University of Southern California Dissertations and Theses

Bone marrow derived mesenchymal stem cells promote survival and drug resistance in tumor cells

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Bone marrow derived mesenchymal stem cells promote survival and drug resistance in tumor cells

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BONE MARROW DERIVED MESENCHYMAL STEM CELLS PROMOTE

SURVIVAL AND DRUG RESISTANCE IN TUMOR CELLS

by

Scott Anthony Bergfeld

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
(PATHOBIOLOGY)

December 2013

Copyright 2013 Scott Anthony Bergfeld

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Dedication

For my wife.

iii

Acknowledgements

I would like to thank my mentor, Dr. Yves DeClerck, for his guidance and
support throughout the course of my studies. I would also like to thank the other
members of my committee, Dr. Florence Hofman, Dr. Shahab Asgharzadeh, and Dr.
Gregor Adams, for their advice on furthering my professional development.
Further thanks go to Dr. Richard Sposto and Jemily Malvar (CHLA Hematology-
Oncology Statistics Core) for statistical advice and linear regression analysis, Dr. Rex
Moats (Saban Research Institute Small Animal Imaging Core) for BMMSC iron-labeling
technique, and Dr. Lucia Borriello for preparation of survival curves. I also thank Dr.
Alan Epstein for advice on cell lines, Dr. Laurence Sarte for assistance with cell culture
and mammary fat pad injections, and J. Rosenberg for her help in preparing this thesis.
Finally, thanks to my wife Kat for all her love and support.
Grant Support
My studies were supported by NIH grant T32 GM067587, as well as The Saban
Research Institute Pre-doctoral Training Award.

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Table of Contents

Dedication ii

Acknowledgements iii

List of Figures v

Abbreviations vi

Abstract viii

Chapter 1: Introduction 1
Tumor Microenvironment in Cancer 1
Definition of MSC 2
Role of MSC in Bone Metastasis 12
Role of MSC in Primary Tumors 14
Summary 20
Table I-1. Pro- and Anti-Tumorigenic Functions of MSC 22

Chapter 2: Protective Effect of MSC on Primary Tumor Cell Apoptosis 23
Introduction 23
Material and Methods 24
Results 31
Discussion 42

Chapter 3: Tracking MSC Recruitment by Tumors in vivo 49
Background 49
Specific Aims 53
Preliminary Data 54
Experimental Approach: Aim 1 59
Experimental Approach: Aim 2 61
Limitations 62
Significance 63

Chapter 4: Role of MSC in Cancer Therapy 64
Targeting MSC in Cancer Therapy 64
MSC as Therapeutic Vectors 65

Chapter 5: Conclusions 72
Future Directions 72
Summary 75

Bibliography 76
v

List of Figures

Figure I-1. Reciprocal Interactions between Tumor Cells and MSC 21

Figure II-1. Bone Marrow Stromal Isolates Display Mesenchymal Cell Markers 32

Figure II-2. BMMSC Enhance Tumor Cell Growth by Suppressing Apoptosis 34

Figure II-3. BMMSC Enhance Survival in Drug-Treated Tumor Cells 37

Figure II-4. BMMSC CM Protects Tumor Cells from Drug-Induced Apoptosis 38

Figure II-5. BMMSC Exhibit a Chemotactic Response to Tumor Cells 40

Figure II-6. BMMSC Suppress Apoptosis in Drug-Treated Tumors in vivo 41

Supplemental Figure II-1. BMMSC do not Enhance Cell Cycle Progression 47

Supplemental Figure II-2. BMMSC Suppress Apoptosis in LL/2 Tumor Cells 48

Figure III-1. Iron Particles Increase BMMSC Detection and Preserve Protection 54

Figure III-2. BMMSC Iron Labeling Decreases Signal Density and Relaxation Time 56

Figure III-3. Iron-loaded BMMSC Produce Diffuse Areas of Negative MR Signal 57

Figure III-4. DsRed-expressing BMMSC are Detectable by in vivo Fluorometry 59

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Abbreviations

Akt Protein Kinase B
ANOVA Analysis of Variance
AP Alkaline Phosphatase
a-sma alpha-Smooth Muscle Actin
bFGF basic Fibroblast Growth Factor
BMMSC Bone Marrow MSC
BMP Bone Morphogenic Protein
BMPR BMP Receptor
CAF Cancer-Associated Fibroblast
CAM Cell Adhesion Molecule
CFU-F Colony Forming Unit Fibroblast
CM Conditioned Media
COX-2 Cyclooxygenase 2
DAPI 4',6-diamidino-2-phenylindole
DC Dendritic Cell
DHS Denatured Horse Serum
DMEM Dulbecco Modified Eagle Media
EC Endothelial Cell
ECM Extracellular Matrix
EGF Epidermal Growth Factor
EPC Endothelial Progenitor Cell
ERK Extracellular-regulated kinase
FCS Fetal Calf Serum
FFPE Formalin-fixed Paraffin-embed
FITC Fluorescein Isothiocyanate
GDF15 Growth Differentiation Factor15
GFP Green Fluorescent Protein
HCAM Hyaluronic Acid CAM
HCC Hepatocellular Carcinoma
HGF Hepatocyte Growth Factor
HSC Hematopoietic Stem Cell
HUVEC Human Umbilical Vein EC
ICAM Intercellular CAM
IFN Interferon
IMDM Iscove’s Modified DMEM
iNOS inducible NO Synthase
i.v. Intravenous
LFA-1 Lymphocyte Function Antigen 1
LNC Lipid Nanocapsule
LNGFR Low Nerve Growth Factor Rcpt
MAPC MultipotentAdult ProgenitorCell
MAPK MitogenActivated ProteinKinase
MCP1 Monocyte Chemoattractant Prot1
MDR Multiple Drug Resistance
MLC Myosin Light-Chain Kinase
MMP Matrix Metalloproteinase
MRI Magnetic Resonance Imaging
MRP Multidrug Resistance Protein
MSC Mesenchymal Stem Cell
NCAM Neural CAM
NK-cell Natural Killer Cell
NSCLC Non-Small Cell Lung Carcinoma
NT-3 Neurotropin-3
OPG Osteoprotegerin
ORO Oil Red o
PAR Protease Activated Receptor
PARP1 PolyADP-ribose polymer 1
PBS Phosphate Buffered Saline
PDGF Platelet-derived Growth Factor
PDGFR PDGF Receptor
PET Positron Emission Tomography
PFA Paraformaldehyde
PGE2 Prostaglandin E2
PI Propidium Iodide
PKR Protein Kinase R
PLA-NP Polylactic Acid Nanoparticle
PPAR PeroxisomeProliferateActivRcpt
Rcpt Receptor
RFP Red Fluorescent Protein
ROCK Rho-Associated Kinase
ROS Reactive Oxygen Species
S1P Sphingosine 1 phosphate
Sca-1 Stem Cell Antigen 1
SD Standard Deviation
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SDF-1 Stromal-derived Factor 1
SPIO Super-paramagnetic Iron Oxide
STAT3 SignalTransduceActivTrancript3
TAF Tumor-Associated Fibroblast
TGF-B Transforming GrowthFactorBeta
TLR Toll-like Receptor
TME Tumor Microenvironment
TNF Tumor Necrosis Factor
TRAIL TNF Apoptosis Inducing Ligand
Treg Regulatory T-cell
TUNEL TdT dUTP nick end labeling
uPA Urokinase PlasminogenActivator
uPAR uPA Receptor
VCAM Vascular CAM
VEGF VascularEndothelialGrowthFact
VEGFR VEGF Receptor
VLA-4 Very Late Antigen-4
XIAP X-link Inhibitor of ApoptosisProt

viii

Abstract

Bone marrow mesenchymal stem cells (BMMSC) are recruited to primary tumors
and have been reported to display pro-tumorigenic as well as anti-tumorigenic activities.
We hypothesize that circulating BMMSC are incorporated into tumor sites and protect
tumor cells from therapy-induced apoptosis. Adherent stromal cells isolated from murine
bone marrow that expressed phenotypic and functional characteristics of BMMSC were
tested for their effect on 4T1 murine mammary adenocarcinoma and LL/2 Lewis lung
carcinoma cells. Primary BMMSC stimulated the expansion of 4T1 cells in 3D co-
cultures and conditioned medium from these cells increased the viability of 4T1 and LL/2
cells in 2D cultures. Analysis of apoptosis in 4T1 cells exposed to BMMSC conditioned
medium under low serum concentrations (0.5 to 1%) revealed a 2-fold reduction in
apoptosis as determined by Annexin V expression and caspase-3 and caspase-9 activity.
Furthermore, exposure of 4T1 and LL/2 cells to BMMSC conditioned medium increased
the viability of 4T1 and LL/2 cells when treated with paclitaxel or doxorubicin at
therapeutic concentrations. This protective effect was accompanied by significant
reductions in caspase-3 activity and Annexin V expression. We then demonstrated that
BMMSC are attracted specifically by 4T1 and LL/2 cells in vitro, and in vivo migrate into
the invasive front of 4T1 tumors implanted in mice. When co-injected with 4T1 cells in
the mammary fat pad of mice subsequently treated with doxorubicin, they inhibit drug-
induced apoptosis by 42 percent. Overall, our data identify BMMSC as an important
mediator of tumor cell survival and treatment resistance in primary tumors.

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Chapter 1 - Introduction
Tumor Microenvironment in Cancer
In the classical model of tumor development, genetic changes within transformed cells
lead to deregulated growth, local invasion, and metastatic spread. Recently, however, the
particular characteristics of the tissue environment that harbors the developing lesion,
called the tumor microenvironment (TME), have been identified as an important source
of tumor promoting activity. These mechanisms include the extracellular matrix (ECM),
the network of support proteins which maintains tissue architecture and drives cell
growth by adhesion-based signaling in injured tissue, as well as non-transformed
inflammatory and connective tissue cells that enhance growth by soluble and contact-
dependent signals. Of particular interest to our laboratory is a tissue environment known
to exhibit significant tumor stimulating potential: the bone marrow.
The bone marrow is a unique environment for transformed cells, especially those that
have metastasized from distal sites. It is the home of progenitor cells that generate the
subsets of inflammatory cells that migrate to primary tumors and enhance growth and
metastasis. These bone marrow resident progenitors require a supportive niche which not
only attracts metastatic tumor cells but also enhances their survival and engraftment. The
support cells which produce and maintain the metastatic niche are derived from bone
marrow-resident progenitors as well. Furthermore, the bone matrix itself is a source of
growth factors that can be released by resident bone remodeling cells upon stimulation by
metastatic cells. As such, modulation of lineage differentiation in bone marrow-derived
cells is an important aspect of tumorigenesis in the both peripheral tissues and the
marrow compartment.

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The various populations of bone marrow-derived cells stem from two lineages.
Hematopoetic cells, including lymphocytes, erythrocytes, megakaryocytes, and myeloid
cells, are generated from hematopietic stem cells (HSC). Endothelial cells also share a
common progenitor with hematopoitic cells, the angioblast, which generates HSC and
endothelial progenitor cells (EPC). The second lineage of bone marrow-resident cells,
which is the subject of this manuscript, is the mesenchymal lineage. These are
connective tissue producing cells which generate bone, fat, and cartilage in the marrow,
as well as collagen rich reticular fibers in inflamed tissue, and are derived from
mesenchymal stem cells (MSC)(1). Osteoblasts, which produce bone, and MSC are
crucial for maintenance of the HSC niche but can also provide metastatic tumor cells with
an engraftment site that promotes dormancy and therapeutic resistance. Native HSC
must compete for the same attachment space and supportive microenvironment as
metastatic cells, resulting in significant HSC dislodgement in bone metastatic lesions (2).
The participation of MSC and their derivatives in the inflammatory tumor
microenvironment can thus have dramatic effects on tumor growth and survival, which
are now the subject of ongoing investigation in our laboratory.
Definition of MSC
Location
One of two major stem cell populations in the adult bone marrow, mesenchymal
stem cells produce the myriad of bone marrow stromal cell types (including osteocytes,
adipocytes, pericytes, and reticular fibroblasts), and provide microenvironmental
regulation of hematopoietic stem cell quiescence and proliferation(3-5). The rarity of this
cell type, which represents only 0.01 to 0.001% of all mononuclear cells in the bone

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marrow, has made identification of a native MSC niche difficult (6-7). Several locations
for the in vivo MSC niche within the bone marrow have been proposed. These include
the periosteal niche, with MSC found adjacent to osteoblasts, the pericytic niche, with
MSC found in contact with pericytes or reticular fibroblasts, and the perivascular niche,
with MSC adjacent to sinusoidal endothelial cells. Surface marker expression studies
point to the perivascular niche as the true “home” for MSC (8), which allows their
progeny easy access to the circulation.
Although the hematopoietic stem cell and its progeny have been well studied, the
true “stem” cell responsible for multipotent stromal production remains elusive. The
term “MSC” itself is ambiguous, denoting not only the classically defined mesenchymal
stem cell (3), but also stromal stem cells, multipotent stromal cells, mesenchymal
progenitor cells, and multipotent adult progenitor cells (MAPC)(9). The identification of
MSC in other connective tissues such as dental pulp and fat further impedes a clear
phenotypic definition (10). MSC likely represent a heterogeneous cell population with a
shifting percentage of mulitpotent and committed progenitor cells.
Isolation and Characterization of MSC
Multipotent bone marrow stem cells from mice were originally identified by
Friedenstein through in vitro functional markers, as these cells displayed adherence to
tissue culture plastic, produced colony forming unit - fibroblasts (CFU-F), and showed
differentiation into adipocytic, osteocytic, and chondrocytic lineages (11). They were
also capable of in vivo stromal differentiation following implantation (12). However, the
similarity of these cells to Caplan’s MSC, which is the true progenitor cell of all
connective tissue types in the bone marrow and periphery, is still unclear, leading to their

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labeling as mesenchymal stromal cells (13). Since their original identification, new
techniques for isolation of mulitpotent bone marrow stromal cells have been developed,
including alterations to culture media supplements and serum percentage, growth on
various substrates such as collagen and fibronectin, and depletion of hematopoietic cell
contaminants by surface marker based negative selection (9,14). These methods allow
enrichment of a fibroblastic spindle-cell population, and although a heterogeneous
mixture of spindle cells, star-shaped cells, and large flattened cells is frequently obtained,
a characteristic pattern of surface marker co-expression indicative of self-renewal and
multipotence is universally demonstrated (15-17). Additional surface markers reveal
subpopulations of MSC that are differentially committed to various stromal cell
types(18).
Human and murine MSC generally do not express hematopoietic markers CD34
and CD45, although subpopulations of cells that express a low level of these markers
have been identified in freshly isolated MSC (19-20). In culture, CD34 and CD45
positivity tends to disappear after a few passages (21), which may indicate a
contaminating hematopoietic population and not MSC-derived cells. Human MSC are
positive for the surface markers CD44, CD73, CD90, CD105, CD106, and STRO-1 (22).
Murine MSC express all these markers except STRO-1, and also express stem cell
antigen-1 (Sca-1) (5). These markers are used in combination to purify MSC from bone
marrow isolates since other cell types found in the bone marrow share their expression
(23). Additionally, certain multipotent marrow stromal cells do not display these markers
in vivo. As a result, sorted MSC populations may not contain all multipotent bone
marrow stromal subtypes.

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The absence of MSC-specific genes and markers is another hurdle faced in the
characterization of these cells (23). The trypsin-resistant antigen denoted STRO-1
remains the best choice, as it is not found in peripheral tissues, in the hematopoietic
compartment, or in mature mesenchymal cells, though it is expressed on endothelial
progenitors (24). Unfortunately, the structural and functional characteristics of STRO-1
have yet to be determined. This aspect, combined with STRO-1 negativity in mice,
severely hampers its suitability for pre-clinical studies (25). Recent efforts have been
devoted to identification of surface markers with high expression by MSC versus
alternative marrow subtypes. These candidate markers include Low-affinity Nerve
Growth Factor Receptor (LNGFR), which is expressed on neural cells, and CD49a, an
alpha integrin subunit commonly associated with inflammatory cells. Both of these
molecules have been shown to enrich a homogeneous population of multipotent bone
marrow cells (18,25-27).
Functional characteristics of MSC can be used to distinguish these cells from
other adherent cell types, namely epithelial cells. MSC are extremely motile, expressing
high levels of contractile proteins like vimentin, adhesion molecules that regulate
migration like LFA-1 (CD11a) and VLA-4 (CD49d), and the dynamic adhesion molecule
N-cadherin. Native epithelial cells are non-motile, expressing E-cadherin and gap
junctional proteins that encourage aggregation, although this behavior can change in
response to injury. Epithelial cells are also a source of basement membrane collagen IV,
while MSC produce wound-related collagen I and collagen III.

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Differentiation Pathways
Genes activated in mature cells of the three classical bone marrow lineages
(adipocyte, osteocyte, and chondrocyte) elucidate MSC phenotypic differentiation at the
molecular level (9). Cell surface interactions guide MSC into chondrogenic
differentiation (28), producing high levels of cartilage proteoglycan when cultured in
pellets versus monolayers. Downregulation of integrin a5B1, a fibronectin receptor,
during chondrocyte maturation highlights the alternative ECM interactions which
separate MSC from their progeny (29). Bone formation depends on transcription factors
Runt-related transcription factor 2 (RUNX-2) and OSTERIX, which signal for osteoblast
differentiation (10,30). There is also evidence that these genes are basally expressed by
undifferentiated MSC in culture, allowing maintenance of osteoblastic differentiation
capacity in late passages. Peroxisome proliferator-activated receptor (PPAR) gamma
proteins and C/CAAT enhancer binding proteins drive adipocyte differentiation (31).
Physical constraints play a large role in steering MSC toward osteoblastic or adipogenic
fates. Adipocytes are produced by tightly packing MSC, while osteoblastic cells require
more space (32). Mature osteoblasts maintain some multipotency, as evidenced by
observations of in vitro adipocytic trans-differentiation. The age-related accumulations
of adipocytes in bone marrow paired with a concomitant loss in osteoblasts, provides
evidence for bone marrow stromal trans-differentiation in vivo as well (33).
Expression pathways driving differentiation toward more peripherally-located
stromal subtypes have also been elucidated. Transcription factors GATA-4 and Nkx2.5
create cardiac myocytes, and MyoD and myogenin create skeletal myocytes (34). MSC
subjected to mechanical strain generate smooth muscle tissue, another example of cell-

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surface regulation of MSC phenotype (35). Alpha-smooth muscle actin polymerization is
required for pericyte differentiation, downstream of Rho-associated kinase (ROCK) and
myosin light chain (MLC), as well as smooth muscle differentiation of MSC (36).
The in vitro production of non-mesenchymal lineages from MSC has been
demonstrated by several groups, although the ability of these cells to functionally
integrate in vivo is questionable (37). Verfaille described generation of stromal,
endothelial, hepatocytic cells in vitro from bone marrow isolates (38). Further studies
showed generation of ectodermal, mesodermal, and endodermal lineages in vivo, leading
to the term MAPC (39). MAPC could in fact be an MSC progenitor, but recent
difficulties in repeating the original experiment have prevented further examination (9).
Functional neurons and skeletal myocytes were generated in vivo by Shiota, using 3D
MSC spheroid implantation (40). Pluripotent markers Oct-4 and Sox-2 could not be
detected in the spheres, suggesting that they are a more terminally differentiated type of
stromal cell.
Soluble signals in differentiation
MSC growth and differentiation are heavily influenced by signal molecules
present in the extracellular mileau. Osteocytes, chondrocytes, and adipocytes can be
generated by bone morphogenic proteins (BMPs), a TGF-B family derivative (41).
Signaling through alternative receptors BMPR-IA and -IB produces either osteoblasts or
adipocytes (42). Furthermore, BMPs can suppress proliferation in undifferentiated MSC
(43). Opposite effects are seen with TGF-B1, leading to enhanced proliferation and
limiting differentiation in MSC (44-46). This only occurs later in MSC differentiation
since TGF-B1 drives chondrogenesis and growth of osteoprogenitors (47,48).

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Conflicting signals produced by TGF-B proteins are due to their activation of Smads that
block BMPs as part of a regulatory loop (46). MSC phenotype is also regulated by
canonical and non-canonical Wnt signaling (49). MSC growth is driven by canonical
Wnt3a (50) and limited by non-canonical Wnt5a or Wnt-3a overload (51,52). Wnt3a
suppresses generation of mature osteocytes but has the opposite effect when its signaling
is high (50,52). Canonical Wnt enhances pre-osteoblast generation while preventing
maturation (53). Additionally, canonical Wnt3a suppresses adipogenesis. Chondrocyte
maturation is enhanced by Wnt4 and blocked by Wnt5a (54). Wnt4 additionally prevents
chondrogenesis unless TGF-B is also present (55,56). MSC also generate myocytes in
response to Wnt (57). Suppression of PPAR-γ limits adipogenesis and facilitates
osteogenesis, an inhibition produced by IL-1 and TNF-a signalling (58). Finally,
Platelet-Derived Growth Factor (PDGF) drives differential effects on MSC phenotype
using the receptors PDGFR-α and PDGFR-β, which act on the aforementioned ROCK/α-
SMA pathway. Engagement of PDGFR-α increases α-SMA expression and
polymerization, stimulating pericyte and smooth muscle differentiation, while PDGFR-B
inhibits this effect (36).
MSC Recruitment
Tissue repair, inflammation, and neoplasia are just a few of the processes that
encourage engraftment of circulating MSC (59). Tumor tropism of MSC can be
examined by cell trafficking assays in vitro and in vivo. A number of modalities, such as
intravenous injection, utilizing fluorescence, bioluminescence, and magnetic resonance,
can be used to track ex-vivo expanded MSC (60-62). No studies have truly established
migration of endogenous MSC into tumors, but some have shown that labeled MSC

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home to tumor stroma following bone marrow engraftment in sublethally irradiated mice
(63,64).
MSC recruitment into tumors follows a similar pattern to tissue repair,
particularly recruitment of activated inflammatory cells. Vascular Endothelial Growth
Factor (VEGF) recruits endothelial cells, and in murine glioma can recruit MSC (65).
MSC also show graded responses to leukocyte and endothelial activating TGFB-1, IL-8,
and neurotropin 3 (NT-3) (66). Other immunomodulators are shown to attract MSC,
such as epidermal growth factor (EGF), basic Fibroblast growth factor (bFGF),
hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF) (67,68).
Chemokines play a significant, but not always predictable, role in MSC migration. CCL
2 (Monocyte Chemoattractant Protein-1) strongly attracts MSC (69), while CXCL12
(Stromal-Derived Factor-1) binding to CXCR4 shows mixed results, as blocking this axis
is sufficient to impair MSC migration in some studies but not in others (68, 70-72).
Tumor cells secrete many chemoattractants that promote the migration of MSC (73) and
MSC express receptors of all four chemokine subfamilies: CC, CXC, CX(3)C, and C.
Dose-dependent migration of MSC induced by chemokines like CCL2/MCP1, CCL25
(Thymus Expressed Chemokine), CXCL8 (IL8), CXCL12 /SDF1α, and CXCL13
(BCA1) has been demonstrated in chemotaxis assays in vitro (74,75). Sphingosine 1
phosphate (S1P), a bioactive lipid that acts via G-protein-coupled receptors, also exerts
strong chemoattraction on MSC through Matrix Metalloproteinase (MMP)-mediated
signaling events and the RhoA/ROCK and MEK1/ERK intracellular pathways (76).
HGF could be another mechanism, as it is expressed only by apoptotic cells in injured
tissues, and interacts with the c-Met receptor which is expressed by MSC. Blocking HGF

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bioactivity resulted in significant reduction of MSC migration (77). Because many
tumors express HGF, it could be one of the mechanisms attracting MSC as has been
shown in glioma (78). Under hypoxic conditions, breast cancer cells secrete high levels
of IL-6, which serves to activate and attract MSC. IL-6 acts in a paracrine fashion on
MSC, stimulating the activation of both STAT3 and MAPK signaling pathways to
enhance migratory potential and cell survival (79). TNF-α is another potential
recruitment factor. In myocardial infarct it is capable of potentiating MSC migration as
well as inhibiting MSC migration as an indirect consequence of osteoprotegerin (OPG)
induction, which might result in a suboptimal recruitment of circulating MSC (80). LL-
37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein
18 which is present in many tumors, also stimulates the migration of MSC. LL-37
facilitates ovarian tumor progression through recruitment of MSC to serve as pro-
angiogenic factor-expressing tumor stromal cells (81). Irradiated tissues and irradiated
tumors also are potent chemoattractants for MSC. Irradiated 4T1 cells have increased
expression of cytokines like MCP-1, TGF-β1, VEGF and PDGF-BB. Interestingly, the
chemokine receptor CCR2 is upregulated in MSC exposed to irradiated tumor cells and
inhibition of CCR2 leads to decrease of MSC migration in vitro (82). Thus, not only the
production of a chemoattractant by tumor cells but also the expression of its
correspondent receptor in MSC are altered by radiation therapy. Interestingly, there is
recent evidence that the production of some of these chemokines is controlled by
miRNA. Using microarray and bioinformatics approaches, Luet al. identified six
miRNAs with differential expression in damaged liver tissue. They found that miR-27b

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could directly interacts with the 3'UTR of SDF-1α to suppress SDF-1α protein expression
compared to normal C57BL/6 murine liver tissue (83).
Apart from leukocyte recruitment molecules, several factors which play a role in
ECM remodeling at injury sites are shown to be trophic for MSC. High expression of
Urokinase Plasminogen Activator (uPA) and its receptor (uPAR) on solid tumor lines
leads to strong induction of MSC migration. Additionally, uPA itself was found to
directly stimulate MSC migration (84). MMP1 is also crucial for MSC homing. Though
it is not directly chemotactic, MMP1-mediated cleavage of the g-protein Protease
Activator Receptor 1 (PAR1) on MSC imparts sensitivity to recruitment signals, as
disrupting the cleavage event inhibits tumor-directed migration (85). Tumor cell surface
molecules also contribute to the recruitment capacity of the extracellular environment, as
extracts from fractionated lesions stimulated robust MSC migration (70).
The question whether MSC from the bone marrow are released and recruited by
primary tumors has, however, not been entirely elucidated. Several laboratories,
including ours, have shown that MSC injected in the tail vein of mice can be found in
primary tumors. However the number of these cells is generally small and in vivo
kinetics studies have shown that viable donor MSC injected i.v. in mice are present in the
lungs up to a maximum of 24 h after infusion, after which they disappear (86). Direct
evidence of MSC recruitment by human tumors is also lacking at the present time.
However, there is some indirect evidence. MSC have been isolated and expanded in vitro
from several fresh, enzymatically digested tumor tissues, including MSC-like cells from
human esophageal carcinoma (hEC-MSC) and adjacent non-cancerous tissues (hECN-
MSC). These cells express several MSC markers such as CD13, CD29, CD44 and

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CD105 (87). Other groups have shown that MSC can be isolated as CD29+, HLA+ and
CD105+ cells from gastric tumors in both tumoral tissues and adjacent non-tumoral
tissues (88,89). A recent study in 15 patients with non-small cell lung cancer (NSCLC)
also demonstrated that MSC can be isolated not only from tumor tissue but also from
corresponding normal lung tissue. These MSC were characterized and selected according
to their mesenchymal-multilineage differentiation capability. When compared to MSC
from normal tissue, tumor-derived MSC showed accelerated growth kinetics and reduced
sensitivity to cisplatin (90).
The question whether, under pathological conditions of cancer progression, MSC
are actively recruited by tumor cells and are educated into TAF and other tumor-
associated mesenchymal cells has not been entirely resolved. Recent studies suggest that
MSC are recruited, but these preliminary studies await important confirmation and
validation in larger numbers of tumors. Whether MSC also are recruited by normal tissue
and contribute to the formation of a premetastatic niche is another interesting question
that has not been explored so far.
Role of MSC in bone metastasis
MSC are responsible for creating the bone marrow microenvironment that drives tumor
growth, bone metastasis, and drug resistance. Osteogenic MSC derivatives produce
chemoattractant proteins SDF-1 and MCP-1which chemoattract HSC as well as
metastatic leukemia, mammary carcinoma, and myeloma (91-93). Adhesion-dependent
and adhesion-independent mechanisms mediate the cross-talk between tumor cells and
MSC. Adhesion between metastatic cells and MSC drive tumor progression in studies of
multiple myeloma (94). HCAM (CD44), VLA-4 (CD49/CD29), ICAM-1(CD54),

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NCAM (CD56), VCAM-1 (a ligand for α4β1 integrin), and LFA-3 (CD58) are found on
human myeloma and allow binding to stromal cells (94-96) and ECM (97). IL-6 is
produced by MSC following contact with myeloma cells, leading to osteolysis (98).
Contact between stromal cells or fibronectin and myeloma also promotes tumor cell
survival and drug resistance (99). STAT3 signaling, driven by IL-6, is responsible for this
effect (100), with cyclo-oxygenase-2 (Cox-2) driven PGE2 production creating a
synergistic enhancement (101). VCAM-1 and α4β1 integrin induce myeloma adhesion to
MSC, increasing the production of osteoclast activating factors (98).
Metastasis to the bone marrow is also driven by contact-independent signals,
produced by tumor cells, which stimulate MSC. This is true of neuroblastoma (102), a
pediatric malignancy that often metastasizes to the bone marrow (70.5%) and the bone
(55.7%) (103). Neuroblastoma cells induce MSC to produce pro-tumorigenic IL-6 (104).
This stimulation of MSC IL-6 expression requires neuroblastoma cells to produce soluble
signals, as opposed to cell–cell or cell–ECM contact in myeloma.
MSC represent a low percentage of cells found in bone marrow, so their ability to
enhance metastatic tumorigenesis may be limited. It thus bears investigating if MSC can
still enhance tumor growth once they have differentiated into connective tissue subtypes
(osteoblasts, chondrocytes, adipocytes, muscle cells), with preliminary evidence
providing confirmation of this effect. Adipocytes drive tumor growth and protection
from chemotherapy (105), and osteoblasts enhance homing and survival of metastatic
leukemia cells by production of the Wnt antagonist sFRP-1, which drives daunomycin
resistance (105,106). It remains to be seen if chondrocytes and myoblasts will have a
similar effect on tumors.

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Role of MSC in primary tumors
After homing to the tumor microenvironment, MSC alter the stromal composition
and growth rate of the lesion. Tumors possess dense, reactive stroma, similar to the
granulation tissue of healing wounds. This stroma is primarily comprised of mesodermal
cell types, including fibroblasts, myofibroblasts, endothelial cells, and pericytes (107).
Though the majority of the stromal cells are likely recruited from local tissue sources, in
vivo tracking of labeled MSC indicate that these bone marrow derived progenitors
differentiate into fibroblasts, pericytes, and possibly endotheilial cells within the tumor
mass (108). Recently, a new stromal subtype in solid tumors has been described. These
Tumor Associated Fibroblasts (TAF) display characteristics of myofibroblasts, such as α-
SMA and vimentin, but also express fibroblast activation markers, ECM remodeling
enzymes, and various growth factors (73,109). MSC exposed to tumor conditioned
media display TAF markers, as do MSC co-injected with tumor cells in subcutaneous
models.
Proliferation
The influence of MSC derivatives on the growth of tumor cells shows significant
variance between models. In xenogenic studies, MSC display both stimulatory and
inhibitory effects on tumor cell proliferation. Intravenously-injected MSC inhibited the
growth of Kaposi’s sarcoma cells in nude mice. Co-culture of MSC and sarcoma cells in
vitro also reduced proliferation, driven by inhibition of AKT (Protein Kinase B) in tumor
cells that were in contact with MSC (110). In a study of human glioma, co-
administration of MSC and glioma cells in vivo reduced tumor volume and vascular
density, an effect that was not observed when glioma cells were injected with

15

immortalized normal human astrocytes. This growth suppression was confirmed using
both patient-derived glioma cells and glioma cell lines in vitro (78). Opposite effects on
proliferation were observed in an ovarian carcinoma model, in which co-injection of
human ovarian carcinoma cells with MSC isolated from ovarian carcinoma tumors
enhanced tumor growth in vivo. Enhanced production of BMP-2, -4, and -6 by ovarian
carcinoma-derived MSC played a role in this proliferative effect, which could be partially
blocked by BMP inhibition (111). MSC also supported faster growth of human colon
carcinoma (112) and generated larger foci of human ovarian carcinoma in nude mice
(73). Both MSC and MSC conditioned media drive ovarian carcinoma cell growth in 3D,
an effect dependent on MSC-derived IL-6. MSC increased production of VEGF, TGF-B,
and IL-6 when stimulated by ovarian carcinoma conditioned media (73). Furthermore,
MSC enhanced the growth of human osteosarcoma in nude mice, and MSC conditioned
media sped osteosarcoma cell proliferation in vitro (70). CCL5 was necessary for this
effect, as tumor growth could be inhibited by an anti-CCL5 antibody (113). Syngeneic
tumor models show similarly contradictory effects of MSC – tumor interactions. In a
subcutaneous 4T1 model, i/v MSC did not increase tumor mass (114). MSC co-injection
did, however, stimulate faster tumor onset in A17 mesenchymal tumors, which represent
a stromal population from Her2/neu mammary carcinoma (115). Co-implantation of rat
osteosarcoma cells with rat MSC also enhanced tumor formation and growth rate in vivo
(116). Similarly, mammosphere formation by 3D cultured breast carcinoma cells was
enhanced in the presence of MSC (117).

16

Survival
MSC differentially alter apoptotic signaling in various tumor models, and can
induce tumor cell survival or death. Our laboratory has provided evidence that MSC
protect neuroblastoma cells from drug-induced apoptosis and that this effect is in part
mediated by IL-6, which is overexpressed by MSC when in the presence of tumor cells
(104,118). Downstream of IL-6 is the STAT3-dependent expression of many survival
proteins like Bcl-XL, Bcl2, Mcli and survivin (87). Another recent study points to
reactive oxygen species (ROS) as an additional pathway by which MSC provide a
protective environment for tumor cells by affecting their metabolism. MSC-derived
stanioclacin1 (STC1) promotes the survival of lung cancer cells by uncoupling oxidative
phosphorylation, reducing intracellular ROS, and shifting their metabolism towards a
more glycolytic metabolic profile consistent with the Warburg effect (119). Adipocytes
derived from MSC block the activity of all-trans retinoic acid and attenuates therapy-
induced apoptosis in promyelocytic leukemia cells (120). STAT-3 and MAPK activation
were required for this effect and could be induced by leptin secreted by MSC-derived
adipocytes. H
2
O
2
oxidative stress in neuroblastoma cells could also be blocked by MSC
(121). Intravenous injection of cord blood or adipose MSC increased cell death in human
mammary carcinoma by enhancing PARP-1 and caspase-3 cleavage (122). Apoptotic
proteins p21 and caspase-3 were also found to be upregulated in murine hepatoma and
lymphoma cells exposed to MSC (123).
Angiogenesis
MSC contribute to the generation of tumor vasculature by both cell-mediated and
paracrine effects. The secreted protein profile of MSC includes several proangiogenic

17

factors such as VEGF, angiopoetin, IL-6, IL-8, TGF-B, PDGF, and FGFs -2 and -7 (67).
MSC show increased production of TGF-B, VEGF, and IL-6 in response to tumor
conditioned media, likely as a function of TAF differentiation (73). Additionally, the
inflammatory cytokine TGF-A, important in the tissue repair response, drives VEGF
production by activating MEK and PI3-K in MSC (124,125). In vivo, VEGF contained
in MSC conditioned media induced sprouting in cultured HUVEC. VEGF production by
intravenously-injected MSC also generated increased vessel density in human pancreatic
carcinoma xenografts. This effect was blocked by transducing MSC with VEGF siRNA
before injection (126). Notably, VEGF, TGF-B, and IL-8 are involved in MSC homing
as well as angiogenesis, highlighting their role in the cross-talk between tumor cells and
recruited MSC.
Tumor vasculature is associated with a number of MSC derivatives. MSC-
derived pericytes and mural cells are localized around tumor blood vessels following
labeling and implantation (127). Certain endothelial cells may be MSC-derived, based on
the formation of tube structures by Matrigel-implanted MSC and the induction of
endothelial markers on MSC exposed to angiogenic signals (128). Only a small
percentage of VEGF-stimulated MSC acquire endothelial markers, suggesting a limited
contribution of MSC-derived endothelial cells to the overall tumor vasculature (126).
Inhibitory effects of MSC on angiogenesis have also been noted. MSC form gap
junctions with endothelial cells using connexin 43, though the nascent networks break
down if too many MSC are present (129). MSC-produced ROS trigger apoptosis in
endothelial cells when cultured together in matrigel, and antioxidants prevent this death

18

response. Further, implantation of MSC in subcutaneous melanoma enhanced caspase-3
cleavage and reduced growth of endothelium (129).
Immunosuppression
The immunomodulatory ability of MSC is also likely to play a role in tumor
development, although this interaction has been less well studied (130). MSC can inhibit
T-cell proliferation (131,132), DC maturation (133), and NK- and B-cell activation (134).
MSC also enhance regulatory T-cell generation (Treg) (10,135). However, this
immunosuppression is not necessarily pro-tumorigenic, largely depending on how a
particular lesion responds to prolonged inflammation. B16 melanoma tumors could be
induced in an allogeneic recipient by co-injection with MSC (136), due to enhanced Treg
production and reduced levels of cytotoxic T-cells. MSC exhibit extensive anti-
proliferative properties against lymphocytes under different conditions (137) and
stimulate the early formation of tumors in mouse adenocarcinoma cells (Renca)
implanted in syngeneic mice (138). The mechanisms behind the immunosuppressive
effect of MSC are complex. MSC express TLR which enhance their immunosuppressive
phenotype. Immunosuppression mediated by TLR is dependent on the production of
kynurenines by the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase-1 (IDO-
1). Induction of IDO-1 by TLR involves an autocrine IFN-β signaling loop, which is
dependent on protein kinase R (PKR) but independent of IFN-γ (139). MSC are also a
source of several soluble immunosuppressive factors, such as IL-10, TGF-β and
prostaglandin E2 (PGE2). Expression of these factors is in part mediated by galectin-1
and galectin-3, which are constitutively expressed and secreted by human bone marrow
MSC (140). Inhibition of galectin-1 and galectin-3 gene expression with small

19

interfering RNAs abrogates the suppressive effect of MSC on allogeneic T cells (141).
Conflicting effects of immunosuppression mediated by MSC have been identified in
clinical studies. MSC injected into cancer patients simultaneous with hematopoeitic
stem cell transplantation did not affect the growth of breast or hematologic lesions (142-
144). Separate studies indicated an increased chance of relapse in cancer patients
transplanted with both hematopoietic cells and MSC (145).
Metastasis
MSC have been shown to influence the metastatic potential of transformed cells
and participate in the formation of secondary lesions. Tropism of MSC toward metastatic
tumors has been visualized through several modalities, and the recent use of MRI to
detect i.v. injected, iron-labeled MSC in lung xenographs of human memory carcinoma
demonstrates the clinical potential of in vivo MSC tracking (146). MSC regulate the
dissemination of tumor cells at both the primary tumor and targeted secondary organs,
likely through different mechanisms depending on the site. MSC differentiation is also
altered in the microenvironment of primary and metastatic lesions. In a comparison of
syngeneic subcutaneous and metastatic mammary carcinoma, MSC became osteoblasts in
metastatic lung tumors and adipocytes in the primary tumors (114).
Despite their recruitment potential, MSC show inconsistent effects on the growth
of metastatic lesions. MSC did not affect lung metastasis of syngeneic mammary
carcinomas following intravenous injection. In xenogenic studies of orthotopic
mammary carcinoma, injection of cord blood or adipose MSC reduced metastatic lung
modules, although adipose MSC had no effect on growth if administered the same day as
tumor induction. Co-injection of tumor cells with adipose and cord blood MSC limits

20

metastasis (114). On the other hand, co-injection of human mammary carcinoma and
MSC enhanced metastasis (147). Explants of the MSC-conditioned metastatic tumor cells
did not display the same invasive potential when re-implanted without MSC. This effect
was driven by CCL5/CCR5 and could be blocked by anti-CCL5 antibody treatment or
induction of tumor cells with CCR5 siRNA. Nude mice injected with orthotopic
osteosarcomas showed greater levels of metastasis when injected i.v. with MSC (70).
Summary
Pro-tumorigenic properties of MSC are presented in Figure 1, while tumor promoting
versus tumor inhibiting effects are summarized in Table 1. Although tumor growth
enhancement is the typical result of the MSC-tumor crosstalk, it is still uncertain whether
this pro-tumorigenic stimulus is primarily driven by increased proliferation or increased
survival. The alterations in MSC signaling which lead to reductions in tumor cell
survival also remain unexplored. In our current study, we sought to determine whether
observed growth enhancements in tumor cells exposed to MSC were due to increased
proliferation or reduced apoptosis.

21

Figure I-1. Reciprocal interactions between tumor cells and MSC in the bone marrow
and the tumor
Circulating tumor cells are attracted in the bone marrow niche by several cytokines like
SDF-1. When in the bone marrow, tumor cells find a sanctuary and interact with MSC
via integrin-dependent mechanisms, via the production of soluble factors like Gal-3BP
and via exosomes. MSC respond by producing cytokines like IL-6, IL-8, MCP-1, growth
factors like TGF-B and VEGF as well as exosomes. MSC are also recruited by primary
tumors through the production by tumor cells of factors like MCP-1, SDF-1, CCL-25, IL-
8, CXCL13, S1P, HGF, LL-37 that attract MSC. When in tumors, MSC can become TAF
expressing FSP-1, FAP, MMP-11, IL-6 and collagen that affect tumor cell proliferation,
survival, immune-escape, angiogenesis, EMT and the formation of a desmoplastic stiff
matrix. The bone marrow is thus not only a site of metastasis but also a ‘remote-
controller” of the primary tumor.

22

Pro-tumorigenic Anti-tumorigenic
CAF differentiation Contact dependent - Akt inhibition
growth inhibition - MAPK inhibition
- Cell fusion
Angiogenic stimulation - VEGF, bFGF, TGFβ, IL-/8,
IL-6, angiopoeitin
Vascular disruption - ROS generation
- Caspase-3 cleavage
Immunosuppression - IL-6, IL-10, TGFβ, PGE2,
iNOS, IDO, HGF
Drug sensitization - ERK1/2 inhibition
Contact independent - IL-6, CCL5, TGFβ, EGF,
growth stimulation BMP2, SDF-1, CXCL7
- ADAM12
- Estrogen receptor
- ERK1/2
- Exosomes
Pro-apoptotic stimulation - PARP-1 cleavage
- Caspase-3 cleavage
- p21 upregulation
- Caspase-3,8 upreg.
- Bax upregulation
- Bcl-2 downreg.
- Microvesicles
- Cell fusion
Contact dependent - Cell fusion
growth stimulation - Cancer stem cell renewal

Anti-apoptotic - STAT3
stimulation - MAPK
- PI3K/Akt
- Antioxidant enzymes
- Tumor autophagy
- Cell fusion
- Survivin

Vascular stability - Endothelial/pericyte diff.

Invasive/metastatic - CCL5, NRG1, IL-6, VEGF,
stimulation TGFβ, SDF-1
- CXCR2, CXCR3
- EMT activation
- Estrogen receptor
- ADAM10
- MMP1,3,13

Drug resistance - IL-6, PGE2, GDF15
- STAT3
- Fatty acids

Table I-1. Pro and anti-tumorigenic functions of MSC

23

Chapter 2 – Protective Effect of MSC on Primary Tumor Cell Apoptosis
Introduction
The process of tumorigenesis, previously thought to stem primarily from genetic
changes within transformed cells, is now known to also depend on extracellular signals
from non-cancerous cells present in the tumor microenvironment (TME) (148). Among
the normal cells that contribute to the TME are bone marrow-derived myeloid cells
whose function and contribution to neoplastic growth has been best understood (78).
Recently, there has been increased evidence that bone marrow-derived mesenchymal
cells also contribute to tumorigenesis and metastasis, but their role has been less well
characterized (149). Mesenchymal cells produce local, terminally differentiated
fibroblasts and are descended from multipotent bone marrow-derived mesenchymal
stromal cells (BMMSC) (1). Within the bone marrow microenvironment, BMMSC and
osteoblasts provide a supportive niche for homing of hematopoietic stem cells (HSC) and
tumor cells which promotes quiescence and survival (4). These cells also serve to
generate the various connective tissue lineages (adipocyte, osteoblast, chondrocyte,
myocyte, neuron, fibroblast) found in the marrow and in distal organs, and are actively
recruited to sites of injury, inflammation and neoplastic transformation.
The kinetics of BMMSC recruitment from the marrow niche, as well as their
distribution in primary tumors, are still poorly characterized. BMMSC display a
chemotactic response to chemokines, growth factors, and extracellular matrix (ECM)
proteases produced by tumor cells, which mimics the activity of inflammatory HSC and
is mechanistically indistinguishable from the generalized wound healing response (150).

24

Though BMMSC exhibit potent effects even in small numbers, studies of tumor-recruited
BMMSC suggest that their migration is inefficient and difficult to quantify in vivo (151).
However, once recruited to tumor sites, BMMSC differentiate into tumor-associated
fibroblasts (TAF), which produce mitogenic and angiogenic factors and display potent
ECM remodeling capabilities (109). Cytokines secreted by BMMSC are also known to
modulate immune responses within the TME, creating immunosuppressive effects which
drive tumor progression (152). Concordantly, introduction of BMMSC into tumor
bearing mice by intravenous injection or co-injection shows a net positive effect on tumor
growth in a majority of studies (70, 153). However, anti-tumorigenic effects, driven by
increased caspase-3 and PARP-1 cleavage, have also been reported (123). Most
published work on the MSC-tumor interaction has focused on proliferative, angiogenic
and immunoregulatory effects. Previous studies conducted in our laboratory have
identified a pro-survival effect of human BMMSC on metastatic human neuroblastoma
cells in the bone marrow microenvironment that promotes drug resistance (104, 118).
This observation provides the basis for our present examination of a novel role of these
mesenchymal cells and their derivatives within primary tumors, rather than the bone.
I hypothesized that circulating BMMSC are incorporated into primary tumor sites
and protect tumor cells from spontaneous and therapy-induced apoptosis via the
production of soluble factors, similar to the role of native BMMSC in promoting
metastatic tumor cell survival in the bone marrow microenvironment.
Material and Methods
Reagents and cells
The murine cell lines 4T1 mammary carcinoma, LL/2 Lewis lung carcinoma and

25

NIH3T3 fibroblasts were purchased from ATCC (American Type Culture Collection).
Cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) or RPMI-1640
(4T1) containing 10% fetal calf serum (FCS) and supplemented with penicillin and
streptomycin. BMMSC culture medium was Iscove’s Modified Dulbecco Medium
(IMDM) containing 15% FCS and 15% denatured horse serum (DHS) supplemented with
hydrocortisone (10
-6
mol/L), 2-mercaptoethanol (10
-4
mol/L), and penicillin-
streptomycin. StemPro osteocyte/chondrocyte differentiation medium and adipocyte
differentiation medium were from Gibco. Anti-mouse CD34-PE, CD45-PerCP, CD31-
PE, VEGFR2-PE, and CD44-FITC were purchased from BD (Becton Dickinson).
Paclitaxel was purchased from Sigma, and doxorubicin from GensiaSicor.
Bone marrow mesenchymal stromal cells
Murine BMMSC were obtained from 6-8 week old Balb/cJ mice (Jackson
Laboratories) using a protocol adapted from Kirshner, et al. 2008 (154). In brief, animals
were sacrificed and both femurs, iliac bones, and tibias were used. The extremities of
each bone were removed with scissors and the marrow cavity was flushed a first time
with phosphate buffered saline (PBS) using a 27-gauge needle to remove non-adherent
hematopoietic cells, and the material was discarded. Each bone was then flushed a
second time with PBS, using the needle to simultaneously scrape the endosteal surface of
the marrow cavity. Flow through material from the second flush was collected, subjected
to hemolysis, and spun to remove debris. Pooled bone marrow cells from each animal
were plated in BMMSC culture medium in one well on a 12-well culture dish coated with
collagen I and fibronectin, both at 5 µg/cm
2
. Bone marrow cells were cultured for 24
hours and non-adherent cells removed by gentle washes two times. Adherent cells were

26

grown for 3-6 weeks to allow the development of a confluent stromal monolayer and then
harvested for routine passaging in regular plastic tissue culture dishes.
Fluorescent cell labeling
BMMSC were labeled for detection by fluorescence microscopy using the PKH26
Red Fluorescent Cell Linker Kit (Sigma). Briefly, 10
7
BMMSC were rinsed with PBS,
loosely pelleted, and suspended in 1 mL PBS for staining with 8 µL red fluorescent dye.
Cells were stained for 90 seconds, followed by treatment with 2 mL BMMSC culture
medium and an additional 60 seconds of staining. Cells were pelleted and rinsed three
times with culture medium, with a fresh tube used for each rinse. Cells were rinsed two
times with PBS, plated on a 10 cm dish in culture medium, incubated for 24 hours at
37°C, and harvested for use.
Flow cytometry
BMMSC were harvested with PBS-based cell dissociation buffer (Gibco) and
suspended in PBS containing 5% FCS (v/v) and 0.1% sodium azide (w/v). Analysis was
performed using a FACS Caliber flow cytometer and data were analyzed using
CellQuestPro software.
Osteocyte/adipocyte staining
For analysis of spontaneous differentiation, BMMSC were plated in 48-well dishes,
allowed to expand for 14 days, and fixed in 4% paraformaldehyde (PFA) prior to staining
for either alkaline phosphatase activity (AP), using a detection kit (Millipore), or for
triglyceride deposits, using oil red O (ORO; Sigma). For directed osteocyte
differentiation, BMMSC were treated with osteocyte differentiation medium for 21 days,
fixed in 4% PFA (w/v), and stained with alizarin red (Sigma) to detect calcium deposits.

27

For directed adipocyte differentiation, BMMSC were plated in 12-well dishes, treated
with adipocyte maintenance medium for 7 days, switched to adipocyte induction medium
for 7 days, fixed in 4% PFA, and stained with ORO.
BMMSC conditioned medium.
For generations of conditioned medium (CM), BMMSC were plated in 10 cm dishes
and grown to a confluent monolayer. Cells were rinsed with PBS and treated with two
changes of BMMSC culture medium containing 0.5% FCS (v/v) and 0.5% DHS (v/v)
(1% total serum) or 0.25% FCS (v/v) and 0.25% DHS (v/v) (0.5% total serum) for 30
minutes each. Medium was replaced with 10 mL culture medium containing 0.5% or 1%
total serum. After 3-4 days exposure to BMMSC, the CM was collected and spun to
remove cellular debris and stored at 4°C prior to testing. CM was also generated from
NIH3T3 cells by a similar method.
Cell proliferation and apoptosis assays
For 2D proliferation assays, tumor cells were cultured in 6-well plates at 5x10
4
per
well in the presence of 1% serum (v/v) CM or IMDM containing 1% serum (v/v). Cells
were harvested by trypsinization and counted in a hemocytometer. For 3D proliferation
assays, 4T1 cells were suspended in 4.5 mg/mL high growth factor Matrigel (BD
Biosciences) and plated on 24-well plates at 1x10
4
per well. After polymerization, 0.4
µm pore size transwell inserts containing 1x10
4
BMMSC in serum free medium were
added to the wells. The total number of tumorspheres (>50 µm diameter) in each well
was counted after 21 days. For apoptosis assays, cells were cultured in 6-well plates at
2.5x10
4
(4T1) or 1x10
5
(LL/2) per well in the presence of 1% serum (v/v) CM or 1%
serum (v/v) control medium. Cells were harvested by trypsinization, rinsed twice with

28

PBS, and suspended in Annexin V binding buffer. Annexin V and PI staining were
performed as per the manufacturer’s instructions (Annexin V-FITC apoptosis detection
kit I, BD Biosciences). Apoptotic cells were defined as Annexin V-FITC positive.
Caspase-3, caspase-9, and caspase-8 activity was determined using the appropriate
ApoTarget colorimetric protease assay kit (Invitrogen) on aliquots containing 50 µg
protein.
Drug resistance assay
Tumor cells were plated in opaque white 96-well culture plates at 1x10
4
per well and
treated with paclitaxel or doxorubicin for 72 hours (LL/2) or 96 hours (4T1) in the
presence of 1% serum (LL/2) or 0.5% serum (4T1) CM or BMMSC culture medium.
CellTiter-Glo reagent (Promega) was then added to each well at a 1:1 ratio with the cell
medium, and luminescent reporter activity was measured via GloMax Multi Detection
System (Promega), using Instinct software (Promega). For determination of caspase-3/7
activity, tumor cells were treated with paclitaxel or doxorubicin for 24 hours in the
presence of 1% serum (LL/2) or 0.5% serum (4T1) CM or BMMSC culture medium.
Caspase-Glo 3/7 reagent (Promega) was then added to each well at a 1:1 ratio with the
cell medium, and luminescent reporter activity was measured as above described. For
apoptosis induction, 4T1 cells were cultured in 6-well plates at 3x10
5
per well and
treated with 0.5 µmol/L paclitaxel or doxorubicin for 16-24 hours in the presence of 0.5%
serum (v/v) CM or 0.5% serum (v/v) control medium. Cells were harvested by
trypsinization, rinsed twice with PBS, and suspended in Annexin V binding buffer.
Annexin V and PI staining were performed as per the manufacturer’s instructions
(Annexin V-FITC apoptosis detection kit I, BD Biosciences).

29

Transwell migration assay
For transwell migration studies, 8x10
3
BMMSC were plated in 24-well, 8.0 m pore
size transwell inserts and exposed to serum-free tumor cell CM or control medium added
to the lower chamber of the transwell dish. After migration, transwell inserts were
removed and the upper side of the filter was swabbed to remove non-migratory cells.
Transwell filters were then fixed/stained with the Hema3 Stat Pack (Protocol) as per the
manufacturer’s instructions. Filters were cut free and mounted on slides in a drop of
immersion oil for microscopic examination.
BMMSC iron labeling
BMMSC were grown in 10 cm dishes to form a low-density monolayer (1-2x10
6

cells per dish). Cells were rinsed with PBS and treated with 6.5 mL serum-free IMDM
supplemented with 2U/mL heparin (Sigma). Within 15 seconds, 60 µg/mL protamine
sulfate (APP Pharmaceuticals) was added to medium, with gentle agitation of the dish.
Within another 15 seconds, 100 µg/mL Feraheme (AMAG Pharmaceuticals) was added
to medium, with simultaneous agitation of the dish. Cells were incubated for 2 hours at
37ºC and 5% CO2, followed by addition of 6.5 mL BMMSC culture medium. After 24
hours, cells were rinsed with PBS, followed by a second rinse with PBS containing
10U/mL heparin, followed by a third rinse with PBS. Cells were harvested by
trypsinization for use in vivo.
Animal experiments
Studies in mice were performed under a protocol approved by the Institutional
Animal Care and Use Committee at Children’s Hospital Los Angeles (Protocol 41-11).
For tumor recruitment studies, Balb/cJ mice were injected s.c. with 6x10
5
4T1 cells in the

30

left flank. On day 12 of tumor growth, mice received ~2x10
6
PKH26-labeled BMMSC
via tail vein. Forty-eight hours after BMMSC injection, mice were sacrificed and tumors
extracted for cryopreservation. For early-stage tumor recruitment studies, Balb/cJ mice
were injected s.c. with 5x10
5
4T1 cells in the left flank. Four days after tumor cell
injection, mice received 3x10
5
iron-loaded or control BMMSC via tail vein. Six days
after BMMSC injection, mice were sacrificed and tumors extracted for formalin fixation
and paraffin embedding. For drug resistance studies, Balb/cJ mice were injected with
1x10
6
4T1 cells in the right 4
th
mammary fat pad and 1x10
6
4T1 cells plus 2x10
5

BMMSC (5:1 ratio) in the contralateral fat pad. On days 3 and 4 after tumor cell
injection, mice received doxorubicin (5 mg/kg) by i.p. injection. On day 5 after tumor
cell injection, mice were sacrificed and tumors extracted for cryopreservation.
Histology
Prussian blue staining was carried out on formalin-fixed, paraffin-embedded (FFPE)
sections (6 µm) of 4T1 tumors from mice injected with iron-loaded and control BMMSC.
Sections were deparaffinized, rehydrated, and treated with a solution of potassium
hexacyanoferrate (II) trihydrate (Sigma) in HCl for 20 minutes. Slides were
counterstained with nuclear fast red (Sigma), dehydrated, cleared in xylene, and mounted
in Cytoseal. Frozen sections (6 µm) of 4T1 tumors from mice injected with PKH26-
labeled BMMSC were mounted in Vectashield medium for fluorescence with DAPI
(Vector Laboratories) and examined for red fluorescent signal. Frozen sections of
orthotopic 4T1 tumors from mice co-injected with BMMSC were stained for apoptotic
cells using the Cell Death Detection Kit (Roche), as previously reported (155).

31

Statistical analysis
Comparisons between two groups were performed by unpaired Student t test with
unequal variances assumed, while multiple group comparisons were performed by 2-way
ANOVA or linear regression analysis. T tests and ANOVA were performed using Excel
(Microsoft), and linear regression analysis was performed using Stata 11 software
(StataCorp). Two sided p values are reported.
Results
BMMSC characterization
Murine mesenchymal cells harvested from the bone marrow and expanded in vitro
were tested for their stem cell potential by a combination of surface marker staining and
functional studies (Fig. 1). FACS analysis of BMMSC revealed positive staining for
CD44, an established mesenchymal marker at both early (<10) and late (10-20) passages
(23). The cells were negative for the endothelial markers CD31 and VEGFR2 at early
and late passages, along with the myeloid marker CD45. Weak positive staining for the
hematopoietic marker CD34 was observed at early passages, potentially due to the
presence of some myeloid cells that were not eliminated in the washing procedure. Late
passage BMMSC were negative for CD34, which parallels the loss of contaminating cell
types over subsequent passages (Fig. 1A). The tested cells thus displayed characteristic
BMMSC markers by cell surface phenotype.
To establish whether these cells also retained their pluripotent function, they were
cultured for an extended period and tested for spontaneous generation of both adipocytes
and osteoblasts. Analysis of a single BMMSC culture over multiple passages showed
relatively stable levels of adipogenesis, while early osteoblastic differentiation potential

32

decreased at around passage 8 (Fig. 1B). Late passage BMMSC were able to generate
mature osteocytes, characterized by alizarin red-positive calcium deposits, when cultured
in osteoblast induction medium (Fig. 1C), and cultures of BMMSC treated with

Figure II-1. Primary murine bone marrow stromal isolates display mesenchymal cell
markers
A, FACS analysis of BMMSC surface markers at early (<10) and late (>10) passages.
Upper panel: representative histograms for early passages (open histogram represents the
autofluorescent control). Lower panel: percent of cells positive for indicated marker at
early and late passages. B, spontaneous osteoblast/adipocyte differentiation of BMMSC
in culture. Upper panel: light micrographs of early passage BMMSC cultured for 2-3
weeks and stained for alkaline phosphatase (AP) activity (osteoblasts) or lipids by Oil
Red O (ORO, adipocytes). Scale bar = 250 µm (left) and 100 µm (right). Lower panel:
percent of BMMSC with positive staining for AP and ORO over passages in culture. The
data represent the mean ±SD of positive cells counted in five 40x fields. C, mature
osteoblast differentiation of BMMSC cultured 3 weeks in osteogenesis medium vs.
control medium and stained with Alizarin red. D, early adipocyte differentiation of
BMMSC cultured for 2 weeks in maintenance medium or switched to adipogenic
medium after 1 week, and stained for the presence of lipids with ORO (Scale bar = 100
µm).

33

adipogenesis induction medium showed an increased percentage of ORO-positive cells
versus controls, which generated smaller numbers of more mature adipocytes (Fig. 1D).
The observed spontaneous and inducible differentiation of osteoblasts and adipocytes in
these experiments confirmed the identity of the tested cells as BMMSC.
BMMSC enhance tumor cell expansion
In order to determine the effect of BMMSC on tumor cell expansion, 4T1 murine
mammary adenocarcinoma cells were first grown in the presence of BMMSC in 3D
cultures of Matrigel. This experiment revealed that Matrigel-embedded 4T1 cells grown
in the presence of BMMSC yielded increased numbers of tumorspheres when compared
to BMMSC-free controls (p = 0.0006) (Fig. 2A). Using 2D cultures of 4T1 grown in the
presence of 1% serum CM from BMMSC, we then observed a two-fold increase in cell
number when compared to regular medium containing 1% serum (p = <0.001 by
ANOVA) (Fig. 2B). Similarly, LL/2 cells exposed to 1% serum CM from BMMSC
showed significantly increased numbers of viable cells versus controls, maintaining a
two-fold difference from day 2 on (Fig. 2B). The data thus indicated that BMMSC
increase the viability of tumor cells by a mechanism that does not require direct cell-cell
contact and is primarily mediated by soluble factors present in the CM. To determine if
this increase in tumor cell expansion stemmed from enhanced proliferative potential vs.
changes in spontaneous apoptosis, 4T1 cells cultured in the presence or absence of
BMMSC CM were examined for proliferation by cell cycle analysis. The data revealed
that 4T1 cells grown in the presence of 1% serum CM showed no significant increase in
the percentage of cells in S-phase or G2/M-phase at 2 days, but showed a significant
decrease in the percentage of S-phase and G2/M-phase cells at 4 days, suggesting in fact

34

some inhibitory effect on proliferation (Supplementary Fig. S1A). Using BrdU labeling,
however, we did not find any significant difference in this percentage of cells in G2 phase

Figure II-2. BMMSC enhance tumor cell growth by suppressing spontaneous
apoptosis
A, Matrigel-embedded 4T1 cells cultured for 21 days in the presence or absence of
BMMSC in a transwell chamber were examined for the formation of tumorspheres. The
data represent the mean ±SD of 4T1 tumorspheres in quadruplicate wells from one of two
experiments showing similar results. Inset: light micrographs of 4T1 spheroids in the
central field of representative control and co-culture wells. Scale bar = 500 µm. B, 4T1
cells (left panel) and LL/2 cells (right panel) were cultured in 2D for 4 days in the
presence or absence of CM from BMMSC. The data represent the mean ±SD of viable
cells at indicated times from triplicate wells. C, 4T1 cells were cultured in 2D for 5 days
in the presence or absence of CM from BMMSC or NIH3T3 cells. Top panel:
representative analysis of apoptosis by flow cytometry at day 5. Lower panel: the data
represent the mean ±SD of Annexin V-FITC positive cells at indicated times in triplicate
wells from one of two experiments showing similar results. D, 4T1 cells were cultured
for 5 days in the presence or absence of CM from BMMSC and examined for caspase
activity at the indicated times. The data represent the mean ±SD of caspase-3 (top),
caspase-9 (middle), and caspase-8 (bottom) activity at day 3, 4, and 5 from triplicate
wells from one of two experiments showing similar results.

35

in BrdU-positive 4T1 cells in the presence of BMMSC CM versus regular medium
(Supplementary Fig. S1B). These data thus suggested that the increase in viability
observed when tumor cells were cultured in the presence of BMMSC CM was not due to
a positive effect on proliferation.
BMMSC protect tumor cells from spontaneous apoptosis
I therefore explored whether BMMSC could affect tumor cell survival. 4T1 grown
in 1% serum CM or control medium for 3-5 days were stained with Annexin V-FITC and
PI and examined by flow cytometry (Fig. 2C). We observed significant reductions in the
percentage of Annexin V(+) cells in cultures exposed to BMMSC CM for 3 (16.8% CM
vs. 24.8% control; p = 0.012), 4 (21.8% CM vs. 46.1% control; p = 0.002), and 5 days
(7.4% CM vs. 19.1% control; p = 0.0004). This effect was specific to BMMSC since we
did not observe a statistically significant decrease in spontaneous apoptosis when cells
were cultured in 1% serum CM from NIH3T3 cells (16.4% NIH3T3 CM vs. 19.1%
control; p=0.21). Experiments utilizing LL/2 cells revealed a similar reduction in the
percentage of Annexin V(+) cells in the CM-treated samples versus controls that reached
statistical significance by day 5 (51.8% CM vs. 63.6% control; p = 0.033 (Supplementary
Fig. S2A). Consistent with a protective effect of BMMSC CM on apoptosis, an analysis
of cell lysates from 4T1 cells indicated a statistically significant decrease in the level of
caspase-3 activity in cells exposed to BMMSC CM for 5 days, compared to controls
(44.5% of control) (Fig. 2D). Similar data were observed with LL/2 cells
(Supplementary Fig. S2B). The data thus suggest that the observed increase in viability
in tumor cells cultured in the presence of CM from BMMSC was primarily the result of a
protective effect against apoptosis induced by serum starving. In order to determine if

36

this protective effect stemmed from a suppression of intrinsic or extrinsic apoptotic
signaling, we examined the effect of BMMSC CM on caspase-9 and -8 activity. These
data revealed a statistically significant decrease in caspase-9 activity (44.2% of control)
and a non-significant inhibition of caspase-8 activity (25% of control) in the presence of
BMMSC CM at day 5, consistent with a primary protective effect on intrinsic apoptosis
(Fig. 2D). The data thus indicated that CM of BMMSC increased the viability of tumor
cells primarily by having a protective effect on intrinsic apoptosis.
BMMSC protect tumor cells from drug-induced apoptosis
I then asked whether BMMSC would also protect tumor cells from drug-induced
apoptosis. For these experiments, 4T1 and LL/2 cells were exposed to apoptosis-
inducing drugs in the presence or absence of CM containing 1% total serum (LL/2) or
0.5% total serum (4T1). 4T1 cells cultured in the presence of BMMSC CM and treated
with paclitaxel or doxorubicin (concentrations ranging from 0.00001 to 100 µmol/L)
showed a significantly greater survival than control cells cultured in regular medium at
therapeutic concentrations (Fig. 3A), as confirmed by linear regression analysis (p ≤
0.001). Similarly, LL/2 cells treated with paclitaxel or doxorubicin at therapeutic
concentrations showed a significantly greater survival when cultured in the presence of
BMMSC CM than in the presence of regular medium (Fig. 3B), (p ≤ 0.016). Consistent
with a protective effect of BMMSC on drug-induced apoptosis, we observed a
significantly lower level of caspase-3/7 activity in 4T1 cells cultured in the presence of
BMMSC CM than controls when exposed to paclitaxel (0.1 to 10 µmol/L) or doxorubicin
(5 to 10 µmol/L) (Fig. 4A) (p ≤ 0.02). LL/2 cells treated with BMMSC CM also showed
significantly lower levels of caspase-3/7 activity than controls in the presence of

37

paclitaxel (5 µmol/L) or doxorubicin (1 to 10 µmol/L) (Fig. 4B) (p ≤ 0.019). The
protective effect of BMMSC CM on drug-induced apoptosis was confirmed by flow
cytometry analysis of 4T1 cells grown in 0.5% serum CM or control medium for 16-24
hours in the presence of 0.5 µmol/L paclitaxel or doxorubicin and stained with Annexin
V-FITC and PI (Fig. 4C). This analysis indicated a significant reduction in the
percentage of Annexin V(+) cells in paclitaxel-treated cultures exposed to BMMSC CM
for 16 hours (11.8% CM vs. 29.7% control), although this difference was no longer

Figure II-3. BMMSC enhance survival in drug-treated tumor cells
A, cell viability of 4T1 cells (1x10
4
) cultured in the presence or absence of 0.5% serum
BMMSC CM and treated with paclitaxel (upper panel) or doxorubicin (lower panel) for
96 hours. The data represent the mean ±SD of surviving cells for indicated drug
concentrations in triplicate wells from one of three experiments showing similar results.
B, cell viability of LL/2 cells (1x10
4
) cultured and treated as indicated in A for 72 hours.
The data represent the mean ±SD of surviving cells for indicated drug concentrations in
triplicate wells from one of two experiments showing similar results.

38

significant after 24 hours of paclitaxel treatment (23.3% CM vs. 31.1% control).
Doxorubicin-treated 4T1 cells exposed to BMMSC CM also showed a significant
reduction in Annexin V(+) cells at both 16 hours (3.6% CM vs. 12.2% control) and 24
hours (6.4% CM vs. 17.1% control). The data thus demonstrated that BMMSC had a

Figure II-4. BMMSC CM protects tumor cells from drug-induced apoptosis
A, 4T1 cells (1x10
4
) cultured in the presence or absence of 0.5% serum BMMSC CM and
treated with paclitaxel (upper panel) or doxorubicin (lower panel) at indicated
concentrations for 24 hours were examined for caspase-3/7 activity. The data represent
the mean ±SD fold-change from 4T1 cells cultured in the presence of regular medium
(controls) in triplicate wells. They are representative of two experiments showing similar
results. B, LL/2 cells (1x10
4
) cultured as indicated in A were examined for caspase 3/7
activity. The data represent the mean ±SD fold-change from controls in triplicate wells
from one of two experiments showing similar results. C, 4T1 cells cultured as indicated
in A were examined for Annexin V expression by flow cytometry after 16 and 24 hours.
Left panel: the data represent the mean ±SD of Annexin V-FITC positive cells in
triplicate wells. Right panel: representative analysis by flow cytometry at 16 hours.

39

pro-survival effect on tumor cells that not only protected them from serum starvation-
induced apoptosis but also from drug-induced apoptosis.
BMMSC migrate toward tumor-derived products in vitro
I next asked the question whether BMMSC would be attracted by tumor cells. We
initially explored this possibility by testing the effect of tumor cell CM on BMMSC
migration in vitro in a transwell migration assay. This experiment indicated a robust
migration of BMMSC upon exposure to serum free 4T1 and LL/2 CM for 2 days (Fig.
5A). Further experiments utilizing serum free 4T1 and LL/2 CM also demonstrated
significant BMMSC recruitment after only 3 hours of exposure to tumor-derived
products. This effect seemed in part tumor specific since 3 hours of exposure to serum-
free NIH3T3 CM resulted in a migratory response that was only 28% of the response
observed in the presence of 4T1 CM. These experiments demonstrated that BMMSC
were preferentially recruited by tumor cells.
BMMSC are recruited at the invasive front of developing tumors
I then asked the question whether BMMSC would preferentially home to primary tumors
in vivo. For these experiments, 4T1 tumor-bearing mice were injected i.v. with PKH26-
labeled BMMSC on day 12 after tumor cell injection. An analysis by fluorescent
microscopy revealed the presence of isolated deposits of single, red fluorescent cells in
frozen sections of primary tumor and organs (lung, liver and kidney) from mice injected
with labeled BMMSC. Additionally, foci of PKH26-positive cells were found near the
margins of some tumor sections (Fig. 5B). To further examine the localization pattern of
tumor-recruited BMMSC in vivo, 4T1 tumor-bearing mice were injected i.v. with iron
nanoparticle-labeled BMMSC on day 4 of tumor growth. Histological evaluation of 4T1

40

tumor sections by Prussian blue staining revealed the presence of iron-containing cells
primarily at the invasive front of the tumors (Fig. 5C) that was distinguishable from
extracellular iron present in the tumor stroma at areas of hemorrhage and necrosis.

Figure II-5. BMMSC exhibit a chemotactic response to tumor cells in vitro and in vivo
A, BMMSC (8x10
4
per well) were cultured for 3 hours or 48 hours in a transwell assay in
the presence or absence of serum-free CM from 4T1, LL/2, and NIH3T3 cells. Left
panel: the data represent the mean ±SD number of migrated BMMSC from triplicate
filters from one of two separate experiments showing similar results. Right panel: light
micrographs of the bottom side of representative transwell filters from 48 hour assays.
Scale bar = 100 µm. B, fluorescence analysis of frozen sections (6 µm) of 4T1 tumors
and indicated organs harvested on day 14 post injection of tumor cells (6x10
5
injected
s.c. in the left flank) and day 2 post-injection of PKH26-labeled BMMSC (1.86x10
6
via
tail vein). Cy3/DAPI overlay for tumor, liver, lung, and kidney are shown. Scale bar =
50 µm. C, histological analysis of iron deposits by Prussian blue staining on sections
from FFPE 4T1 tumors (6 µm) harvested on day 10 of tumor growth (5x10
5
injected s.c.
in the left flank) and 6 days post-injection of iron-labeled BMMSC (3x10
5
via tail vein).
Scale bar = 250 µm (left) and 30 µm (right). T = tumor; N = normal tissue.

41

BMMSC suppress drug-induced apoptosis in vivo
I finally asked whether exogenous BMMSC would protect tumors from chemotherapy-
induced apoptosis in vivo. To examine this possibility, mice injected with 4T1 cells in
the presence or absence of BMMSC (ratio tumor cell:MSC 5:1) were treated with
doxorubicin on days 3 and 4 and tumors were analyzed on day 5 for the presence of
apoptotic cells (Fig. 6). This analysis revealed a significantly reduced number of
TUNEL-positive cells in sections of 4T1 tumors co-injected with BMMSC, when
compared to 4T1 tumors injected alone (p = 0.003). The data thus indicate that, in vivo,
BMMSC also protected tumor cells from drug-induced apoptosis.

Figure II-6. BMMSC suppress apoptosis in drug-treated tumors in vivo
4T1 cells (1x10
6
) were injected in the mammary fat pad of Balb/cJ mice in the absence or
presence of 2x10
5
BMMSC. Three days post-injection of tumor cells, mice were treated
with doxorubicin for two consecutive days, and tumors were harvested on day 5 and
examined for the presence of apoptotic cells. A, fluorescent micrographs of
representative tumor sections from control and tumors co-injected with BMMSC, stained
for the presence of apoptotic nuclei. Top: TUNEL and bottom: overlay with DAPI.
Arrowheads indicate the presence of apoptotic nuclei. Scale bar = 50 µm. B, the data
represent the mean ±SD of TUNEL-positive cells counted in a total of 10 32x fields in
each of 5 tumors.

42

Discussion
My studies identify a novel role for recruited BMMSC in primary tumors, namely a
protective effect on drug-induced apoptosis by soluble BMMSC-produced factors, which
is separate from their protective function in the bone marrow. BMMSC are reported to
promote tumorigenesis through production of a number of soluble factors, many of which
operate synergistically. However, since these pro-tumorigenic pathways largely function
through growth stimulation or immune modulation, our examination of apoptosis in the
BMMSC-tumor crosstalk brings a new aspect to the interaction between tumor cells and
MSC.
Our primary murine bone marrow stromal cells exhibit self renewal, adherence to
tissue culture plastic, and lineage differentiation, similar to the multipotent mesenchymal
cells originally identified by Friedenstein in bone marrow explants (11). However, it is
uncertain whether our cells represent a true population of mesenchymal stem cells,
rigorously defined by Caplan as the progenitor of all connective tissue lineages (3), or if
they are part of a subpopulation of multipotent cells alternately called stromal stem cells,
multipotent stromal cells, mesenchymal stromal cells, or multipotent adult progenitor
cells (9). My examination of adipocytic and osteogenic differentiation was sufficient to
confirm the mesenchymal origin of our primary cells without conducting a full
characterization of their ability to generate other lineages (chondrocyte, muscle, etc.).
Additionally, the surface marker profile for these primary cells, which were positive for
CD44 and negative for CD34, CD45, CD31, and VEGFR2, confirmed them to be of
mesenchymal origin, as opposed to hematopoietic or endothelial origin, without a full
characterization of their mesenchymal marker expression (23). As mentioned previously,

43

the low level of CD34 found in early passage BMMSC may indicate the presence of non-
adherent myeloid cells or adherent fibrocytes, which express hematopoietic markers but
exhibit a mesenchymal-like phenotype and contractile properties (156). Weak CD34
expression may also represent a transient production of this marker by BMMSC (21).
Given the phenotypic and functional characteristics of our primary cells, they are
appropriately referred to as mesenchymal stromal cells (13).
My current study adds to the body of literature on the effect of BMMSC on tumor
cell growth by specifically pointing to an effect on survival rather than on proliferation.
The growth enhancement effects of BMMSC reported typically involve a stimulation of
tumor cell proliferation, as demonstrated for example in human and rat osteosarcoma (70,
153). Human colorectal carcinoma xenografts also show increased proliferation in the
presence of BMMSC in multiple studies (112,157). Additional growth stimulatory
effects are reported in mammospheres of breast carcinoma cells grown in the presence of
BMMSC (117), and in mammary carcinoma cells exposed to CCL5 produced by
BMMSC (113). However, MSC have also been shown to have a growth-suppressive
effect, reducing the proliferation of Kaposi’s sarcoma cells in vitro and in vivo (110) and
limiting cell cycle progression in human hepatoma and ovarian carcinoma cells, glioma
and neuroblastoma (158-160). These conflicting data on the effect of BMMSC on tumor
cell proliferation may be due to differences in the types of MSC studies and also the
cancer cells. While proliferation promoting activities have been primarily reported in
cancer of epithelial origin, proliferation inhibitory activities have been shown in neural
tumors. Polarization of BMMSC into an anti or pro-tumorigenic function, similar to the
one observed in T cells and macrophages, may also explain the discrepancy in BMMSC

44

functions (161).
The novelty of the data presented in this thesis relates to the observation that the
positive effect of BMMSC on tumor cell viability was not due to an effect on
proliferation but rather on survival, an effect that has not been much explored so far.
Several published studies have in fact reported a pro-apoptotic effect of BMMSC on
tumor cells. For example, intravenous injection of MSC has been shown to increase
PARP-1 and caspase-3 cleavage in mammary carcinoma xenografts (122), and murine
hepatoma and lymphoma cells were reported to increase the production of caspase-3 and
p21 proteins when exposed to BMMSC in vitro and in vivo (123). Additionally, direct
injection of BMMSC in rat gliomas increased tumor cell expression of pro-apoptotic
caspase-3 and Bax while downregulating anti-apoptotic Bcl-2 (162). The disparity
between these findings and our own observation of BMMSC-mediated suppression of
tumor cell apoptosis may stem from our use of serum depletion and drug-induced cell
death models. It should be pointed out that these previous studies have examined
apoptosis of tumor cells exposed to MSC under unstressed conditions, suggesting that
MSC can increase baseline apoptotic signaling in tumors. An important difference in our
studies is that we have examined the effect of BMMSC under stress conditions such as
low serum and drug treatment. My data clearly demonstrate that under these conditions
BMMSC promote rather than inhibit survival, a role that is highly relevant to clinical
chemotherapeutic interventions.
The use of co-injection of BMMSC with 4T1 tumor cells in the mammary fat pad
represents a helpful model to examine the interaction between BMMSC and tumor cells
in vivo, although it has the limitation that BMMSC were not recruited from the bone

45

marrow by primary tumors. However similar co-injection models have been used to
examine other aspects of BMMSC-tumor interaction in vivo. Karnoub et al. found that
co-injection of human mammary carcinoma xenografts with BMMSC increased the
metastatic spread of tumor cells (147), an effect linked the production of CCL5 by
BMMSC, while Suzuki et al. reported in Lewis lung carcinoma tumors co-injected with
BMMSC an enhancement of tumor growth, driven by increased angiogenesis 163).
These co-injected tumors, artificially enriched with BMMSC, developed stromal
elements with functional properties similar to TAF, which are known descendants of
BMMSC (63,64,73,164). My current study leaves open the question of whether BMMSC
and BMMSC-derived TAF would play a similar protective role on apoptosis as they are
recruited from the bone marrow.
The observed protective effect of BMMSC on primary tumor cell survival was
contact-independent, as has been found in previous studies of metastatic tumor cells in
the bone marrow. Production of GDF15 by bone-marrow resident BMMSC protects
multiple myeloma cells from melphalan, bortezomib and lenalidomide (165), and several
laboratories including ours have identified a chemoprotective effect of BMMSC on tumor
cells driven by Il-6, which increases in a STAT3-dependent manner, the expression of
anti-apoptotic Bcl2, BclXl, survivin and XIAP, as well as multidrug resistance proteins
MDR and MRP (87,166-168). The identity of the protective factor(s) involved in our
observed suppression of tumor cell apoptosis is currently investigated by our laboratory
and preliminary data points to soluble proteins. It should be noted that other signaling
molecules besides proteins could also be involved in this protective effect, as fatty acids
released by BMMSC in response to platinum-based therapies have been shown to protect

46

murine colon carcinoma and Lewis lung carcinoma tumors from platinum-induced
cytotoxicity (169).
Finally, my data indicate that our primary BMMSC displayed a migratory response
to tumor cells in vitro and in vivo. However, the mechanism behind this chemoattraction
is still being investigated, with preliminary studies eliminating SDF1/CXCR4 and CD44
as the molecules involved (data not shown). MCP-1 is another potential recruitment
factor for BMMSC, as this chemokine has been shown to stimulate BMMSC migration
into murine mammary tumors in vivo (69). Alternatively multiple factors may be
involved since BMMSC have been shown to exhibit migration in response to a large
variety of growth factors and cytokines (66, 67).
In summary, in this thesis I provide data identifying BMMSC as an important
mediator of tumor cell survival and drug resistance, and evidence that BMMSC could
exert this function not only in the bone marrow as previously assumed but also in the
primary tumor.

47

Supplemental Figure II-1. BMMSC do not enhance cell cycle progression in tumor
cells
A, 4T1 cells were cultured in 2D for 2 or 4 days in the presence or absence of CM from
BMMSC and stained with propidium iodide. Top, the data represent the mean +/- SD of
cells in each cell cycle phase in triplicate wells from one experiment. Bottom,
representative analysis by flow cytometry. B, 4T1 cells were cultured in 2D for 8hr or 2-
4 days in the presence or absence of CM from BMMSC, pulsed with BrdU, and stained
with propidium iodide and anti-BrdU(FITC). Top, the data represent the mean +/- SD of
BrdU+ cells in triplicate wells from one experiment. Bottom, representative analysis by
flow cytometry.

48

Supplemental Figure II-2. BMMSC suppress baseline apoptosis in LL/2 tumor cells
A, LL/2 cells were cultured in 2D for 3-5 days in the presence or absence of CM from
BMMSC. Top, the data represent the mean +/- SD of AnnexinV-FITC positive cells in
triplicate wells from one experiment. Bottom, representative analysis by flow cytometry.
B, LL/2 cells were cultured for 5 days in the presence or absence of CM from BMMSC.
The data represent the mean +/- SD of caspase-3 activity in triplicate wells from one
experiment.

49

Chapter 3 – Tracking MSC Recruitment by Tumors in vivo
Background
Non-invasive imaging of cell biodistribution is increasingly vital in cell
transplantation studies, allowing detailed analysis of cell migration kinetics over time. A
handful of imaging modalities that are already in clinical use are suitable for this task,
including magnetic resonance imaging (MRI), positron emission tomology (PET), and
optical imaging (170). Of these, it is MRI that has shown the greatest potential for
resolving soft-tissue architecture in 3 dimensions and tracking recruitment dynamics of
labeled cells (171,172).
In vivo, MRI has already proven useful in the study of inflammatory disorders,
including tumors, and stem cell transplantation therapy. Commercially available iron
nanoparticles persist inside labeled cells for weeks, and do not affect function or viability
of the recipient cells (173). Used as an MR contrast agent, superparamagnetic iron oxide
(SPIO) particles, like ferumoxide, strongly decrease T2 and T2* relaxation times, which
is seen as a negative contrast or shadow on weighted images. This property has led to
development of numerous methods to increase cellular uptake of these particles. Iron-
labeled cells have been tracked by in vivo MR in pre-clinical models of tissue repair
(174), and clinical studies have also recently been undertaken (175). Large phagocytic
cells, including macrophages and human endothelial cells, have been tracked over time at
the single cell level (176,177). Additionally, Andersen’s group showed that iron-labeled
stem cells could be directly imaged in the neovasculature of a murine glioma. In vivo
migration and incorporation of the labeled cells into tumor vasculature was visualized by
negative-contrast MRI (178). Another group used MR to track the migration and

50

engraftment of ferumoxide labeled neural stem cells into damaged brain tissue following
intracranial injection into patients with traumatic brain injury (179).
Mesenchymal stem cells, a progenitor cell population with demonstrable ability to
home and engraft at sites of injury, have been utilized in iron-based MRI cell trafficking
studies. MSC labeled with ferumoxides can migrate to heart (180), liver (181), and
tumor tissue (110), findings that were validated by histologic evaluation. Further studies
of tumor recruitment showed chemoattraction and incorporation of iron labeled MSC in
glioma vasculature, as mentioned previously (178). However, reliance on exogenously
administered BMMSC for tumor recruitment studies has led to a dearth of information on
the migratory behavior of native, bone-marrow resident BMMSC in response to tumor
signals.
MR detection of iron labels has shown promise in a number of pre-clinical models
of BMMSC-mediated tissue repair. The presence of iron in an injured mouse spinal cord
could be detected by MR following direct implantation of iron-labeled MSC, although
signals produced by direct implantation of free iron nanoparticles or dead iron-labeled
MSC could not be distinguished from those produced by live MSC (182). Migration of
iron-labeled human BMMSC into ischemic rabbit brain tissue could also be tracked by
MR following implantation of labeled cells into the contralateral hemisphere (183). Iron-
labeled BMMSC injected into infarct sites in rat myocardium could be detected for up to
4 months by MR. Additionally, the volume of signal voids produced by the engrafted
cells shrank over time in mildly damaged myocardium while remaining constant in
severely damaged myocardium, due to prolonged repair activity of iron-labeled BMMSC
(184). This was confirmed in a separate study of spinal cord injury, where areas of

51

negative contrast density produced by implantation of iron-labeled MSC also shrank over
time (185). Iron-labeled MSC implanted in the subventricular zone of rats did not show
migration into neurogenic cell niches, in contrast to iron-labeled neural stem cells (186).
However, a mechanical lesion to the olfactory bulb did stimulate migration of labeled
MSC out of the subventricular zone, highlighting the dedicated role of MSC in tissue
repair.
In vivo studies of inflammatory disease can also benefit from iron-labeled cell
tracking. Progression of a skin infection in mice exposed to S. aureus was visualized
using iron oxide particles bound to the bacterial cell wall (187). Recruitment of
inflammatory cells into the tumor microenvironment has also been visualized by MR. In
a subcutaneous prostate tumor model, accumulation of iron-labeled NK cells at the tumor
site was visualized by MRI after s.c. injection but not after i.v. and i.p. injection, although
the presence of tumor localized, labeled NK was confirmed by histology for all three
injection routes (188). Furthermore, iron-positive signals could be detected in
subcutaneous breast carcinomas co-injected with iron-labeled BMMSC for up to 1 month
following implantation, and trafficking of intravenously injected iron-positive BMMSC
into lung metastasis of breast carcinomas was also demonstrated by MR (189).
Uptake of iron by endogenous cells at injury sites may complicate tracking of
iron-labeled cell engraftment and survival. In a model of limb ischemia, MR signals
produced by intramuscular injection of iron labeled MSC were sustained at the transplant
site for a greater length of time in immunocompetent mice compared to nude mice, owing
to uptake of iron particles by inflammatory macrophages (190), and systemically
administered iron nanoparticles persisted at sites of crush-induced nerve injury following

52

uptake by inflammatory macrophages (191). Iron nanoparticle accumulation at
inflammatory sites following systemic administration was also observed in a murine
model of experimental autoimmune encephalitis (192). These studies highlight the
ongoing difficulties in utilization of iron-labels for in vivo imaging which are only now
being addressed.
New advancements in detection of iron labeled cells by MR have mostly stemmed
from modifications of contrast agents, allowing enhanced cellular uptake and retention
(193,194). One such compound, liposome-bound gadolinium, was found to be a more
sensitive marker of transplanted cell viability than iron nanoparticles, as iron-labeled cells
produce a persistent magnetic signal void even after cell death (195). Negative-contrast
density can be used to identify iron-labeled cells in MR scans, but the feasibility of these
studies is still limited by a number of issues. Distinguishing signal voids of labeled cells
from nonspecific background tissue signals can be quite difficult, and the volume of
regions of negative density may not be fully representative of the area of labeled cell
infiltration. Furthermore, alterations in magnetic field density due to tissue artifacts, like
those found at air or liquid interfaces, can also complicate labeled cell detection. In a
number of studies, including my own, negative contrast produced by SPIO particles can
be difficult to distinguish from non-specific losses of signal intensity in regions of
irregular blood flow, vascular leakage, or air pockets (196,197).
My experiments will address the lack of detailed kinetic analysis of BMMSC-
tumor recruitment in the published literature and establish a time course for optimal
integration of these circulating cells into tumors. These findings will be of great benefit to
those seeking to develop BMMSC as vectors for targeted delivery of anti-cancer

53

compounds. Additionally, use of novel MR-based imaging will allow more sensitive
detection of isolated populations of labeled BMMSC than would be possible with other
live imaging modalities, such as bioluminescence. This technique is thus ideally suited
for further studies tracking the migration of low numbers of bone marrow engrafted
BMMSC from their endogenous environment into solid tumors, whereas previous studies
have only demonstrated recruitment of heterogeneous populations of engrafted bone
marrow cells using ex vivo examination of fluorescent cell deposits in tumor sections.
Hypothesis
I hypothesize that native BMMSC migrate into the peripheral circulation and are
recruited into primary tumors, and that tumor engraftment of both endogenous and
exogenous MSC can be visualized by MR detection of iron nanoparticle labels.
Specific Aims
I propose the following two aims for the present study:
Aim 1: Recruitment of IV-injected BMMSC. We will examine the real-time
kinetics of BMMSC recruitment into subcutaneous tumors by magnetic resonance-based
detection of intracellular iron-nanoparticle labels. Tumor localization of labeled BMMSC
will be confirmed by Prussian blue staining of iron deposits, as well as by in vivo
fluorometric detection of a transgenic RFP label.
Aim 2: Recruitment of bone-marrow engrafted BMMSC. We will examine
tumor-homing of BMMSC injected into the bone marrow of mice bearing a subcutaneous
lesion. In vivo BMMSC migration will be tracked by MR and fluorometry, and
BMMSC-derived stromal elements identified by fluorescent and histochemical markers
ex vivo.

54

Preliminary Data
Iron-labeled BMMSC are detectable by MRI and retain protective functions
To confirm specificity of iron nanoparticle detection, agarose suspensions of
labeled and unlabeled cells were scanned by T2* MRI. Single echo images revealed
strong negative contrast density in tubes containing iron-labeled BMMSC compared to
unlabeled BMMSC, despite a 50% increase in the number of cells suspended in the
unlabeled tube (6x105 unlabeled vs. 4x105 labeled) (Figure III-1A). Using a two-fold
dilution series of agarose suspensions of iron-labeled BMMSC, we observed a dose

Figure III-1. Iron-particles increase BMMSC detection and preserve protective effects
A, Iron-loaded (4x10
5
) and control BMMSC (6x10
5
) were suspended in 200uL agarose
and scanned by MR. B, Two-fold dilutions of iron-loaded BMMSC (0.5x10
5
-4x10
5
)
were suspended in 200uL agarose, along with a blank tube, and scanned by MR. C, 4T1
cells were cultured in 2D for 4 days in the presence or absence of CM from iron-loaded
BMMSC. The data represent the mean +/- SD of AnnexinV-FITC positive cells in
triplicate wells from one experiment.

55

dependent increase in negative contrast density in tubes with increasing numbers of cells
(0.5-4 cells per mL) (Figure III-1B). I then explored whether iron-laneling would disrupt
the protective effect of BMMSC on tumor cell survival. 4T1 cells grown in 1% serum
CM from iron-labeled BMMSC or control medium for 3-5 days were stained with
Annexin V-FITC and PI and examined by flow cytometry (Figure III-1C). We observed
significant reductions in the percentage of Annexin V(+) cells in cultures exposed to iron-
positive BMMSC CM for 4 days (21.3% CM vs. 31.5% control; p = 0.032). These data
confirmed that BMMSC-bound iron nanoparticles do not affect BMMSC functions and
are detectable by MRI.
Signal density and T2* relaxation times correlate with iron-labeled cell numbers
I then asked whether increased numbers of iron-labeled cells would show greater
perturbations in the magnetic spectra of T2* weighted images. For these experiments, we
suspended 3.6x105 and 7.2x105 iron-labeled cells in 50uL agarose-PBS sequentially in
the same tube, with a 50uL suspension of 3.6x105 unlabeled cells separating the labeled
cell layers. T2* imaging revealed an increase in negative contrast density across the cell
layers from lowest to highest cell number (Figure III-2A). T2* relaxation time for the
least concentrated suspension of labeled cells was much lower than that for the unlabeled
suspension, as expected, and T2* relaxation time for the most concentrated labeled cell
layer was lower than that of the least concentrated (Figure III-2B). Additionally, signal
density for both labeled layers was lower than that of the unlabeled layer at each echo
time (Figure III-2C). The data indicate that the presence of iron-labeled cells can be
detected by negative perturbations in T2* relaxation time, as well as dose-dependent
differences in signal density.

56

Iron-labeled MSC recruited to tumor margins produce diffuse areas of negative signal
density
Preliminary bioluminescence imaging of subcutaneous 4T1 tumors of mice
injected i.v. with luciferase-expressing MSC showed significant uptake of luciferase-
positive cells by the lung one day after i.v. injection. Lung-localized signals vanished
after 4 days but re-emerged 1 week later, accompanied by the appearance of a strong
luciferase-positive signal from the region of the tumor. Both of these secondary signals

Figure III-2. BMMSC iron-labeling decreases MR signal density and T2* relaxation
time
A, Iron-loaded (3.6x10
5
,7.2x10
5
) and control BMMSC (3.6x10
5
) were suspended in 50uL
agarose, layered, and scanned by MR. B, T2 relaxation times derived from MR scans of
layered suspensions from A. C, Signal intensity from MR scans of layered suspensions
from A, with data from three successive echoes shown.

57

diminished after 1 week and were gone by the following week (Figure III-3A). Taken
together, these data indicate localized proliferation of luciferase positive cells lodged in
tumor and lung tissue. In a follow-up experiment, MR imaging of subcutaneous 4T1
tumors of mice injected i.v. with iron-loaded MSC revealed diffuse, discontinuous areas

Figure III-3. Iron-loaded BMMSC produce diffuse areas of negative signal density in
tumors A, Bioluminescence analysis of 4T1-bearing mice beginning 13 days after tumor
induction (2x10
5
injected s.c. in the left flank) and 1 day after intravenous injection of
luciferase-positive BMMSC (2x10
6
via tail vein). B, MR analysis of 4T1-bearing mice
on day 10 of tumor growth (5x10
5
injected s.c. in the left flank) and 6 days post-injection
of iron-labeled BMMSC (3x10
5
via tail vein). C, Histological analysis of iron deposits by
Prussian blue staining on sections from FFPE 4T1 tumors (6 µm) harvested on day 10 of
tumor growth (5x10
5
injected s.c. in the left flank) and 6 days post-injection of iron-
labeled BMMSC (3x10
5
via tail vein). Scale bar = 250 µm (left) and 30 µm (right).

58

of magnetic signal density at the leading edge of the tumor just beneath the dermis,
possibly indicating deposition of iron-positive cells. This contrasts with MR scans of
4T1 tumors of mice injected with control MSC, which showed clear, continuous areas of
signal density in the same region, indicative of a nonspecific background signal produced
by fresh bleeding (Figure III-3B). It should be noted that background signal density
within these subcutaneous tumors was very heterogeneous, making iron-specific signals
extremely difficult to distinguish from nonspecific artifacts. Histological evaluation of
FFPE sections of 4T1 tumors by Prussian blue staining revealed high levels of
nonspecific iron deposition at the invasive front, which is characteristic of hemosiderin
leakage from lysed red blood cells in areas of fresh bleeding. In addition to this
background signal, sections of 4T1 tumors of mice receiving iron-loaded MSC displayed
strong iron-positive staining localized to the cell bodies of a number of individual stromal
cells at the leading edge (Figure III-3C). These cell-specific signals were clearly
distinguishable from nonspecific staining, which is more diffuse and localized between or
around cells. No cell-specific iron signals were found in the core of either iron-loaded or
control MSC-injected 4T1 tumors. The data indicate that circulating iron-labeled
BMMSC migrate to the leading edge of subcutaneous tumors but are difficult to
positively identify by live MR imaging.
RFP-positive MSC are detectable by in vivo fluorometry
In addition to magnetic resonance imaging, I tested whether fluorometric
detection could be used to identify labeled cells in vivo. Red fluorescence was identified
in cell suspensions of MSC expressing RFP under the control of the B-actin promoter,
while nude mice showed no detectable red fluorescence under the tested parameters

59

(Figure III-4A). When injected subcutaneously into the flank of nude mice, fluorescent
signals produced by the RFP-positive MSC could be detected by fluorometric scanning
(Figure III-4B). These data indicate that in vivo fluorometric detection of labeled cells
can be used to validate recruitment events identified by other imaging modalities like
MR.
Experimental Approach: Aim 1
Rationale
In vivo studies of tumor-directed migration have so far been unable to establish a dose-
dependent engraftment curve for IV- injected BMMSC, indicating a highly
inefficient process. Our own preliminary studies have demonstrated engraftment of i.v.-

Figure III-4. DsRed-expressing BMMSC are detectable by in vivo fluorometry
A, Fluorescence analysis of 2 suspensions of dsRed-positive BMMSC (1x10
6
cells/mL) in
1 and 0.1 mL PBS, along with an unmodified nude mouse. B, Fluorescence analysis of a
nude mouse subcutaneously injected with 1x10
5
DsRed-positive BMMSC in 100uL PBS.

60

injected BMMSC into subcutaneous 4T1 tumors by use of a fluorescent label.
Unfortunately, these experiments yielded only a few tumor sections with verifiable
BMMSC deposits. Further attempts to track luciferase-expressing BMMSC in vivo by
bioluminescence were similarly unsuccessful, as labeled cells were only identifiable after
undergoing significant proliferation due to a high threshold of detection. However, in
vivo experiments using magnetic resonance imaging to track BMMSC labeled with iron
nanoparticles showed dramatic levels of tumor homing in mice that received intravenous
BMMSC at an early stage of subcutaneous tumor growth. Interestingly, the iron-labeled
BMMSC were found to localize to the invasive front of the tumor rather than its core.
These studies represented our first attempt to visualize cell trafficking in vivo by MR,
allowing us to identify minute populations of tumor engrafted BMMSC without
sacrificing the test animals. The sensitivity of detection of iron-labeled cells by MR
makes it an ideal modality for live tracking of small, enriched populations of BMMSC
that would be undetectable by bioluminescence, owing to the significant attenuation of
luciferase reporter signal as it passes through live tissue. We will validate our in vivo
BMMSC migration model by light and fluorescence microscopy and, additionally, refine
our MR analysis of iron-labeled BMMSC engraftment to achieve a greater understanding
of their recruitment kinetics and distribution.
Design
Intravenous recruitment model: 4T1 tumor cells will be subcutaneously
injected above the left scapula of syngeneic balb/c mice and allowed to grow for
approximately two days. Labeled BMMSC will then be introduced by intravenous tail
vein injection.

61

In vivo imaging: BMMSC will be prepared for live imaging by labeling with iron
nanoparticles, which does not affect viability or tumor-protective biological functions.
Migration of labeled BMMSC into primary tumors will be tracked by MR imaging at 1,
2, 3, and 6 days post-BMMSC injection and compared to label-free controls. Magnetic
density data will be used to create a 3 dimensional reconstruction of the tumor site to
examine localization of iron-labeled cells. Additionally, iron-labeling of dsRed-
expressing BMMSC will allow validation of MR signal data with live fluorometric scans
taken at 1, 2, 3, and 6 days post-BMMSC injection.
Ex vivo microscopic evaluation: Subcutaneous 4T1 tumors will be extracted and
FFPE sections collected 6 days post-injection of labeled BMMSC. Sections will be
stained with Prussian blue to identify intracellular iron deposits, and
immunohistochemistry performed for markers of CAF (a-smooth muscle actin, fibroblast
activation protein). Additionally, frozen tumor sections will be collected for detection of
intracellular red fluorescent protein transgenically expressed by BMMSC.
Experimental Approach: Aim 2
Rationale
Identification of native BMMSC in the stroma of primary tumors has so far been
impossible, as there is no established single marker for this cell population. Studies
performed outside our lab have identified bone marrow-derived cells in the tumors of
mice transplanted with heterogeneous populations of fluorescently labeled bone marrow
stromal cells. Though compelling, these studies do not provide reliable evidence of native
BMMSC recruitment into tumors since the labeled cells did not originate from a pure

62

population of BMMSC. We will test the in vivo tumor homing activity of labeled
BMMSC transplanted into the marrow of tumor-bearing mice by MR and microscopy.
Design
Orthotopic recruitment model: 4T1 tumor cells will be subcutaneously injected
into the left flank of syngeneic balb/c mice and allowed to grow for approximately two
days. Labeled BMMSC will be then be injected into the right femur.
In vivo imaging: BMMSC will be prepared for live imaging by labeling with iron
nanoparticles. Migration of labeled BMMSC into primary tumors will be tracked by MR
imaging at 1, 2, 3, 6, 14, and 21 days post-BMMSC injection and compared to label-free
controls. Magnetic density data will be used to create a 3 dimensional reconstruction of
the tumor site to examine localization of iron-labeled cells. Additionally, iron-labeling of
dsRed-expressing BMMSC will allow validation of MR signal data with live
fluorometric scans taken at 1, 2, 3, 6, 10, 14, 17, and 21 days post-BMMSC injection.
Ex vivo microscopic evaluation: Subcutaneous 4T1 tumors will be extracted and
FFPE sections collected at 21 days post-BMMSC implantation. Sections will be stained
with Prussian blue to identify intracellular iron deposits, and immunohistochemistry
performed for markers of CAF (a-smooth muscle actin, fibroblast activation protein).
Additionally, frozen tumor sections will be collected for detection of intracellular red
fluorescent protein transgenically expressed by BMMSC.
Limitations
Use of iron nanoparticles may produce non-specific phantom signals during in vivo MRI
analysis should iron-labeled BMMSC die and release their contents into the surrounding
tissue. By comparing dsRed-expressing cells with iron-marked cells, we will be able to

63

determine their viability. Additionally, it is possible that dsRed-expressing BMMSC will
not produce an appreciable level of in vivo fluorescence until they have expanded locally,
making sensitive detection of early-stage BMMSC migration difficult. By combining
these two imaging modalities in our study we hope to overcome both the problem of
nanoparticle specificity, using expression of the dsRed vector to confirm cell-specific
signaling, and the problem of fluorescent sensitivity, using the robust MRI signal of iron
to increase detection of small numbers of cells. As mentioned previously, the results of
our study may not apply to the human system, due to our use of murine cells. While this
weakness could be overcome by using human tumor cells and BMMSC in an
immunocompromised mouse, we feel that our experimental model requires an intact
immune system to properly characterize the role of BMMSC in tumor inflammation.
Significance
We will utilize novel MRI-based in vivo imaging to track the fate of implanted BMMSC
and their recruitment by implanted tumors. The sensitivity of this labeling method will
allow use of small, enriched populations of BMMSC for bone-marrow engraftment,
which will provide more reliable evidence of tumor-targeted BMMSC homing than
previous transplantation studies employing heterogeneous populations of bone-marrow
derived cells. This work is part of a collaborative effort between our lab and the Moats
lab that aims to develop new imaging technologies at CHLA to facilitate investigations
into the cellular and developmental biology of pediatric disease. Such advances beyond
the current limitations of bioimaging, which permit only low-resolution/low-
magnification views, will be necessary to explore cellular dynamics in a live system at
the single cell and even gene transcriptional level.

64

Chapter 4 – Role of MSC in Cancer Therapy
The unique characteristics of MSC have led to a number of therapeutic applications. Co-
implantation of these cells in a murine HSC transplantation model can successfully
reduce GVHD, although clinical studies have not been able to recapitulate this effect, as
mentioned previously (142-145). MSC also show beneficial effects in pre-clinical
models of joint damage and ischemic heart injury, enhancing the regeneration of cartilage
and cardiac muscle tissue. It should be noted that implanted MSC did not generate
identifiable chondrocytes or functioning cardiomyocytes in these studies, indicating that
the exogenous cells enhanced the repair activity of local cell populations by soluble or
contact-dependent signals instead of directly contributing to the pool of differentiated,
tissue-specific cells. Finally, the homing of MSC to sites of injury makes these cells
ideal candidates for use as targeted delivery vectors for anti-cancer therapy, as will be
discussed in this chapter.
Targeting MSC in Cancer Therapy
The question whether MSC can themselves serve as an anti-cancer treatment target
remains presently unanswered. Animal experiments indicate, however, that MSC can be
targeted in vivo with drugs like imatinib (Gleevec) that blocks PDGFR-mediated
signaling. Mice xenografted with KM12 cells and MSC developed rapidly growing
tumors. Treatment of these tumor-bearing mice with imatinib increased survival
significantly. Moreover, the ability of MSC to migrate to tumor stroma was impaired by
imatinib and the number of MSC surviving in the tumor microenvironment was
significantly decreased (198). It is presently unknown if chemotherapeutic damage to
tumor-recruited MSC will have a beneficial effect on tumor growth, although it has been

65

suggested that use of DNA-damaging agents in anti-cancer therapy could lead to
malignant transformation of MSC and the development of mesenchymal tumors (199).
Blocking TGF-β may be another approach to inhibit MSC. TGF-β signaling is essential
for differentiation of human BM-MSC to TAF in the TME and their protumorigenic
effects. Thus, blocking the TGF-β/Smad pathway may have an anti-MSC effect in
addition that contributes to its overall immunostimulatory effect. Small molecule
inhibitors of TGF-β are currently in clinical trials (200,201) . Zoledronic acid (ZA), a
nitrogen-containing bisphosphonate approved by the FDA for patients with bone
metastasis, significantly reduces activation of AKT and ERK in MSC, along with their
production of cytokines like IL-6 and RANTES and their ability to migrate, suggesting
that the anti-tumor effect of ZA may include a direct effect on MSC (202). Cox-2
inhibitors have also been shown to inhibit osteogenesis in MSC, suggesting that under
inflammatory conditions they may inhibit the formation of the osteoblastic niche.
Accordingly, the osteogenic potential of MSC is inhibited and delayed by treatment with
high-dose non-steroid anti-inflammatory drugs (203,204).
MSC as Therapeutic Vectors
The colonization of tumors by MSC can be exploited for the targeted delivery of a
number of therapeutic compounds, including oncolytic viruses, biological molecules and
pro-drugs, and packaging compounds.
MSC in gene therapy
Genetically modified MSC have demonstrated antitumoral activity in a number of
preclinical studies. MSCs engineered to express the herpes simplex virus-thymidine
kinase are useful in targeted cancer suicide gene therapy, as they promote the conversion

66

of the prodrug ganciclovir to its active form specifically within the tumor
microenvironment (205-207). Similarly, retrovirus transduction of adipose-derived MSCs
with the enzyme cytosine deaminase also allowed targeted activation of the prodrug 5-
fluorocysteine, creating a strong anti-tumour effect in vivo (208). MSCs designed to
overexpress the Sodium-Iodide symporter (NIS) have been used in both in vivo cell
tracking and anticancer therapy, as these cells demonstrate enhanced uptake of
radioactive iodine-based compounds (209). In a murine model of hepatocellular
carcinoma (HCC), the recruitment of intravenously-administered, NIS-expressing MSCs
to HCC xenografts was successfully tracked by 123I-scintigraphy and 124I-PET imaging.
Administration of 131I to HCC tumor-bearing mice following injection of NIS-
expressing MSC was shown to delay HCC tumor growth (210).
A recent application of this technology is the genetic engineering of MSCs to
express proteins that enhance their ability to target the tumor microenvironment. In vitro
studies of MSC adhesion under shear flow conditions, used as a model of cell recruitment
from the circulation, demonstrated that artificial expression of sialyl Lewis X on MSCs
increased cell rolling on a P-selectin-coated surface. As P-selectin is upregulated on
endothelial cells found in tumors and inflammatory sites, this technique may have
potential applications in improving MSC engraftment in vivo (211). Other groups have
indicated that MSC can be modified to specifically home to factors produced at tumor
sites which do not normally recruit these cells. In an in vivo study utilizing GL261
gliomas and B16 melanoma, which do not induce native MSC migration, MSCs
engineered to overexpress the epidermal growth factor receptor displayed enhanced
migration towards both tumor types (212). Binding of transforming growth factor alpha

67

and epidermal growth factor, produced by GL261 and B16 tumors, was responsible for
induction of MSC recruitment.
MSC for Delivery of Biologicals
MSC may provide an important tool for delivery of biological compounds which
do not normally reach levels sufficient to drive anti-tumor effects in the tumor
microenvironment. Co-injection of MSC expressing IFNβ with glioma (213), lung (214),
or pancreatic tumor cells (215) has been shown to suppress tumor growth and increase
animal survival. Administration of IFNβ systemically or at a site distant from the tumor
showed no effect on growth, suggesting that localized production was necessary to
achieve therapeutic benefit. Additionally, MSC engineered to secrete IL-12 reduced the
growth of subcutaneous mammary carcinoma tumors when co-implanted with tumor cells
in matrigel plugs(216). This effect also required localized production of Il-12, as no
effect on growth was observed when MSC were implanted in a matrigel plug on the flank
opposite from the tumor. Similar to IFN-β, bone marrow-derived MSCs expressing
interferon (IFN)-α increased apoptosis in a melanoma lung metastasis model, slowing
profileration and reducing the tumor burden (217). Enhanced apoptosis, accompanied by
S-phase cell cycle arrest, was also observed in other tumor models, such as glioma (218),
melanoma (214), and prostate cancer (219), following IFN-α delivery by MSCs.
Additionally, IFN-α released from MSCs suppressed in vitro proliferation of leukemic
cells (220).
MSC can be also used to suppress endogenous growth-promoting pathways that
drive tumorigenesis. In a lung cancer model, intravenously-injected MSCs expressing
NK4, a hepatocyte growth factor antagonist, induced cancer cell apoptosis and

68

suppressed angiogenesis and lymphangiogenesis, leading to increased animal survival
(221). Lentivirus-transduced MSC expressing pigment epithelium-derived factor, which
stimulates terminal differentiation, reduced hepatocellular carcinoma tumor growth, lung
metastases, and angiogenesis following intravenous injection (222). Tumor infiltration
by M1 polarized inflammatory cells is another endogenous enhancer of tumor growth that
can be targeted by MSC. iNOS, a powerful immunosuppressant involved in M2
polarization, has also shown significant anti-tumor effects when delivered by genetically
modified MSC, reducing fibrosarcoma tumor growth in a mouse model (223).
Delivery of exogenous cytokines that enhance anti-tumoral immunity is another
application of engineered MSC that has shown pre-clinical efficacy. Intravenous
injection of adenovirus-transduced MSC expressing IL-2 or IL-12 stimulated
inflammatory cell infiltration of subcutaneous mammary carcinoma tumors, leading to
reduced tumor growth (224,225). This effect required the participation of a fully
functioning immune system, as no changes in tumor growth were observed in
immunodeficient animals upon injection of MSC (225). Levels of Il-12 were increased in
both the tumor and serum of MSC receipiant mice, and delivery of IL-12 did not result in
systemic toxicity. Injection of Il-12-expressing adenovirus increased serum IL-12 but not
intratumoral IL-12 in recipient mice, and also induced systemic toxicity (226). Thus,
local delivery of cytokines by modified MSC may be better tolerated by the host than
viral delivery, avoiding unacceptable systemic inflammation.
TRAIL is another extremely useful cytokine for therapeutic delivery. This protein
induces caspase-mediated apoptosis in cells that overexpress its receptor, such as tumor
cells, while healthy tissues, including MSC, are resistant to TRAIL-mediated effects due

69

to low expression of TRAIL receptors (227). Intravenously-administered MSCs secreting
TRAIL have been shown to reduce the growth of xenotransplanted lung carcinoma cells
(228). Similar anti-tumor effects were observed in human breast and cervical cancers in
nude mice (229), as well as in xenotransplanted gliomas (230,231). In a study utilizing
lentivirus-transduced MSC expressing TRAIL under the control of a tet promoter,
injection of doxycycline following administration of engineered MSC led to a complete
reduction in lung metastasis of subcutaneous murine breast carcinomas (232). Co-
injection of TRAIL-expressing MSC in subcutaneous mammary carcinomas was only
able to significantly reduce tumor weight when TRAIL-expression was induced by
doxycycline on the day of inoculation (day 0). Doxycycline treatment of established
tumors co-injected with engineered MSC (day 25) had no effect on tumor weight,
suggesting that early tumors are a more appropriate target for MSC-delivered cytokines.
MSC for Virus Delivery
MSC can also be used for delivery of tumor-targeted oncolytic viruses. These
replication-competent viruses lyse infected tumor cells as they reproduce, allowing
further infection and lysis of nearby cells. Targeting and delivery are crucial in this
process, as the virus is capable of killing both normal and transformed cells. Injection of
adenovirus-carrying MSC led to better survival in mice bearing syngeneic ovarian
carcinoma than did systemic injection of the adenovirus alone (233), with similar results
observed using xenotransplanted human glioma (234). Another study found that MSC-
delivered adenovirus effectively transduced and limited the growth of orthotopic human
breast and lung carcinomas, even without significant tumor infiltration by MSC (235).
Virus-delivery by MSC has also shown promise in a clinical setting. Four patients with

70

metastatic neuroblastoma resistant to front-line therapies received several doses of
autologous MSC carrying oncolytic adenovirus. Complete remission was observed in one
patient after therapy, with no sign of recurring disease after three years (236). Systemic
toxicity was not observed, making this an extremely safe therapeutic approach.
MSC for delivery of packaging compounds
Engineered packaging compounds loaded with therapeutic agents can also be
targeted to tumors by MSC. These intracellular packages, called nanoparticles (NP), are
composed of materials that promote uptake by cells, using either surface charge-mediated
endocytosis or lipophilic diffusion through the plasma membrane. Two types of NP,
poly-lactic acid (PLA-NP) and lipid nanocapsules (LNC) were loaded with coumarin-6 to
demonstrate their potential as delivery vehicles. These NP were efficiently internalized
into MSC and did not affect cell viability or differentiation (237). Furthermore, NP-
loaded MSC were able to migrate toward an experimental human glioma model. In a later
study utilizing LNC loaded with the anti-cancer drug ferrociphenol, MSC internalized the
nanoparticles without suffering cytotoxic effects due to their contents. LNC-loaded MSC
produced a cytotoxic effect on U87MG glioma cells in vitro, and intratumoral injection of
these MSC also reduced growth of xenotransplanted gliomas in nude mice (238). More
specialized packaging compounds can also be used to enhance tumor-specific targeting
by modified MSC. Cells loaded with magnetic nanoparticles can be directed to target
lesions in vivo by use of a magnetic field (239). Application of this technology has been
limited, however, by attenuation of magnetic field strength in deep tissues. Huang’s
group has designed a magnetic pole in which the magnetic field density can be focused at

71

a certain distance from the pole, allowing NP-loaded MSC to be enriched at the focal
point (240).

72

Chapter 5 – Conclusions
In this thesis, I have discussed emerging roles of MSC in promoting tumor growth and
survival in the bone marrow and primary tumors, as well as the controversy surrounding
their reported pro- and anti-tumorigenic effects. I have obtained our own experimental
evidence that the MSC-tumor interaction is pro-tumorigenic and operates through
suppression of the intrinsic apoptotic pathway. I have also discussed the recruitment
potential of MSC, along with recent attempts to modify these cells for delivery of
therapeutics. MSC are the subject of numerous investigations seeking to develop targeted
anti-cancer therapies, some of which show promise in pre-clinical studies. MSC
engineered to express inhibitory cytokines, extracellular ligands, or drug-activating
enzymes suppress the growth and chemotherapeutic resistance of xenotransplanted
tumors. With their robust tumor-homing capacity, MSC also provide a suitable vehicle
for delivery of oncolytic viruses and anti-cancer compounds packaged in microsomal
vesicles, such as chemotherapeutic agents and pro-drugs. However, the results of my
current study reinforce the discrepancy in pro- vs. anti-tumorigenic behavior of MSC that
is still frequently encountered in the in vivo literature, and suggests that our current
understanding of MSC function is too limited to justify their use in cell-based
therapeutics at the clinical level.
Future Directions
A number of unanswered questions regarding the role of MSC in primary tumors bear
further study: (1) What is the protective factor responsible for suppression of apoptosis
in tumor cells exposed to MSC? (2) What are the kinetics of MSC recruitment into solid

73

tumors following intravenous injection? (3) What are the kinetics of recruitment of
native MSC from the bone marrow into solid tumors?
1. Identification of protective MSC-produced factor
For my immediate studies, Western blotting will be used to further characterize
the apoptotic regulatory proteins (Bax, Bcl2, Bcl-xL) which are activated or suppressed
in tumor cells exposed to MSC. As these proteins are downstream of signaling molecules
which drive tumor-promoting effects of MSC in the bone marrow, such as IL-6, their
involvement in MSC-mediated protection within primary tumors seems likely.
For future studies, protein and mRNA-based screening will be utilized to identify
the MSC-produced pro-survival factor which mediates suppression of tumor cell
apoptosis. This technology is widely available in the form of commercial kits which can
be used to query production of a broad panel of cytokines and growth factors known to
play a role in tumorigenesis. Similar screening experiments, focused on chemokines and
cytokines, will also be used to reveal recruitment factors responsible for tumor-induced
migration of MSC.
2. Tracking exogenous MSC recruitment
My next step in identifying the kinetics of MSC engraftment and survival in target
tissues will be to repeat our MRI-based in vivo detection experiments using multi-echo
MR scanning, rather than single echo, to produce higher resolution images and generate a
time course for loss of iron-labeled cells from the implantation site. For these studies, I
will utilize intracranial injections into the brain, a target tissue which produces far less
MRI background signal than subcutaneous tumors. High resolution brain scans will be
used to create 3D reconstructions of ferromagnetic signal density within the live animal,

74

allowing me to accurately pinpoint populations of labeled cells and gauge their
persistence within the injection site.
Based on the success of our in vivo MSC recruitment model, which yielded robust
deposition of iron-labeled cells in the wound margin of developing tumors, future
attempts will be made to develop our MRI-based imaging of iron-labeled MSC.
Detectability of these cells can be enhanced by optimization of the iron-labeling protocol
to increase iron uptake. Cell labeling will be repeated using different concentrations of
heparin, protamine sulfate, and iron nanoparticles to generate maximal levels of complex
formation and MSC binding.
3. Tracking recruitment of endogenous MSC by tumors
In order to examine the feasibility of tracking iron-labeled MSC from the bone
marrow into tumors, I will utilize intrafemoral injections of labeled MSC into healthy
mice, followed by immediate (within 24 hours) imaging of the femoral region by MR. A
short time course will be necessary for these experiments as exogenous MSC do not
typically survive well in the marrow cavity post-injection, requiring partial ablation of
native bone marrow stromal cells to successfully engraft.
Future studies will attempt to identify native MSC in the stroma of primary
tumors. This task has so far been impossible, as there is no established single marker for
the MSC population. Studies performed outside our lab have identified bone marrow-
derived cells in the tumors of mice transplanted with heterogeneous populations of
fluorescently labeled bone marrow stromal cells. Though compelling, these studies do not
provide reliable evidence of native MSC recruitment into tumors since the labeled cells
did not originate from a pure population of MSC. Despite these uncertainties, we

75

hypothesize that native MSC migrate into the peripheral circulation and are recruited into
primary tumors. We will test subcutaneous tumors for recruitment of native, bone
marrow-resident MSC using an intrafemoral engraftment model. Sublethally irradiated
mice will receive intrafemoral injections of iron-labeled MSC. After the cells have been
allowed to engraft, subcutaneous tumors will be induced and intermittently scanned by
MR to track the deposition of labeled cells.
Summary
In summary, I have identified MSC as an important mediator of tumor cell
survival and drug resistance, and further studies of the mechanisms responsible for their
recruitment in primary tumors and their protective effect on transformed cells could be
easily translated into therapeutic interventions that disrupt the pro-tumorigenic influence
of stromal cells in the tumor microenvironment.

76

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Abstract (if available)

Abstract Bone marrow mesenchymal stem cells (BMMSC) are recruited to primary tumors and have been reported to display pro-tumorigenic as well as anti-tumorigenic activities. We hypothesize that circulating BMMSC are incorporated into tumor sites and protect tumor cells from therapy-induced apoptosis. Adherent stromal cells isolated from murine bone marrow that expressed phenotypic and functional characteristics of BMMSC were tested for their effect on 4T1 murine mammary adenocarcinoma and LL/2 Lewis lung carcinoma cells. Primary BMMSC stimulated the expansion of 4T1 cells in 3D co-cultures and conditioned medium from these cells increased the viability of 4T1 and LL/2 cells in 2D cultures. Analysis of apoptosis in 4T1 cells exposed to BMMSC conditioned medium under low serum concentrations (0.5 to 1%) revealed a 2-fold reduction in apoptosis as determined by Annexin V expression and caspase-3 and caspase-9 activity. Furthermore, exposure of 4T1 and LL/2 cells to BMMSC conditioned medium increased the viability of 4T1 and LL/2 cells when treated with paclitaxel or doxorubicin at therapeutic concentrations. This protective effect was accompanied by significant reductions in caspase-3 activity and Annexin V expression. We then demonstrated that BMMSC are attracted specifically by 4T1 and LL/2 cells in vitro, and in vivo migrate into the invasive front of 4T1 tumors implanted in mice. When co-injected with 4T1 cells in the mammary fat pad of mice subsequently treated with doxorubicin, they inhibit drug-induced apoptosis by 42 percent. Overall, our data identify BMMSC as an important mediator of tumor cell survival and treatment resistance in primary tumors.

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Asset Metadata

Creator Bergfeld, Scott Anthony (author)

Core Title Bone marrow derived mesenchymal stem cells promote survival and drug resistance in tumor cells

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Pathobiology

Publication Date 10/04/2013

Defense Date 06/18/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag apoptosis,drug resistance,mesenchymal stem cells,OAI-PMH Harvest,tumor microenvironment

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor DeClerck, Yves A. (committee chair), Adams, Gregor B. (committee member), Asgharzadeh, Shahab (committee member), Hofman, Florence M. (committee member)

Creator Email bergfeld@usc.edu,sbergfeld@chla.usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-335114

Unique identifier UC11295857

Identifier etd-BergfeldSc-2080.pdf (filename),usctheses-c3-335114 (legacy record id)

Legacy Identifier etd-BergfeldSc-2080.pdf

Dmrecord 335114

Document Type Dissertation

Format application/pdf (imt)

Rights Bergfeld, Scott Anthony

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA