the cytosol becomes saturated, DNR occupies lipid
droplets in the adipocytes. Reports on doxorubicin distribution in tissues show the highest
concentrations in the lung, kidney, spleen, small intestine, lymph nodes, and liver, and the lowest tissue
concentrations in adipose tissue, skin, and skeletal muscle(163–165). Similar findings were reported on
DNR distribution in rabbits(155). This pattern is consistent with the initial rapid distribution of
anthracyclines into tissues highly perfused with blood. It was observed in rabbits that within 4 hours of
intravenous injection, 95% DNR was cleared from the plasma(155). Interestingly, even though only a
small concentration of DNR was detected in the adipose tissue, there was a substantial accumulation of
33
daunorubicinol. The ratio of daunorubicinol to DNR was higher in adipose tissue than in brain, heart,
lung, and spleen, indicating a high level of metabolism of DNR in the adipose tissue. The metabolism of
DNR by adipose tissue may impact in vivo pharmacokinetics, especially in individuals with increased
adiposity.
There has been evidence that adiposity alters the pharmacokinetics of anthracyclines. Specifically, body
weight was found to be inversely related to doxorubicin clearance and positively correlates with
elimination half-life(165). However, this particular study only measured doxorubicin and doxorubicinol
concentrations in patients’ serum, which demonstrated multiexpotential decline. There were no
explanations for the observed increase in elimination half-life in obese patients. The liver is a major site
of anthracycline metabolism, therefore, studies have speculated that decreased liver functions in
obesity contributed to the prolonged half-life, and patients with abnormal liver function tests have been
shown to have lower doxorubicin clearance and increased toxicity(166,167). However, findings from our
current study offer a new hypothesis. Adipocytes may be accumulating and metabolizing DNR, slowing
the elimination of the drug. Preliminary data from our HPLC measurements show that 3T3-L1 adipocytes
have the capacity to convert DNR into daunorubicinol, and can sequester both in large quantities
intracellularly. We also found BV173 cells co-cultured with 3T3-L1 adipocytes had significantly lower
intracellular DNR (Fig. 4.2 and preliminary HPLC data).
MDR-1 expression has been reported to be associated with drug resistance in many types of
cancer(158,160,161,168). However, we did not find an increased expression in the leukemia cells co-
cultured with adipocytes, although the MDR-1 surface expression increased overall with DNR treatment
in BV173 cells. Since adipocytes were depleting DNR from the media, the ALL cells’ drug exposure was
decreased, which would not lead to increased MDR-1. This is consistent with our DNR efflux experiment
results, where there was no change in ALL cells alone or in co-culture with adipocytes.
In summary, our data showed that adipocytes are important players in the microenvironment of
leukemia treatment. One of the mechanisms of protection offered by the adipocytes is sequestering
DNR. Further studies are needed to investigate the role of adipocytes in DNR metabolism in vivo
comparing obese versus normal weight individuals.
34
Chapter 5:
Adipocytes protect ALL cells from chemotherapy by reducing oxidative stress
5.1 Abstract
Adipocytes play an important role in cancer progression and treatment. In this chapter, the crosstalk
between acute lymphoblastic leukemia cells (ALL) and adipocytes is investigated. In vitro co-culture
experiments showed that adipocytes could protect ALL cells from oxidative stress induced by drugs and
irradiation. Microarray analysis of adipocytes exposed to ALL showed an upregulation in the Nrf2-
mediated oxidative stress response. Microscopy and qPCR of selected downstream genes indicated an
increase in intracellular ROS and activation of Nrf2 in adipocytes. The oxidative stress response in
adipocytes, elicited by ALL or cobalt chloride, led to secretion of soluble factors, which protected ALL
cells from daunorubicin (DNR). Blocking the oxidative stress response with chemicals such as buthionine
sulfoximine and auranofin reversed the DNR protective effects. Exogenous supplementation of
antioxidants to ALL cells rescued DNR induced cell death. Collectively, our investigation shows that ALL
cells elicit an oxidative stress response in adipocytes, leading to secretion of survival factors by
adipocytes, and protection of ALL cells against DNR.
5.2 Introduction
Over the past decade, the field of obesity-associated cancer research has gained significant interest.
Many solid tumors reside in locations rich with adipocytes, including breast, ovarian, pancreatic, colon,
and prostate cancers. These cancer types have thus been the main focus on teasing apart the
mechanisms of how obesity/adipocytes influence cancer progression and treatment. However, obesity
has been shown to be associated with poorer event-free survival in acute lymphoblastic leukemia
(28,29). We and others have demonstrated that leukemia cells can be found in the adipose tissue
(57,125,126). Therefore, ALL is also in close proximity to adipocytes. We have previously shown that
adipocytes provide survival advantages to ALL cells by promoting pro-survival genes like Bcl2 and Pim2
and providing essential amino acids like asparagine and glutamine (56,169). Here, we propose to
investigate the role of adipocytes in alleviating oxidative stress caused by chemotherapy treatment in
ALL cells.
Oxidative stress is induced by normal cellular processes like oxidative phosphorylation, where free
radicals and hydrogen peroxide are formed. Cancer cells exhibit elevated intracellular reactive oxygen
species (ROS) due to their metabolic and signaling aberrations. The transcription factor nuclear factor
erythroid 2-related factor 2 (Nrf2) is one of the most important regulators of oxidative stress (170,171).
Nrf2 activates genes involved in the response to oxidative stress, including heme oxygenase-1 (HO-1)
and the two subunits of glutamate cysteine ligase, modifier (GCLM) and catalyzing (GCLC), which are
involved in glutathione synthesis (72–74,170).
There is evidence in literature that regulation of oxidative stress is crucial for leukemia cell drug
resistance. In acute myeloid leukemia cells, cytarabine and daunorubicin both induce HO-1 mRNA and
protein expression in a dose-dependent manner, and silencing HO-1 increased the sensitivity of the cells
35
to both cytarabine and daunorubicin (172). In chronic myeloid leukemia cells, inhibiting HO-1 reversed
imatinib resistance (173). It has been reported that bone marrow stromal cells can directly support
primary ALL cell growth in vitro by providing cysteine, which maintains glutathione levels in ALL cells and
results in protection against oxidative stress (174). On the other hand, inducing oxidative stress in
leukemia cells have been shown to be an effective strategy for counteracting drug resistance and
selectively killing the cancer cells (83,84,175). For example, chaetocin, a mycotoxin that imposes
oxidative stress by inhibiting thioredoxin reductase-1, has been shown to effectively overcome bone
marrow stromal induced imatinib resistance in murine hematopoietic cell line TonB210 with and
without BCL-ABL expression (175). Aurarofin, an oral gold-containing triethylphosphine used in the
treatment of rheumatoid arthritis, have been reported to induce ROS levels resulting in cell death in
primary chronic lymphoblastic leukemia cells, including those with the biologic and genetic features that
are associated with poor clinical outcomes (84).
Given the close interaction between adipocytes and ALL cells, we speculate a metabolic symbiosis
relationship between the two cell types, similar to those reported between breast cancer cells and
cancer associated fibroblasts(176,177). In this chapter, we hypothesize that adipocytes confer
chemotherapy resistance in ALL cells in part by alleviating oxidative stress caused by chemotherapy.
5.3 Results
5.3.1 Adipocytes protect ALL cells from oxidative stress-induced cell death
We have previously reported that adipocyte protects murine ALL cells against daunorubicin, an
anthracycline, in a co-culture system (56). Daunorubicin, as mentioned in Chapter4, is a widely used
chemotherapy. It is also a high ROS inducer (71). We reported in Chapter 4 that both 3T3-L1 and ChubS7
adipocytes protected BV173, RS4;11, and Nalm6 significantly from daunorubicin treatment, compared
to no feeder and pre-adipocytes. Therefore, we investigated here whether adipocytes prevented DNR
induced ROS.
Glutathione is one of the most abundant antioxidants in cells. See schematic of glutathione synthesis
below. The heterodimer, γ-glutamulcysteine ligase (GCL) catalyzes the rate-limiting reaction in
glutathione synthesis. The messenger RNA (mRNA) levels of the two subunits, GCLC and GCLM, are
increased in animal hepatic and kidney tissues following exposure to a wide variety of oxidative stress
inducers(178–180).
To test whether DNR elicits an oxidative stress response in ALL cells and whether the presence of
adipocytes dampens the response, we measured 8093 and BV173 mRNA expressions of GCLC and GCLM.
8093 and BV173 were treated with DNR with and without co-culture of adipocytes (3T3-L1 and ChubS7,
respectively). Daunorubicin treatment significantly increased the mRNA level of both GCLC and GCLM,
36
while the increase was reversed in the co-culture with adipocytes (Fig. 5.1). Interestingly, GCLC and
GCLM mRNA both decreased significantly in 8093 co-cultured with adipocytes without DNR treatment,
implyingalleviation of the basal level of oxidative stress, as shown in HepG2 cells(181). A similar pattern
of GCLC and GCLM expressions was also observed in Nalm6 (data not shown).
0
1
2
3
4
F o ld C h a n g e
p = 0 .0 6
*
* * *
D a u n o
A d ip o
G C L C G C LM
8 0 9 3
–
–
+
–
–
+
+
+
–
–
+
–
–
+
+
+
0
1
2
3
G C L C G C LM
*
*
D a u n o
A d ip o
–
–
+
–
–
+
+
+
p = 0 .0 8
–
–
+
–
–
+
+
+
B V 1 7 3
*
p = 0 .0 6
Figure 5.1 GCLC and GCLM gene expressions are suppressed in ALL cells in the presence of adipocytes
despite DNR treatment. GCLC and GCLM gene expressions by qPCR in 8093 (left) and BV173 (right)
treated with DNR (35nM and 100nM, respectively), adipocytes, or both for 24 to 48 hours. β-actin was
included as control. * p<0.05, ** p<0.01 compared to no daunorubicin no adipocyte controls.
We used 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA) to directly measure Nalm6 intracellular
ROS during DNR treatment. While DNR induced detectable intracellular ROS within 6 hours in a dose-
dependent manner, the presence of adipocytes significantly decreased the ROS
high
percentage while the
presence of pre-adipocytes did not change the percentage at all (Fig. 5.2).
0 8 0 1 6 0 2 4 0 3 2 0
0
2 0
4 0
6 0
N F
F
A
D N R (n M )
% R O S
h ig h
*
Figure 5.2 Adipocytes alleviate DNR-induced ROS in ALL cells. Measurement of % ROS
high
population in
Nalm6 cells treated with different doses of DNR for 6 hours over no feeder (NF), fibroblasts (F), or
adipocytes (A). *p<0.05 comparing A versus NF.
Caspase 3 and cleaved caspase 3 protein expressions were evaluated in BV173 cells treated with DNR
with and without 3T3-L1 adipocytes as an indicator of apoptosis. As expected, DNR treatment
significantly increased cleaved caspase 3 to caspase 3 ratio (Fig. 5.3, p<0.001 compared to no DNR and
no adipocyte), while cells treated with DNR in co-culture with adipocytes expressed a significantly
37
decreased ratio (p<0.05). Interestingly, cells co-cultured with adipocytes without DNR treatment also
expressed significantly decreased ratio compared to the baseline condition.
C a s p a s e 3
C le aved
C a s p a s e 3
0 .0
0 .5
1 .0
1 .5
2 .0
C le a v e d /C a s p a s e 3 D N R
A dipo
* * *
*
*
–
–
+
–
–
+
+
+
D N R
A dipo
–
–
+
–
–
+
+
+
Figure 5.3 Adipocytes prevent DNR-induced cleaved caspase 3 expressions in ALL cells. Representative
blot of caspase 3 (37kDa) and cleaved caspase 3 (19 and 17kDa) in BV173 treated with DNR, adipocytes,
or both is shown on the left. Quantification of the ratio of cleaved over total caspase 3 is shown on the
right. * p<0.05, *** p<0.001 compared to no daunorubicin no adipocyte control.
To test whether the protection from adipocytes is specifically due to alleviation of oxidative stress, we
tested known oxidative stress inducers. Chaetocin is a fungal-derived toxin that inhibits thioredoxin
reductase-1, leading to increased cellular oxidative stress. We found 3T3-L1 adipocytes significantly
protected all three ALL cell lines from chaetocin induced cell death (Fig. 5.4 A, BV173 and Nalm6 p<0.01
for F vs. A, p<0.001 for NF vs. A; RS4;11 p<0.05 for NF vs. A and F vs. A). We also found that co-culturing
irradiated BV173 (12 Gy and 18 Gy) with F and A significantly protected from irradiation-induced cell
death (Fig. 5.4 B). Though in this case, adipocytes did not offer significantly more protection than
preadipocytes.
3 T 3 -L 1
B V 1 7 3 N a lm 6
0
2
4
6
V ia b le C e lls (x 1 0
5
)
* *
* * *
* *
* * *
R S 4 ;1 1
0 .0
0 .6
1 .2
1 .8
*
*
1 2 G y 1 8 G y
0 .0
0 .4
0 .8
1 .2
V ia b le C e lls (x 1 0
5
)
*
*
*
* *
3 T 3 -L 1
A B
N F
F
A
B V 1 7 3
Figure 5.4 Adipocytes protect ALL cells from oxidative stress. A. 3T3-L1 pre-adipocytes (F) and adipocytes
(A) co-cultured with ALL cells with chaetocin treatment (BV173 and Nalm6 100nM, RS4;11 50nM). B.
3T3-L1 pre-adipocytes and adipocytes co-cultured with BV173 after irradiation. Cell counts were done
with trypan blue exclusion at 72 hours. * p<0.05, ** p<0.01, *** p<0.001 compared to no feeder (NF) or
fibroblasts (F).
38
5.3.2 ALL cells induce oxidative stress in adipocytes
To study the effect of leukemia cells on adipocytes, we performed a gene expression microarray. We
exposed adipocytes to leukemia cell conditioned media (LCM). We found 3528 gene expression changes
in the adipocytes, with many genes in the oxidative stress response pathway upregulated (Table 5.1
shows the top 10 most upregulated genes). Ingenuity Pathway Analysis (IPA) identified Nrf2-mediated
oxidative stress response to be one of the most affected pathways in adipocytes, with alternations in 47
genes down-stream of the pathway. Therefore, we decided to further investigate whether ALL cells
induce oxidative stress in adipocytes.
Table 5.1. Top 10 upregulated genes
Gene symbol Name Fold change P-value
Mt2 Metallothionein 2 4.6 0.02
Aldh1l2 Aldehyde dehydrogenase 1 family, member l2 3.6 0.00004
Adm Adrenomedullin 3.5 0.0006
Trib3 Induced in fatty liver dystrophy 2 3.1 0.002
Eno2 Enolase 2, gamma neuronal 3.0 0.00002
Hmox1 Heme oxygenase (decycling) 1 2.7 0.0001
Ddit3 Dna-damage inducible transcript 3 2.7 0.0003
Myd116 Myeloid differentiation primary response gene 116 2.6 0.0003
Car6 Carbonic anhydrase 6 2.5 0.004
Got1 Glutamate oxaloacetate transaminase 1, soluble 2.5 0.003
We measured adipocyte intracellular ROS by DCFH-DA using fluorescence confocal microscopy (Fig. 5.5
left). We observed an increase in intracellular ROS with exposure to BV173 cells co-cultured in a
Transwell system after 48 hours (Fig. 5.5 right, quantification, p=0.05 Adipo + ALL vs. Adipo). Buthionine
sulfoximine, an irreversible inhibitor of glutamylcysteine synthetase, was used to treat adipocytes (20
mM) as a positive control.
B a s e lin e A L L B S O
0
2
4
6
8
F o ld C h a n g e
p = 0 .0 5
*
Adipo Adipo + ALL in TransWell Adipo + BSO
D C F D IC
39
Figure 5.5 ALL cells induced oxidative stress in adipocytes. Representative fluorescent confocal
microscopy images of 3T3-L1 adipocytes alone, with ALL in TransWells, or treated with 20mM BSO for 48
hours. Top: DCF (green) only, bottom: DIC images. Quantification of images is shown on the right.
As a transcription factor, Nrf2 is normally bound by Keap1 in the cytoplasm and marked for
ubiquitination. Upon activation, Nrf2 dissociates from Keap1 and becomes phosphorylated, which is
then translocated into the nucleus. Therefore, we performed Western blots on the cytoplasmic and
nuclear fractions of 3T3-L1 adipocytes treated with LCM for 24 hours (Fig. 5.6). There was a significant
increase in nuclear Nrf2 with LCM treatment (p<0.05), while cytoplasmic Nrf2 levels did not change.
Interestingly, despite a predicted molecular weight of 68 kDa, Nrf2 runs between 95-110kDa on gels
depending on gel types(182–184). The appearance of the double bands have been shown to be due to
phosphorylation of Nrf2, and the bands directly below the 98kDa bands are nonspecific(185).
N rf2
P 8 4
G A P D H
C yto p la s m ic N u c le a r
0
1
2
3
4
N rf2 F o ld C h a n g e
C o n tro l
+ L C M
*
C N C N
C o n tro l L C M
N o n s p e c ific
6 2 k D a
8 4 k D a
3 7 k D a
9 8 k D a
Figure 5.6 Nrf2 is translocated into the nucleus in 3T3-L1 adipocytes upon LCM treatment.
Representative blot of Nrf2 of 3T3-L1 adipocytes cytoplasmic (C) and nuclear (N) fractions with and
without LCM treatment for 24 hours is shown on the left. P84 was used as a nuclear control, and GAPDH
a cytoplasmic control. Quantifications is on the right.
Using qPCR, we confirmed that when 3T3-L1 adipocytes were exposed to BV173 cells, there is significant
upregulation of HO-1, ADM, and GCLM, and a trend of increase in Mt2 (Fig. 5.7 left). We also confirmed
the increase of HO-1 protein with Western blots (Fig. 5.7 right).
H O -1 M t2 A D M G C L C G C L M
0
2
4
6
8
F o ld C h a n g e
C o n tro l
+ A L L
*
p = 0 .0 8
* *
*
H O -1
G A P D H
D N R
A L L
–
–
+
–
–
+
+
+
40
Figure 5.7 Nrf2 downstream genes are upregulated in 3T3-L1 adipocytes co-cultured with ALL. 3T3-L1
gene expressions were measured by qPCR with (gray bar) and without (white bar) 8093 cells. β-actin
was included as control. Representative blot of HO-1 protein expression in 3T3-L1 adipocytes with DNR,
ALL or both shown on the right. GAPDH was included as loading control.
5.3.3 Oxidative stress in adipocytes leads to secretion of survival factors that protect ALL cells from DNR
We made adipocyte leukemia cell conditioned media (ALCM) using BV173 with either 3T3-L1 or ChubS7
adipocytes, and then we tested the protective effects of the ALCM against DNR treatment in 8093 and
BV173 cells, respectively. Figure 5.8 shows that ALCM significantly protected 8093 (left) and BV173
(right) from DNR treatment compared to both control media and the corresponding adipocyte
conditioned media (ACM). This experiment indicates that the mechanism of protection of adipocytes
against DNR may only be partially explained by the absorption of DNR by adipocytes, as shown in
Chapter 4.
C o n tro l A C M A L C M
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
C h u b S 7
* *
*
C o n tro l A C M A L C M
0 .0
0 .2
0 .4
0 .6
0 .8
3 T 3 -L 1
V ia b le C e lls (x 1 0
5
)
* * *
* *
Figure 5.8 ALL cells stimulate adipocytes to secrete survival factors. 3T3-L1 ACM and ALCM cultured with
8093 cells with DNR treatment (left), and ChubS7 ACM and ALCM cultured with BV173 cells with DNR
treatment (right). Cell counts were performed after 72 hours.
To mimic oxidative stress, we treated 3T3-L1 and ChubS7 adipocytes with cobalt chloride, a well-
established stabilizer of HIF-1α mimicking hypoxia. We found that ACM made with 30µM cobalt chloride
(w/ CoCl
2
) were also protective against DNR compared to regular ACM (Fig. 5.9, A. 3T3-L1 conditioned
media and B. ChubS7 conditioned media). ACM plus fresh CoCl
2
were included as controls. Given that
the mechanism of action of CoCl
2
involves stabilization of HIF-1α, HIF-1α protein levels were measured
in ChubS7 treated with CoCl
2
(Fig. 5.9 C, one representative blot shown), and gene expressions of HO-1,
GCLC, and GCLM were measured in 3T3-L1 adipocytes treated with CoCl
2
(Fig. 5.9 D).
41
H O -1 G C L C G C L M
0
2
4
6
F o ld C h a n g e
C o n tro l
+ C o C l
2
*
*
*
A C M + C o C l2 w / C o C l2
0 .0
0 .5
1 .0
1 .5
C h u b S 7
V ia b le C e lls (x 1 0
5
)
*
3 T 3 -L 1
A C M + C o C l2 w / C o C l2
0 .0
0 .1
0 .2
0 .3
0 .4
0 .5
V ia b le C e lls (x 1 0
5
)
*
A B
C D
H IF -1
G A P D H
C o C l
2
- +
Figure 5.9 Oxidative stress in adipocytes leads to secretion of survival factors for ALL cells. A. 8093 cells
were treated with 35nM DNR while exposed to 1) 3T3-L1 ACM, 2) ACM plus fresh CoCl
2
, and 3) ACM
made in the presence of CoCl
2
for 72 hours. B. BV173 cells were treated with 100nM DNR in
aforementioned conditioned media made with ChubS7. C. Representative blot of HIF-1α in ChubS7
adipocytes treated with CoCl
2
. GAPDH was used as loading control. D. 3T3-L1 HO-1, GCLC, and GCLM
gene expressions by qPCR with CoCl
2
treatment after 24 hours. All CoCl
2
treatments were at 30µM. For
cell counts, viable cells were quantified with trypan blue exclusion at 72 hours. *p<0.05 compared to
ACM (A), ACM plus fresh CoCl
2
(B) or control adipocytes (D).
5.3.4 Glutathione synthesis is partially involved in adipocyte protection of ALL cells
To study the significance of induced oxidative stress response in adipocytes in protecting ALL cells
against oxidative stress, we investigated the importance of antioxidant pathways in adipocytes. BSO is a
blocker of glutathione synthesis and auranofin is a blocker of thioredoxin synthesis. Pretreatment of
adipocytes with 20mM BSO for 24 hours partially reversed their protection of BV173 and Nalm6 against
DNR (Fig. 5.10 A, p<0.05 Adipo vs. Adipo + BSO). Pretreating adipocytes with BSO and auranofin (AUR,
250nM) for 24 hours also reversed protection (Fig. 5.10 B and C). Comparing pretreatment with BSO and
the combination of BSO and AUR on ChubS7 adipocytes, there were no significant differences (Fig. 5.10
C), indicating that there were no additive effects of BSO and AUR.
42
A B
N F
A
A + B S O + A U R
0 .0
0 .5
1 .0
1 .5
B V 1 7 3
V ia b le C e lls (x 1 0
5
)
***
*
N F
A
A + B S O + A U R
0 .0
0 .2
0 .4
0 .6
N alm 6
*
*
C
N F
A
A + B S O
A + B S O + A U R
0 .0
1 .0
2 .0
3 .0
4 .0
B V 1 7 3
V ia b le C e lls (x 1 0
5
)
*
*
* *
N F
A
A + B S O
A + B S O + A U R
0 .0
0 .5
1 .0
1 .5
N alm 6
* *
* *
3 T 3 -L 1 3 T 3 -L 1 C h u b S 7
B V 1 7 3 N a lm 6
0 .0
0 .5
1 .0
1 .5
2 .0
V ia b le C e lls (x 1 0
5
)
*
* *
*
* *
N o F e e d e r
A d ip o
A d ip o + B S O
Figure 5.10 Glutathione synthesis is partially involved in adipocyte protection of ALL cells. A. 3T3-L1
adipocytes (gray bar) and adipocytes pretreated with 20mM BSO (black bar) for 24 hours co-cultured
with BV173 and Nalm6 and treated with DNR. B. 3T3-L1 adipocytes and adipocytes pre-treated with
20mM BSO and 250nM AUR for 24 hours then co-cultured with BV173 and Nalm6 and treated with DNR.
C. Similar experiments to A and B with ChubS7 adipocytes. For all cell counts, viable cells were
quantified with trypan blue exclusion at 72 hours.
We then measured intracellular GSH in 3T3-L1 adipocytes treated with BSO, AUR, and exposed to ALL
and LCM for 24 hours. BSO significantly abolished intracellular GSH levels (Fig. 5.11). Interestingly, AUR
significantly increased GSH levels, likely as a compensatory effect from blocking thioredoxin synthesis.
3T3-L1 adipocytes treated with both BSO and AUR has intracellular GSH levels similar to those treated
with BSO only (data not shown). Exposure to ALL and LCM did not alter intracellular GSH in adipocytes,
which could be because there is GSH conjugates and GSH secretion into the media that are not
accounted for in this assay.
V e h B S O A U R A L L L C M
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
3 T 3 -L 1
G S H /m g
F o ld C h a n g e
*
*
Figure 5.11 3T3-L1 intracellular GSH measurements. 3T3-L1 adipocytes were treated with BSO, AUR, ALL
in TransWells, or LCM for 48 hours. Cells were lysed and GSH levels were measured. Protein
concentrations were measured for normalization. *p<0.05 compared to vehicle control.
5.3.5 Exogenous antioxidants cause ALL daunorubicin resistance
We tested whether exogenous antioxidants could provide protective effects to ALL cells against DNR.
Addition of glutathione (GSH) to BV173 and Nalm6 significantly decreased cytotoxic effects of DNR (Fig.
43
5.12 A, p<0.05 BV173 and p<0.01 Nalm6). By providing ALL cells with N-acetylcysteine (NAC), a precursor
to GSH, BV173 and Nalm6 both became significantly resistant to daunorubicin treatment (Fig. 5.12 B).
Similar effects were observed in RS4;11 (data not shown).
- N A C
0 .0
0 .5
1 .0
1 .5
2 .0
* *
V ia b le C e lls (x 1 0
5
)
- N A C
0 .0
0 .2
0 .4
0 .6
0 .8
*
- G S H - G S H
0 .0
0 .5
1 .0
1 .5
V ia b le C e lls (x 1 0
5
)
*
* *
B V 1 7 3 N alm 6 B V 1 7 3 N alm 6 A B
Figure 5.12 Exogenous antioxidants cause ALL DNR resistance. A.BV173 and Nalm6 treated with DNR or
DNR + GSH (20mM). B. BV173 and Nalm6 treated with DNR or DNR + NAC (10mM). For all cell counts,
viable cells were quantified with trypan blue exclusion at 72 hours.
We confirmed with flow cytometry that ALL cells treated with the combination of NAC and DNR had
significantly greater viability (Fig. 5.13 A, both BV173 and Nalm6) and trended towards less intracellular
ROS (Fig. 5.13 B, BV173 p=0.06) than cells treated with DNR alone.
C o n tro l
D N R
D N R + N A C
0
4 0
8 0
1 2 0
v ia b ility (% )
***
**
C o n tro l
D N R
D N R + N A C
0
5
1 0
1 5
M F I
p = 0 .0 7
p = 0 .0 6
A B V 1 7 3 N alm 6 B
C o n tro l
D N R
D N R + N A C
0
2 0
4 0
6 0
8 0
* *
* * *
C o n tro l
D N R
D N R + N A C
0
2
4
6
8
1 0
*
B V 1 7 3 N alm 6
Figure 5.13 Exogenous antioxidants increase ALL viability and decreases ROS despite DNR treatment. A.
Cell viability and B. Intracellular ROS evaluated using flow cytometry with DAPI and CellROX staining of
BV173 and Nalm6 treated with DNR alone or DNR and NAC.
5.4 Discussion
In this present study, we demonstrated a novel mechanism of adipocyte-mediated drug resistance in
ALL cells. Our findings may explain, in part, the higher relapse rate in obese ALL patients. The study
provides rationale for utilizing ROS-inducing anti-cancer agents to target ALL cells in the bone marrow or
adipose microenvironment. As shown from the Ingenuity Pathway Analysis, ALL cells trigger the Nrf2-
mediated antioxidant response pathway in adipocytes.
44
Oxidative stress is induced by an increase in intracellular reactive oxygen species (ROS), which are
normal byproducts generated by many cellular processes. The amount of intracellular ROS is tightly
controlled by antioxidants, which are products of the oxidative stress response pathways. ROS act as
signaling molecules to promote proliferation and survival(67–69). However, when ROS levels increase,
they could cause protein and DNA oxidation damage and exert oxidative stress on cells. Sustained high
levels of ROS could lead to cell senescence or death. Because of their high proliferation and metabolic
rates, cancer cells are under greater oxidative stress compared to normal cells, and therefore have an
active oxidative stress response pathway(186–189). However, despite having an enhanced antioxidant
system, cancer cells maintain higher ROS levels than normal cells(70). In fact, many chemotherapeutics
induce high levels of oxidative stress, targeting the sensitivity of cancer cells to further accumulation of
ROS(69,71). Anthracyclines, which are routinely given to ALL patients, are one of such chemotherapies.
This is the first report to our knowledge of ALL DNR resistance mediated by adipocytes.
Oxidative stress also plays an important role in adipocyte differentiation, during which time pre-
adipocytes acquire the ability to deposit lipids in lipid droplets(76). The mitochondria are where free
fatty acids are converted to triglycerides, and are a major source of ROS. Numerous reports have been
made on the importance of regulation of ROS in adipogenesis and adipocyte function(77–81). During
differentiation, expressions of certain antioxidant genes, such as superoxide dismutase 2 and catalase,
are induced to help balance intracellular ROS induced by fatty acid oxidation(190).
Adipocytes have been shown to play an active role in both solid tumors and blood cancers to promote
cancer progression. Cancer associated adipocytes are peritumoral adipocytes that exhibit a modified
phenotype and specific biological features(191). We have previously reported the presence of ALL cells
in mouse adipose tissue after retro-orbital implantation(56,57). Based on this finding, we demonstrate
for the first time that adipocytes protect ALL cells from oxidative stress induced by DNR, chaetocin, and
irradiation without cell-cell contact (Fig 5.4). This may be one of the many possible mechanisms of
environment-mediated drug resistance (EMDR), which contributes to minimal residual disease(192).
Adipocyte-derived soluble factors (stable after heat inactivation at 60 C for 30 minutes) have been
reported to protect chronic lymphoblastic leukemia from chemotherapies, which suggests that these are
lipid factors (95,96). However, in our experiments, ACM does not offer any protective effects against
DNR, while ALCM and ACM made with CoCl
2
do (Fig. 5.8, 5.9 A and B).
There is evidence that regulation of oxidative stress is crucial for leukemia cell drug resistance. In acute
myeloid leukemia cells, cytarabine and DNR both induce HO-1 mRNA and protein expression in a dose-
dependent manner, and silencing HO-1 increased the sensitivity of the cells to both drugs(172). It has
been reported that bone marrow stromal cells can directly support primary ALL cells growth in vitro by
providing cysteine, which maintains glutathione levels in ALL cells and results in protection against
oxidative stress(174). This is in agreement to the effects of GSH and NAC observed in our experiments.
On the other hand, inducing oxidative stress in leukemia cells have been shown to be an effective
strategy for counteracting drug resistance and selectively killing the cancer cells(83,84,175). For example,
chaetocin, a mycotoxin that imposes oxidative stress by inhibiting thioredoxin reductase-1, has been
shown to effectively overcome bone marrow stromal induced imatinib resistance in a murine CML cell
line with and without BCR-ABL expression(175).
45
To study the communication between ALL and adipocytes, we used in vitro TransWell co-culture models
in the current paper. The experiments with conditioned media indicate that ALL cells secrete soluble
factors to communicate with adipocytes (microarray analysis). In response, adipocytes secrete soluble
factors to protect ALL cells from DNR treatment (Fig. 5.8). Further investigation is needed to classify the
type and identities of the survival factors present in ALCM and ACM made with CoCl
2
. Similarly, Dirat et
al. established the existence of cross-talk between breast cancer cells and mature adipocytes by
demonstrating that invasiveness of breast cancer cells cultured in adipocyte and tumor cell co-culture
conditioned media is much greater than those cultured in ACM(191). Using IL-6 neutralizing antibodies,
the authors attributed the proinvasive effects of the adipocytes to IL-6.
Given that ALL cells increase ROS in adipocytes and elicit an upregulation of oxidative stress response
genes, we tested whether blocking the adipocyte response could prevent downstream protection from
the adipocytes. We used two oxidative stress inducers, BSO and AUR, both of which block the synthesis
of antioxidants GSH and thioredoxin, respectively. By pre-treating adipocytes with BSO and AUR, we
eliminated the oxidative stress response in adipocytes. When we then co-cultured ALL cells with these
adipocytes and treated them with DNR, the protective effects from adipocytes were diminished (Fig.
5.10). Congruent with our findings, others have demonstrated decreased oxidative stress in cancer cells
in co-culture with stromal fibroblasts and adipocytes and increased oxidative stress in the stromal
cells(94,193–197). The relationship between cancer cells and its stroma is termed “metabolic coupling”,
or “the reverse Warburg effect”(198). The intracellular GSH measurements showed no change in GSH
levels in adipocytes treated with ALL cells or LCM, which is surprising given that GCLM mRNA increased
in adipocytes exposed to ALL cells. It is possible that adipocytes increased GSH production, but
accompanied by GSH export and formation of conjugates with other molecules, which was not
measured with the assays.
With our previous study on ALL infiltration into adipose tissue(57), this study highlights the role of
adipocytes in ALL progression and drug resistance. We have clearly demonstrated for the first time the
crosstalk between ALL and adipocytes, and how the interaction leads to survival of ALL cells. Future in
vivo studies are necessary to better target the ALL cells in the adipose “sanctuary site”. Other
therapeutic targets, including manipulating cancer oxidative stress, should also be considered.
Interestingly, a recent publication demonstrated that combined inhibition of GSH and thioredoxin
antioxidant pathways leads to a synergistic mammary tumor cell death in vitro and in vivo (83). Further
insight into the oxidative stress transfer between ALL and adipocytes is needed to provide ideas for
more effectively targeting ALL cells in adipose tissue.
46
Chapter 6:
Concluding remarks & future directions
“The best work in the pathology of cancer is now done by those who … are studying the nature of the
seed. They are like scientific botanists; and he who turns over the records of cases of cancer is only a
ploughman, but his observation of the properties of the soil may also be useful.”
- Stephen Paget, The Distribution of Secondary Growths in Cancer of the Breast, The Lancet, 1889
6.1 Summary and general discussion
More than a century ago, Stephen Paget published in The Lancet, juxtaposing the phenomenon of
disseminated breast cancer into the liver to that of seeds living and growing only “if they fall on
congenial soil” (199). The assistant surgeon to the West London Hospital and the Metropolitan Hospital
carefully analyzed hundreds of fatal breast cancer cases, and found that metastases in the liver was far
more common than in any other organ, which led to his visionary “seed and soil” hypothesis of
metastasis. His theory was contrary to the popular believe at the time, which stated that cancer cells
traveled through the body in the blood or lymph system and could lodge in any tissue and change the
surrounding cells. It was not until 1980 that the seed and soil hypothesis was brought into the spot light
of cancer research by Hart and Fidler (200).
In the case of ALL, since it is a hematological malignancy, its tumor microenvironment could be multiple
locations depending on the locations of where ALL cells travel to. The most common sites are the bone
marrow, spleen, and the CNS. However, as discussed in Chapter 2, ALL cells are also found in the adipose
tissue. Both the bone marrow and adipose tissue are rich in adipocytes. We measured SDF-1α secretion
from two pre-adipocyte cell lines (3T3-L1 and OP9) and mouse adipose tissue explants, although we did
not detect a difference in obese versus lean adipose tissue. In accordance with that, the adipose tissue
from control and obese leukemia mice had a similar ALL burden per milligram of adipose tissue in most
depots, while the increased amount of adipose tissue likely reflects a higher overall number of ALL cells
in obese mice. Interestingly, serum SDF-1α levels are lower in obese humans (122,123), which could
either reflect a decrease in production and secretion from various tissues or an increase in its clearance
from the blood. The reciprocal relationship between serum SDF-1α level and adiposity is similar to that
seen with adiponectin, which is also secreted by adipocytes, although the mechanism of the regulation
of secretion by adipocytes is not known. Nevertheless, the effect of SDF-1α depends on the local
gradients in capillary beds of tissue. Since circulating ALL cells in obese patients are likely exposed to
more adipose tissue capillary beds, they may have more opportunity to migrate into adipose tissue.
Since we have evidence that both adipocyte cell lines and fat explants from mice co-cultured with ALL
cells confer ALL drug resistance, we developed an in vitro model to study the interaction between the
two cell types.
We adopted the differentiation methods of 3T3-L1 pre-adipocytes from Reed et al (137), which utilizes
an adipogenic hormone cocktail, containing IBMX, dexamethasone, and insulin. We soon discovered
that differentiation efficiency could be extremely variable, depending on the passage number of the
47
cells and the type of plates or flasks we used. Thus, in Chapter 3, we hypothesized that a lower media
volume would increase differentiation efficiency. Using three different pre-adipocyte cell lines, we
showed that higher media volume consistently yielded fewer adipocytes and lipid droplets,
accompanied with lower adipogenic markers during the course of differentiation. There is a common
problem in literature where authors leave out essential details of experimental methods, for example,
the type of plates and volume of media used. Our findings in Chapter 3 illustrate the importance of
attention to detail in research. We not only optimized the protocol for obtaining consistent
differentiation of adipocytes, but also saved money by reducing the reagents used.
Using the in vivo model established, we performed co-culture experiments of adipocytes and ALL cells.
We have previously shown that adipose tissue or adipocytes could alter the distribution and
pharmacokinetics of vincristine (162). In Chapter 4, we examined whether the presence of adipocytes in
co-culture would influence the distribution of DNR in ALL cells. We found that ALL cells indeed
accumulated significantly less DNR intracellularly in the presence of adipocytes. Further investigation
revealed that adipocytes were sequestering DNR and making less of it available in media. In fact, after
48 hours of exposing media containing DNR to adipocytes, the media becomes significantly less
cytotoxic to BV173 cells. The originally EC
90
dose of 100nM DNR was able to support cell proliferation
after the 48 hours exposure to adipocytes. Interestingly, adipocytes did not alter efflux rate of DNR nor
MDR-1 surface expressions in the ALL cells. Thus, in a co-culture system, sequestering DNR is one of the
mechanisms of how adipocytes protect ALL cells.
Previous experiments from our lab have shown that ALL cells secrete soluble factors that initiate
crosstalk with adipocytes, which then leads to adipocyte secretion of survival factors protecting ALL cells
from DNR. The IPA conducted on the microarray data previously obtained revealed that the soluble
factors in LCM upregulated the Nrf2-mediated oxidative stress pathway in adipocytes. Therefore,
chapter 5 investigates this relationship further, in the context of oxidative stress. We found that
adipocytes could protect ALL cells from DNR, CTN, and irradiation induced cell death, all without direct
cell-cell contact. The presence of adipocytes led to decreased GCLC and GCLM gene expressions in ALL
cells, which indicated lowered oxidative stress response in ALL cells. In the meantime, ALL cells cause an
increase in intracellular ROS in adipocytes, and secretion of survival factors, which can also be elicited by
CoCl
2
-induced oxidative stress in adipocytes. Most importantly, we found that blocking the adipocyte
oxidative stress response, for example, with BSO, partially reversed the protective effects.
Obesity and cancer is a rapidly developing field, especially after characterizing adipose tissue as an
endocrine organ instead of a mere energy storage site(201–203). Obesity has been shown to increase
both the incidence and mortality of many different cancers(6), including ALL (22,28). However, there
exists a gap in the molecular mechanism of how obesity, or the adipose tissue, influences leukemia
progression and treatment. This dissertation was to address this gap and develop a better
understanding of the leukemia microenvironment.
6.2 Limitations of the study
One of the greatest limitations of this dissertation is the simulation of in vivo tumor microenvironment in vitro
using co-culture of adipocytes and leukemia cells. TransWells were used because we have previously shown that
48
the communication between the two cell types was mediated by soluble factors(56). We decided to focus on the
TransWell model in order to simplify the experimental paradigm and isolate effects mediated by soluble factors. In
doing so, we have omitted any effects that may be modulated/modified by cell-cell contract, as others have
reported direct cell-cell contact mediated stromal protection of leukemia cells against
chemotherapies(108,204,205). Since ALL cells are known to have a rich contact-mediated interaction with cells in
their microenvironment, there is a high chance that these contact-based interactions could indeed influence the
pathways we have explored. Therefore, in the future these findings should be tested in more physiologically
relevant models, such as direct contact co-cultures, 3D cultures, and ideally, in vivo experiments.
We have not identified the molecules from LCM responsible for increasing the oxidative stress response
in adipocytes nor have we purified the protective factors secreted by adipocytes that cause
daunorubicin resistance in ALL cells. We have performed a cytokine panel analysis on LCM and detected
13 of them to be secreted, including RANTES, IL-RA, MIP-1b, FGF, TNFα, and IL1b. However, the
significance of these molecules in regards to communicating with adipocytes is unclear. We would need
to test them individually and in combinations and study their effects on adipocytes. We should also be
aware that the molecules involved in the crosstalk between the two cell types may not be a cytokine.
There have been reports on cancer cells secreting hydrogen peroxide (206), and how it acts in the tumor
microenvironment, causing metabolic changes in cancer-associated fibroblasts (207). As for the factors
secreted by adipocytes upon LCM or ALL stimulation, we have been trying different methods to purify
them in the hope of identifying them with mass spectrometry.
We have identified the importance of the oxidative stress response pathway in adipocytes in their role
of protecting ALL cells from oxidative stress. However, we were unable to abolish the protective effects
using BSO and AUR. Given that the secretion of survival factors was also induced by CoCl
2,
which induces
oxidative stress by stabilizing HIF-1α, we attempted to knock down HIF-1α in adipocytes. We have not
successfully knocked down HIF-1α without impairing the adipocyte’s normal physiology. Transfection in
3T3-L1 pre-adipocytes have been known to be notoriously difficult, and transfection in 3T3-L1
adipocytes are even more so. We approached this problem with a method called “reverse transfection”,
where adherent cells are digested to become a single-cell-suspension immediately prior to addition of
siRNA and transfection reagents, outlined in Kilroy’s publication (208). Using this method, we were able
to achieve consistent knockdown of HIF-1α by 75%. Unfortunately, the process of dissociating
adipocytes and replating them also decreased their ability to protect ALL cells from daunorubicin.
Therefore, we were unable to test the effects of knocking down HIF-1α on daunorubicin protection. In
the future, we could avoid these problems by isolating pre-adipocytes or adipocytes from HIF-1α knock-
down mice.
It is also possible that other types of stress could stimulate adipocytes to secrete survival factors that
protect ALL cells. In the current study, we only used CoCl
2
to mimic hypoxia, which may have other side
effects. For example, at millimolar doses, CoCl
2
generates ROS, which directly impacts mitochondrial
function, and could lead to activation of both the intrinsic and the extrinsic cell death
pathways(209,210). In order to isolate the effect of hypoxia, further experiments should be conducted
by exposing adipocytes to low oxygen tension. Furthermore, effects of other types of stress, such as
irradiation and heat shock, should be explored.
49
6.3 Future research
6.3.1 Contribution to the field
We have developed a model of interaction between ALL cells and adipocytes, from the migration of ALL
cells into adipose tissue to the secretion of signaling molecules, resulting in survival of the ALL cells, as
illustrated below (Fig. 6.1). Future research on the identity of these signaling molecules is crucial for
development of drugs to target these specific interactions. Once these targets have been identified,
other cancers in close proximity to adipocytes can be tested and this model could potentially be applied
to a variety of cancers. This would ultimately improve the outcome of a large portion of cancer patients.
Figure 6.1 Schematic depicting the interaction between adipocytes and ALL cells. Based on published
literature and findings from the current study, this is a proposed model where adipocytes attract ALL
cells by the SDF-1α/CXCR4 axis. In close proximity, ALL cells secrete cytokines/factors that elicit the Nrf2-
mediated oxidative stress response in adipocytes, causing upregulation in many Nrf2 downstream genes.
In turn, adipocytes secrete survival factors, which alleviate ROS in ALL cells, leading to prevention of
apoptosis in the presence of DNR.
6.3.2 Relevance of the work to the clinical treatment of ALL
The clinical observation that obesity at the time of diagnosis being associated with an increased risk of
relapse and poorer event-free survival may be partially explained by the persistent leukemia cells by
end-induction minimal residual disease (28,31). Although minimal residual disease is clinically measured
in the bone marrow, given that we have observed residual ALL cells in fat pads of mice after vincristine
50
treatment, we may find ALL cells in fat tissue of ALL patients after induction therapy as well. The findings
in this dissertation provide strong rationale for developing novel therapies to target ALL cells migration
into adipose tissue and interaction with adipocytes. On the other hand, these findings also illustrate the
importance of treating obesity, while treating ALL.
51
Chapter 7:
Materials and methods
7.1 Experimental animals and leukemia implants
All mouse experiments were approved by the Children’s Hospital Los Angeles Institutional Animal Care
and Use Committee, and were performed in accordance with the USPHS Policy on Humane Care and Use
of Laboratory Animals. C57Bl/6J mice were raised on a 60 kCal% fat diet (Research Diets, New Brunswick,
NJ) at the Jackson Laboratory (Bar Harbor, ME).
To determine whether leukemia cell migration into adipose tissue is an early event in vivo, GFP positive
8093-ALL cells were transplanted retro-orbitally into 20-week old syngeneic obese and lean C57Bl/6J
mice (10
4
cells per mouse [8], 6 mice per group). Ten days after engraftment, blood was collected from
the submandibular plexus by cheek bleeds, and then mice were sacrificed by cardiac perfusion.
7.2 Tissue harvesting and flow cytometry of GFP+ cells
Mice were euthanized at 20 weeks of age by cardiac perfusion with PBS/Heparin under anesthesia.
Various tissues were removed rapidly and washed in cold PBS. Small pieces of each tissue (100 mg) were
washed twice with RPMI plus 10% FBS, and cultured in the same medium. One day later, the culture
medium was changed with fresh medium. Tissue explants were then cultured for an additional two days
without media change before use for migration or chemotherapy protection assay.
For flow cytometry analysis of leukemia cells, various tissues (brain, lung, liver, muscle, spleen, and
kidney), were removed and washed once with cold PBS then digested with Liberase TM (Roche) as per
their instruction manual to prepare stromal vascular fraction (SVF). Blood was processed with BD Pharm
Lyse (BD Biosciences) according to the instruction manual. Bone marrow cells were collected from
femurs by flushing with PBS and then pelleted at 300g for 5 minutes. Processed blood, bone marrow and
SVF were subjected to flow cytometry analysis using an LSR II Analyzer (BD Bioscience) machine. DAPI
was added to each sample to distinguish live cells. Tissues from a non-transplanted mouse were used as
negative controls, respectively, for setting up GFP+ gating.
7.3 Cell culture
Murine pre-B 8093-ALL and GFP positive 8093-ALL cells have been previously described(56,211). Human
leukemia cell lines RS4;11, BV173, Nalm6, REH, SD1 and K562, and murine fibroblastic 3T3-L1
(embryonic) and OP-9 (bone marrow mesenchymal) cells were purchased from ATCC. The human
primary leukemia cell strains ICN13, BLQ1, UCSF02, US.7 and TXL-2 (108,212) were kindly provided by
Yong-mi Kim and Markus Müschen. ChubS7 (immortalized human pre-adipose cell line) was purchased
from Nestec LTD and cultured as described before(213).
Murine 8093 cells were cultured in McCoy’s 5A (Invitrogen), supplemented with 10% fetal bovine serum
(FBS), sodium pyruvate (1mM), Glutamax (2mM), and gentamicin (10µg/mL). Fresh IL3 (0.66nM) and β-
52
mercaptoethanol (55µM) were added to 8093 cells during every passage. All human cell lines were
cultured in RPPMI 1640 (Invitrogen), supplemented with the same ingredients as above.
3T3-L1 cells were cultured in DMEM high glucose (Invitrogen) supplemented with 10% FBS, sodium
pyruvate (1mM), Glutamax (2mM), and gentamicin (10µg/mL). OP9 cells were cultured in MEM-α
(Invitrogen) supplemented with 20% FBS, sodium pyruvate (1mM), Glutamax (2mM), and gentamicin
(10µg/mL). ChubS7 cells were cultured in DMEM/F12 (Invitrogen) with 10% FBS, sodium pyruvate
(1mM), Glutamax (2mM), and gentamicin (10µg/mL).
Pre-adipocyte (FCM) and adipocyte conditioned media (ACM) were collected after 48 hour conditioning
of 3T3-L1, OP-9 and ChubS7 pre-adipocytes and adipocytes. Adipocyte-leukemia cell conditioned media
(ALCM) was collected after 48 hour conditioning of BV173 and 3T3-L1 or ChubS7 adipocytes in co-
culture. All conditioned media were made with RPMI1640 with 10% FBS, sodium pyruvate (1mM),
Glutamax (2mM), and gentamicin (10µg/mL).
7.4 Adipocyte differentiation
Both 3T3-L1 and OP9 were differentiated into adipocytes using the same protocol. Cells were seeded on
day 0 and grown to confluence in poly-D-Lysine coated wells for 72 hours. On day 3, the media were
changed to Diff media (DMEM high glucose with aforementioned supplements with 15% FBS, 20mM
HEPES), plus the AHC (0.5mM IBMX (Sigma), 150nM porcine insulin (Sigma), and 250nM dexamethasone
(Sigma)). On day 5, the media were refreshed plus insulin only. On day 7, the media were refreshed
again. Mature adipocytes were observed on day 10. To differentiate ChubS7 cells into adipocytes, cells
were seeded in their culture media in poly-D-Lysine coated wells for 72 hours to be grown to confluence.
On day 3, media were changed to serum-free DMEM/F12 with sodium pyruvate, Glutamax, and
antibiotics plus 17µM D-pantothenic acid, 15mM HEPES, 33µM biotin, 1nM triiodothyronine, 10µg/mL
transferrin, 850nM porcine insulin, 500µg/mL fetuin, 1µM dexamethasone, 15.6µM rosiglitazone, and
0.5µM isobutylmethylxanthine (IBMX). On day 5, media were refreshed to serum-free DMEM/F12 with
all of the above supplements except IBMX. Media were refreshed every two to three days from day 5 to
day 24. Mature adipocytes were observed on day 24. All three cell lines were maintained in culture in an
incubator with 95% air and 5% CO
2
at 37°C and 100% humidity.
When differentiating 3T3-L1 and OP9 cells in a 24-well plate, 50,000 cells were plated per well 72 hours
prior to initiation of differentiation. Media volumes used in a 24-well plate were 1.5mL, 650uL, and
325uL. For ChubS7 cells, 65,000 cells were plated per well and the same media volumes were used.
When differentiating 3T3-L1 cells in a 6-well plate, 350,000 cells were plated per well 72 hours prior to
initiation of differentiation, and the media volumes were 5mL, 3.2mL, and 1.6mL. When using T150
tissue culture flasks, cells were plated at 4,000,000 cells per flask, and 20mL media volume was used for
differentiation. For the tilting flask experiment, 4,000,000 cells were allowed to adhere to the
horizontally placed flask for 72 hours. On day 0, the flask was propped to be at a 5° angle and 35mL
media volume was used. Cells that were not covered by media towards the cap of the flask were
scraped off. Differentiating plates and flasks were kept in an incubator with 5% CO
2
and either 21% O
2
(normoxia), 5% O
2
(hypoxia), or 30% O
2
(hyperoxia) at 37°C and 100% humidity.
53
7.5 Migration assays
For migration of mouse ALL, FCM and ACM were made by culturing 3T3-L1 cells in RPMI medium
containing 10% FBS for 2 days. Migration of mouse ALL toward feeder layers were set up with a
confluent monolayer of 3T3-L1 and OP9 pre-adipocytes and adipocytes. RPMI medium containing 10%
FBS was incubated with the feeder layers for 48 hours prior to the assays. For migration of human ALL
cells, we generated serum-free ACM in Opti-MEM medium (Invitrogen, Carlsbad, CA) with differentiated
3T3-L1 adipocytes. Migration assays were all performed in 24-well tissue culture plates, using TransWell
inserts with 5 μm (for 8093-ALL cells) or 8 μm (for human cells) pores (Millipore, Billerica, MA). ALL cells
in RPMI with 10% FBS were seeded into the top chambers. The bottom chambers contained cultured
tissue explants (held down by a 1 mm pore size nylon mesh), pre-adipocyte or adipocyte monolayers, or
conditioned media. After 1.5 hours (8093-ALL cells) or 3 hours (human leukemia cells), viable cells in
each chamber were quantified by trypan blue exclusion. The number of cells (% migration) that
migrated to the bottom chamber was calculated from the total cell count in top and bottom chambers.
In some experiments where differentiated adipocytes were used, leukemia cells were collected from the
bottom chambers by vigorous pipetting, and counted using trypan blue exclusion.
7.6 Oil Red O staining and lipid quantification
On day 12 for 3T3-L1 and OP9 and day 26 for ChubS7, adipocytes were stained with Oil Red O and
Hematoxylin (Sigma) following manufacturer’s protocol. Upon staining, adipocytes were viewed under a
Zeiss Axio Observer microscope and pictures were taken at 200X using AxioVision software. Each volume
condition was done in quadruplicate wells, and a blinded observer took pictures of at least five
randomly chosen fields per well. The pictures were then used for quantification of total lipid content,
and lipid droplet number and size.
7.7 Confocal microscopy
Cells were grown on poly-D-lysine coated coverslips for analysis. Daunorubicin and DCF fluorescence
images were acquired with an LSM 700 confocal system mounted on an AxioObserver.Z1 microscope
equipped with a 63x/1.4 Plan-APOCHROMAT objective lens and controlled with ZEN 2009 software (Carl
Zeiss Microscopy, Thornwood, NY). A 488 nm laser and 560 nm long-pass filter was used for
fluorescence excitation and emission. Transmitted laser light was collected to form a DIC image
simultaneously with the fluorescence image.
7.8 Gene expression analysis
3T3-L1 cells were plated in 6-well plates and differentiated with the three volumes according to the
protocol described. Cells were collected on days 0, 1, 3, 4, 7, 11, and 14 and resuspended in RNAProtect
(Qiagen), then cells were lysed with QIAzol (Qiagen) and RNA were extracted and purified with RNEasy
Mini kits (Qiagen). The quantity and quality of extracted RNA were measured by NanoDrop (Thermo
Scientific). Two thousand nanograms of RNA from each condition were reverse transcribed to cDNA with
a High Capacity 1st Strand Synthesis kit (Applied Biosystems). The expressions of adiponectin, leptin, and
pparγ were quantified with rtPCR using 25ng of cDNA, Power SYBR Green PCR Master Mix (Applied
54
Biosystems), and 200nM primers generated using National Center for Biotechnology Information
Primer-BLAST. Murine adiponectin was amplified using primers for adiponectin, PPARγ, leptin, and β-
actin. Gene expression levels were quantified using the ABI 7900HT Sequence Detection System with
the following thermal profile: 10 minutes at 95°C followed by 49 repeats of 95°C for 15 seconds, 60
degrees for 1 minute, and a final dissociation stage of 95°C for 15 seconds, 60°C for 15 seconds, and
95°C for 15 seconds. Transcript levels were normalized to β-actin or GAPDH.
Gene Species Forward Reverse
AdipoQ murine 5’-GTGCAGGTTGGATGGCAGGCA-3’ 5’-CAGTGACGCGGGTCTCCAGC-3’
PPARγ murine 5’-GCCTTCGCTGATGCACTGCC-3’ 5’-CAGCAACCATTGGGTCAGCTCT-3’
Leptin murine 5’-CCCTGTGGAGGTGAGCGGGA-3’ 5’-CCAGCCACCACGAGCCTTCG-3’
β-actin murine 5’-TTGCTGACAGGATGCAGAAG-3’ 5’-AAGGGTGTAAAACGCAGCTC-3’
GAPDH murine 5’-ACCACAGTCCATGCCATCAC-3’ 5’-CACCACCCTGTTGCTGTAGCC-3’
GCLC murine 5’-ACTGAATGGAGGCGATGTTC-3’ 5’-AGTGATGGTGCAGAGAGCCT-3’
GCLM murine 5’-TCCTTGGAGCATTTACAGCC-3’ 5’-AGAGCAGTTCTTTCGGGTCA-3’
HO-1 murine 5’-CACGCATATACCCGCTACCT-3’ 5’-CCAGAGTGTTCATTCGAGCA-3’
Mt-2 murine 5’-CCGCGTGCTTCTCTCCAT-3’ 5’-ATCGACGAGAGATCGGTTTGA-3’
ADM murine 5’-CTCGCTGATGAGACGACAGTTC-3’ 5’-CTCTGGCGGTAGCGTTTGAC-3’
Txn-1 murine 5’-CTCTGCTACGTGGTGTGGAC-3’ 5’-CCTTGTTAGCACCGGAGAACT-3’
Txnrd-1 murine 5’-CCATGGTCCAGCCCTGAAG-3’ 5’-CCAAGAGGAGTCGGTGTGAC-3’
β-actin human 5’-ACAGAGCCTCGCCTTTGCCG-3’ 5’-CGATGCCGTGCTCGATGGGG-3’
GCLC human 5’-AAACCCAAACCATCCTACCC-3’ 5’- CGAGGGTGCTTGTTTATTGC-3’
GCLM human 5’-TCAACCCAGATTTGGTCAGG-3’ 5’-AGGCTGTAAATGCTCCAAGG-3’
HO-1 human 5’-ATGACACCAAGGACCAGAGC-3’ 5’-GTGTAAGGACCCATCGGAGA-3’
Txn-1 human 5’-CTTGGACGCTGCAGGTGATA-3’ 5’-TCTGAAGCAACATCCTGACAGT-3’
Txnrd-1 human 5’-CCGCCGTAGGTCAGCTAAAG-3’ 5’-ATTCCGAGAGCGTTCCTTCC-3’
Table 7.1 Primer list with forward and reverse sequences.
7.9 Western blots
Total protein was extracted from murine or human leukemia cells or adipocytes with protein isolation
buffer(56). Lysates were sonicated briefly and centrifuged for 10 minutes at 15,000 g at 4 C. Cytoplasmic
and nuclear fractions were prepared from 3T3-L1 adipocytes as following. The cells were washed first
with ice cold PBS then with Buffer A (10mM HEPES at pH7.9, 10mM KCl, 1.5mM MgCl
2
, protease
inhibitor cocktail, 1mM PMSF, phosphatase inhibitor cocktail, and 1mM DTT). Then Buffer A plus 0.1%
NP-40 was added to the cells and incubated on ice for 5 minutes before cells were collected into an
Eppendorf tube with cell scrapers. Samples were vortexed and spun at 14,000g at 4 C for 15 minutes.
The supernatant, which is the cytoplasmic fraction is collected and stored at -80 C. The nuclear pellet is
washed three times with 1mL of ice cold PBS, centrifuged at 300g for 10minutes each time. The cell
extraction buffer (Life Technologies, Carlsbad, CA) is supplemented with 1mM PMSF, protease inhibitor
cocktail, and phosphatase inhibitor cocktail before adding to the nuclear pellet. The samples were then
incubated on ice for 30 minutes, with 1 minute vigorous vortex every 5 minutes. Samples were then
sonicated (Bioruptor® Standard, Diagenode, Denville, NJ) three times with 10x30 second cycles in an ice
bath. Finally, the samples were spun at 14,000g at 4 C for 15 minutes. The supernatant, which was the
55
nuclear fraction, was retained. Protein concentrations were quantified by bicinchoninic assay (Pierce
Biotechnology).
Twenty micrograms of protein was subjected to SDS-PAGE and transferred to a nitrocellulose membrane.
Membranes were blocked in 5% milk and probed simultaneously with CXCR4 and actin antibody (Cell
Signaling Technology) because CXCR4 and actin have clearly distinct molecular weights. In other
experiments, the membrane was probed with primary antibodies specific for IRS-1 (Cell Signaling,
Danvers, MA, USA, #2382), pIRS-1 Y895 (Cell Signaling, #3070), HO-1 (Abcam, San Francisco, CA, #13248)
and glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling, #2118), followed by an incubation with
HRP-conjugated anti-rabbit secondary antibody (Cell Signaling, #7074). Bands were detected using
HyGLO-HRP detection kit (Denville Scientific, South Plainfield, NJ, USA) and developed using ImageQuant
LAS 4000 (GE Healthcare Life Science, Pittsburgh, PA). Densitometric band analysis was performed using
Image J (National Institute of Health, Betheseda, MD, USA).
7.10 ELISA
SDF-1α concentrations in plasma, obese and control mice tissue explant conditioned media, and 3T3-L1
and OP9 FCM and ACM, were measured in duplicate by ELISA (RayBiotech, Norcross, GA).
On day 10 for 3T3-L1 and OP9 cells, conditioned media experiments were carried out in 24-well plates
with 1mL of media per well. After 48 hours of conditioning, media were collected, passed through a
0.22µM filter (Millipore) and stored in -80°C. Adiponectin and leptin concentrations were measured
using ELISA kits (Millipore).
7.11 Insulin inhibition of lipolysis
3T3-L1 adipocytes were serum-starved in serum-free DMEM with 0.2% Bovine Serum Albumin (BSA) for
2hrs. Following starvation, cells were washed twice with Krebs Ringer Phosphate (KRP) buffer and
incubated for 3 hrs in KRP buffer with 4% Free-Fatty Acid (FFA)-Free BSA alone or containing 1µM
Isoproterenol and/or 100nM Insulin. Media FFA content was measured with the NEFA-HR(2) kit (Wako
Pure Chemicals Industries, Ltd., Osaka, Japan).
7.12 Oxidative stress measurement
DCFH-DA (Life Technologies) was used as an indicator of intracellular ROS according to manufacturer’s
protocols. Briefly, cells were incubated with 10 μM DCFH-DA for 15 minutes at 37°C and 100% humidity.
Then the reaction was stopped by washing samples with ice cold PBS or placing the samples on ice. DAPI
was added to distinguish live cells. The samples were also protected from light until analysis using an LSR
II Analyzer (BD). In flow cytometry experiments, FITC was the channel for detection of DCF, and PE for
DNR. Proper compensation controls were included for calculations of spill-overs between the two
channels.
7.13 Intracellular GSH measurement
56
The GSH-Glo
TM
Glutathione Assay (Promega, Madison, WI) was used to measure 3T3-L1 adipocyte
intracellular GSH following manufacturer’s instructions. Protein amount of each sample was quantified
by Pierce
TM
BCA protein kit (Life Technologies, Carlsbad, CA) for normalization of GSH.
7.14 Statistical analysis
All statistical tests were performed with GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Two-
sided student’s t-tests were used to compare control and ACM groups, and tissue explants. Square-root
transformation was used when data were not normally distributed. The data are presented as mean±SD.
A p value equal or less than 0.05 was taken as statistically significant. In all graphs, *p<0.05, **p<0.01,
and ***p<0.001.
Lipid content quantification was done using ImageJ, by generating a histogram of results of a picture
with red pixels only. The summation of pixel numbers of greater than and equal to bin=50 were
calculated and defined as the total number of red pixels in a picture. MetaMorph® Microscopy
Automation & Image Analysis Software (Sunnyvale, CA) was used to quantify the number of lipid
droplets and the size of each droplet.
Repeated measures linear regression was utilized to examine the results of all lipid quantification
experiments. Analysis of total lipid content in three pre-adipocyte cell lines was done treating media
volume as a continuous predictor. Volume and region, in the tilting flask experiment, were treated as a
continuous variable, while oxygen level was treated as a categorical variable. The log
10
transformation
was utilized to lessen the effects of outliers due to the highly skewed distribution of lipid content.
Additionally, the analysis is performed on the rank of the observations to confirm the results of the data
with extreme outliers. The mean droplet size and the total droplet count of each image were analyzed
separately. The log
10
transformed qPCR data was analyzed separately for each gene, but presented in
raw scale. ELISA data was log
10
transformed and analyzed separately for each cell line and cytokine. The
media volume was treated as both a continuous and categorical variable. All analyses were performed
using Stata (version 11) (StataCorp, TX).
Oxygen partial pressures at different depths in 5%, 21% and 30% oxygen were calculated using the
formula below:
where is the amount of oxygen consumed per minute by 200,000 3T3-L1 cells, is the
media height above the cell layer, is the oxygen diffusion constant, is the solubility coefficient of
oxygen, and is the surface area at the air-media interface(214,215).
Flow cytometry of intracellular DNR and surface MDR-1 expression experiments were quantified using
median fluorescence intensity (MFI). MFI was calculated by subtracting signals from control samples and
then divided by the control.
57
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Abstract (if available)
Abstract
Obesity is a serious health problem in both adults and children, and it is associated with increased cancer mortality. Specifically, obesity is associated with an increased risk of acute lymphoblastic leukemia (ALL) relapse. In this dissertation, the interactions between ALL cells and adipocytes are investigated. We first developed an optimized protocol for adipogenesis by discovering the relationship between oxygen tension and adipocyte differentiation. We found that using a lower media volume (resulting in higher oxygen tension at the cell layer) yielded more complete adipogenesis in three different pre-adipocyte cell lines. We then investigated whether adipose tissue attracts ALL cells using mouse and cell co-culture models. Syngeneically implanted ALL cells migrated into adipose tissue within ten days. In vitro, murine ALL cells migrated towards adipose tissue explants and 3T3-L1 adipocytes. Human and mouse ALL cells migrated toward adipocyte conditioned media, which was mediated by SDF-1α. In addition, adipose tissue explants protected ALL cells against daunorubicin (DNR) and vincristine. To further study the mechanism of protection against DNR, we examined the accumulation and metabolism of the drug by adipocytes. We found that the presence of adipocytes significantly lowered ALL intracellular DNR. Using confocal fluorescence microscopy and high pressure liquid chromatography, we detected and quantified DNR and its metabolite, daunorubicinol, in adipocyte lysates. These data suggest that adipocyte protection of ALL cells against DNR may be explained, in part, by the shift in distribution of the drug and metabolism by adipocytes. Since DNR is also a potent oxidative stress inducer, we investigated the role of adipocytes in alleviating oxidative stress in ALL cells. We found that adipocytes could protect ALL cells from oxidative stress induced by drugs and irradiation. Microarray analysis of adipocytes exposed to ALL showed an upregulation in the Nrf2-mediated oxidative stress response. Microscopy and qPCR of selected downstream genes indicated an increase in intracellular ROS and activation of Nrf2 in adipocytes. The oxidative stress response in adipocytes, elicited by ALL or cobalt chloride, led to secretion of soluble factors, which protected ALL cells from DNR. Blocking the oxidative stress response with chemicals such as buthionine sulfoximine and auranofin reversed the DNR protective effects. Exogenous supplementation of antioxidants to ALL cells rescued DNR induced cell death. Collectively, we showed that ALL cells elicit an oxidative stress response in adipocytes, leading to secretion of survival factors by adipocytes, and protection of ALL cells against DNR. In conclusion, this dissertation describes how adipocytes actively attract ALL cells into close proximity, leading to crosstalk between the two cell types via soluble factors and adipocytes removing chemotherapeutics and secreting pro-survival factors in the ALL microenvironment, causing ALL drug resistance.
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Asset Metadata
Creator
Sheng, Susan (Xia)
(author)
Core Title
The role of adipocytes in acute lymphoblastic leukemia cell migration and survival against daunorubicin
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Integrative Biology of Disease
Publication Date
11/19/2015
Defense Date
07/10/2015
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
acute lymphoblastic leukemia,adipocyte,adipogenesis,antioxidants,daunorubicin,drug resistance,migration,OAI-PMH Harvest,obesity,oxidative stress
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application/pdf
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English
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Epstein, Anat (
committee chair
), Asgharzadeh, Shahab (
committee member
), Heisterkamp, Nora (
committee member
), Kim, Yong-Mi (
committee member
), Mittelman, Steven (
committee member
)
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xias@usc.edu,xsheng@chla.usc.edu
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https://doi.org/10.25549/usctheses-c40-200800
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Dissertation
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Sheng, Susan (Xia)
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Tags
acute lymphoblastic leukemia
adipocyte
adipogenesis
antioxidants
daunorubicin
drug resistance
migration
obesity
oxidative stress