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Contribution of cancer associated fibroblasts to cancer progression

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Contribution of cancer associated fibroblasts to cancer progression

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CONTRIBUTION OF CANCER ASSOCIATED FIBROBLASTS TO CANCER
PROGRESSION
By
RONGRONG LI

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

August 2022

Copyright 2022 RONGRONG LI
ii

Acknowledgments
There have been so many who helped me a lot with my master thesis, especially my advisor, Dr.
Yves DeClerck. His support, encouragement and guidance are always along with the processing
of my whole research. His principle and planning have benefited me for life and his enthusiasm
for scientific research deeply influenced me. So, I am incredibly grateful to Dr. DeClerck.
I also acknowledge all members in my laboratory, Dr. Laurence Blavier Sarte, Rie Nakata, Dr.
Kevin Louault and Dr. Ren Ming Yang. Without Laurence and Kevin’s useful suggestions and
supports, I could not finish my research successfully.
I am grateful to many researchers and staff at Children’s Hospital Los Angeles, especially
Esteban Fernandez and Fusheng Yang, for their help with the immunohistochemistry and the
quantification of the data.

iii

Table of Contents
Acknowledgments ………………………………………………………….……………………ii
List of Tables………………………………………………………….…………………………iv
List of figures ………………….…………………………………………………………………v
Abstract………………………………………………………….……………………………….vi
Chapter I. Introduction ……………………………………...…………………………….………1
Chapter II. Heterogeneous Presence of CAFs in Human Cancers ………………………………..4
Chapter III. Immunohistochemistry: Expression of p-SMAD2 in human
Xenotransplanted tumors ………………………………………………….…….…...9
1. Rationale for the experiment…….…………………………………………………9
2. Hypothesis…………….……….……………...……………………………………9
3. Experimental design…………………….……………...…………………………10
4. Materials and Methods…………………….……………...………………………10
5. Results…………………….……………...………………………………………16
Chapter IV. Discussion…………………….……………...……………………………………. 22
References ………………………………………………………………….……………………26

iv

List of Tables
Table 1. List of reagents and concentrations used in immunohistochemistry………………….13
Table 2. Clinical trials of TGF-beta targeting drugs……………………………………………25

v

List of figures
Figure 1. Heterogenous function and phenotype of cancer-associated fibroblasts……………….3
Figure 2. Heat map of the expression of 14 genes(column) among 33 types of cancer(row) …...7
Figure 3. Correlation analysis between the expression of different genes………………………..8
Figure 4. Experimental design…………………………………………………………………...12
Figure 5. Immunohistochemistry method……………………………………………………….13
Figure 6. Quantification of immunohistochemistry images with QuPath software……………..15
Figure 7. Bioluminescence of tumor overtime implanted in NSG mice over four weeks………18
Figure 8. IHC: p-SMAD2-CHLA255…………………………………………………………...19
Figure 9. IHC: p-SMAD2-CHLA136…………………………………………………………...20
Figure 10. IHC: p-SMAD2-SK-N-BE (2) ………………………………………………………21

vi

Abstract
Abstract: Cancer associated fibroblasts (CAFs) and their precursor cells mesenchymal stromal
cells (MSC) are one of the most abundant cell types that exist in the tumor microenvironment
(TME) and have an important role to cancer progress. The heterogeneous nature of CAFs and
their contribution to Transforming Growth Factor beta (TGF-beta) activity in neuroblastoma
(NB) is discussed in this thesis. We analyzed in silico the mRNA expression of several CAF
markers in different types of human cancers and provide data showing that CAF-mesenchymal
stromal cells (CAF-MSCs) and monocytes can increase TGF-beta activity and NB tumor growth.
Keywords: neuroblastoma (NB); tumor microenvironment (TME); cancer-associated fibroblasts
(CAFs); mesenchymal stromal cells (MSCs); immunohistochemistry (IHC); heterogeneity;
biomarkers.
Note: A portion of Chapter II (Heterogeneous Presence of CAFs in Human Cancers) has been
previously published with co-authors on Cancers in October 2020. [Louault, K., Li, R. R., &
DeClerck, Y. A. (2020). Cancer-associated fibroblasts: Understanding their heterogeneity.
Cancers, 12(11), 3108.]

1

Chapter I. Introduction
Over several decades, the concept of “seed and soil” proposed by Stephen Paget has been widely
accepted and the understanding of this concept has greatly improved
1
.The complex and
heterogeneous TME is the “soil” for tumor cells, composed by innate immune cells, adaptative
immune cells, stromal cells, vascular cells, and the extracellular matrix (ECM)
2
. Innate immune
cells include tumor-associated macrophages (TAMs) and natural killer cells
3
, adaptative immune
cells include regulatory T cells and B cells, stromal cells include MSCs and CAFs
4
, vascular
cells include micro-vascular endothelial cells (EC) and pericytes
5
, ECM includes a variety
collagen molecules, glycoproteins and proteoglycans
6
. The TME is also the subject of
environmental changes such as hypoxia, low pH, high pressure and is submitted to the effect of a
large number of growth factors and proteolytic enzymes produced by malignant and non-
malignant cells
7
. As a functional unit, the TME has a biological role throughout stimulating
tumor transformation, enhancing tumor growth and invasion, inducing immune tolerance
8
. Non-
malignant fibroblasts and monocytes in the TME are educated by tumor cells to become CAFs
and TAMs respectively
9
.
Fibroblasts are characterized by their contractility and transformation of the ECM. These cells
are activated and proliferated during inflammation, wound healing and fibrosis but also during
the malignant progression where they were initially reported to promote the growth of epithelial
tumors by L.W. Chung et al and were later named CAFs
10
. CAFs are one of the most abundant
cell types in the TME that support tumor cells and promote tumorigenesis by remodeling ECM
and by secreting cytokines and chemokines
11
. CAFs are fibroblasts in tumor tissue activated by
cancer cells and have the characteristics of myofibroblasts (myCAF). Multiple studies over the
last two decades have indicated that CAFs are not innocent bystanders in tumors and can have
2

either pro- or anti-tumorigenic functions
10
. The multiple pro-tumorigenic functions of CAFs
include stimulation of proliferation, chemoresistance, migration, invasion and metastasis,
angiogenesis, and immunomodulation. CAFs can also have anti-tumorigenic functions such as
inhibition of proliferation, inhibition of Treg cells and inhibition of angiogenesis (Figure 1)
12,13
.
TAMs are also abundant in the TME. In 1961, Monis and Weinberg discovered the presence of
macrophage infiltration in tumor tissues for the first time
14
. The polarization of TAMs is not a
definite process
15
. Due to the complexity of TME and the high plasticity of macrophages, TAMs
can exhibit functional and phenotypic diversity according to cancer types and tumor stages.
TAMs can be polarized towards the type I (M1) cells that display tumor-inhibitory activity, or
type II (M2) cells that have tumor-promoting activity
16
. Type I macrophages are activated by
IFN-γ, lipopolysaccharide and GM-CSF in vitro, and secrete anti-tumorigenic inflammatory
mediators such as IL-1 and IL-12, IL-18, IL-23 and TNF. The key role of type I TAMs is to
destroy pathogens and drive type 1 T helper cell response, which has cytotoxic activity against
microorganisms and tumor cells
17,18
. Type II macrophages are activated by IL-4 or M-CSF to
produce arginase-1. Type II macrophages secrete IL-10 and other anti-tumorigenic -
inflammatory cytokines that participate in angiogenesis, and ECM remodeling, and can also
down-regulate the function of type I macrophages.
17,18

Studies have found that in the TME, CAFs can promote the growth of monocytes and lead the
monocytes differentiate into TAMs. And TAMs can stimulate fibroblasts to secrete TGF-beta,
and activate trans-differentiation of fibroblasts into myofibroblasts, also known as myCAFs.
19

3

Figure 1. Heterogenous Function and Phenotype of Cancer-Associated Fibroblasts. The inner circle
lists subtypes of CAFs reported by various groups of investigators. The middle circle indicates markers
and proteins secreted by each subtype and in the outer circle, the functions attributed for each subtype are
indicated by colored circles representing one among the six CAF functions listed in margin.
10

4

Chapter II. Heterogeneous Presence of CAFs in Human Cancers
Tumor heterogeneity indicates the presence of cellular subpopulations, presenting different
phenotypes and functions. It is an important feature of malignant tumors, which occurs both
within the tumor (intra-tumor heterogeneity) and between the tumor (inter-tumor
heterogeneity)
20
. Even two tumors that originate from the same organ and have similar histology
can respond differently to treatment
21
. The TME contributes to the formation of tumor
heterogeneity, creating a complex system of interactive signaling pathways together with tumor
cells. CAFs are members of a heterogeneous group of stromal cells that differ in their origin,
their phenotype, their function, and their presence in diverse types of cancers.
Although CAFs have been reported to be present in most solid tumors, there is a significant
degree of variability regarding their abundance among cancers, with pancreatic adenocarcinoma,
breast carcinoma, lung adenocarcinoma and kidney clear cell carcinoma, being typically heavily
infiltrated with CAFs, and leukemia, lymphoma and brain tumors being mostly devoid of CAFs.
An in silico analysis of transcriptomics data obtained from Gene Expression Profiling Interactive
Analysis (GEPIA) databases (http://gepia.cancer-pku.cn)
22
for the mRNA expression of several
CAF markers (VIM (vimentin), ACTA2 (aka-SMA), S100A4 (fibroblast specific protein-1, a.k.a.
FSP-1), COL1A2 (collagen type I alpha chain 2), ITGB1 (integrin beta 1, a.k.a. CD29), TNC
(Tenascin C), PDPN (podoplanin), POSTN (periostin), FAP, MFAP5 (microfibril associated
protein 5, a.k.a. MAGP2), PDGFRβ, COL11A1 (collagen type 11 alpha chain 1),
ITGA11(integrin alpha 11), and NG2) performed by us illustrates this point (Figure 2A).
Vimentin (VIM) was highly expressed in all types of cancers, including in acute myeloid
leukemia, suggesting that it is not a specific marker of CAFs. In contrast, four transcripts,
ACTA2, S100A4, COL1A2 and ITGB1, had the highest level of expression in cancers known to
5

have a high proportion of CAFs, whereas low mRNA levels of these four genes were found in
uveal melanoma, acute myeloid leukemia and low-grade brain glioma
23,24
. These data are
consistent with other studies demonstrating that S100A4 and α-SMA (a.k.a. ACTA2) are
overexpressed in most pancreatic adenocarcinoma and breast cancers
25,26
. We also found a
positive correlation (r
2
= 0.55) between the mRNA expression of S100A4, a gene that reflects the
inflammatory function of CAFs
27
and COL1A2, a gene reflecting their effect on the stiffness of
the tumor ECM
28
, suggesting that these two functions of CAFs are closely associated (Figure
2B). We also performed correlative analyses between different markers (Figure 3). POSTN is
expressed more abundantly in invasive breast carcinoma, lung adenocarcinoma and squamous
cell carcinoma, colon adenocarcinoma and head and neck squamous cell carcinoma (HNSCC).
POSTN encodes for periostin, which was highly expressed in these cancers as demonstrated by
immunohistochemistry (IHC)
29
. In contrast, CAF-poor tumors such as low-grade glioma and
acute myeloid leukemia had low POSTN expression. Periostin was initially identified in
osteoblasts, but is also secreted by CAFs. Present in the ECM, it promotes tumor cell adhesion,
proliferation and migration through integrin binding and activation of the YAP signaling
pathway, and through the secretion of cytokines such as IL-6 and TGF- beta that promote
immune escape and EMT
30,31,32
. Podoplanin (PDPN) is a mucin-type transmembrane
glycoprotein and a lymphatic vessel marker. We found PDPN to be abundantly expressed in
mesothelioma, HNSCC, lung squamous cell carcinoma and testicular germ cell tumors. This
observation is consistent with an analysis of 662 tumors, examined by IHC for PDPN
expression, which revealed that PDPN is expressed by stromal α−SMA+, VIM- myofibroblasts.
PDPN expression by CAFs may represent a predictive marker of lymphatic/vascular spread.
33
In
breast cancer, the high presence of PDPN+ CAFs is positively correlated with tumor size,
6

invasive potential, and poor prognosis in patients
34,35
. PDPN+ CAFs also have an unfavorable
prognostic value in lung squamous cell carcinoma
36,37
. MFAP5 was strongly expressed in
fibroblastic cancers such as breast, ovarian, sarcoma, pancreatic and lung cancers. MFAP5 is a
25-kD microfibril-associated glycoprotein present in the ECM that interacts with integrins such
as αVβ3 expressed by ECs and with other ECM molecules such as collagen IV. MFAP5
promotes EC motility and the rearrangement of their cytoskeleton via Notch signaling
38,39
.
Recent studies show a correlation between MFAP5 and ACTA2 expression in CAFs, suggesting
that they may identify a new subtype of CAFs
23,40
. In bladder cancer and oral squamous cell
carcinoma, MFAP5 secreted by CAFs activates NOTCH2/HEY1, ERK and PI3K signaling
pathways directly, promoting the proliferation, migration, and invasion of cancer cells
40,41
. In
murine xenografted models of ovarian cancer, MFAP5 secreted by CAFs upregulates lipoma-
preferred partner (LPP) in ECs via FAK/ERK signaling, which promotes paclitaxel
chemoresistance and angiogenesis
42
. In bladder and breast cancers, high expression of MFAP5 in
CAFs correlated with high-grade malignancy, the presence of metastasis and unfavorable clinical
outcome
40
. In murine models of ovarian and PDAC cancers, inhibition of MFAP5 with
monoclonal antibodies is associated with a decrease in the number of CAFs and micro vessels,
but also with increased sensitivity to paclitaxel
43
.
Although this in silico transcriptomic analysis is informative, it does not take into consideration
the potentially significant heterogeneity in CAF distribution within a single tumor where
different sub-populations of CAFs with pro-and anti-tumorigenic functions could co-exist. This
aspect is illustrated in the recent report by Bartocheck et al. discussed above
44
, and by Öhlund et
al showing that myCAFs are in close proximity to tumor cells, while iCAFs are distant
45
. A
7

deeper understanding of the spatial heterogeneity of CAF in cancer will be obtained with further
studies at the single cell level.

Figure 2. In silico gene expression analysis of 14 genes expressed by CAFs in human tumors. (A).
Heat map representation of the expression of 14 genes (column) among 33 types of cancer (row). Data
was generated by Gene Expression Profiling Interactive Analysis (GEPIA) of The Cancer Genome Atlas
database (TCGA); (B). Correlation analysis between the expression of S100A4 and COL1A2 in cancer.
8

Figure 3. Correlation between different markers.
9

Chapter III. Immunohistochemistry: Expression of p-SMAD2 in
human xenotransplanted tumors
1. Rationale for the experiment
NB is a solid malignant tumor that affects children, and accounts for 10% of childhood cancer in
the USA and Europe
46
. A better understanding of the physiopathology of NB is important for
the development of new treatments of this disease. We have learned that the highly complex
TME plays a critical role in tumor progression. In this chapter we focus on the effect of CAF-
MSCs and monocytes on TME of NB tumors. CAF-MSCs and monocytes have emerged as
important contributors to the tumor stroma, especially in drug resistance and immune escape.
CAF-MSCs and monocytes are responsible for the secretion of many factors that increase the
aggressive phenotype of the tumor such as IL-6, IL-8 and TGF- beta. Data from the laboratory
showed in vitro that in triple co-cultures of NB, CAF-MSCs and monocytes there is a strong
induction of the production of several cytokines and chemokines like TGF- beta, IL-6, IL-8, IL-
10 and CCL2/MCP1. Thus, we designed in vivo experiments to verify these results.
2. Hypothesis
We hypothesized that the addition of human CAF-MSCs and monocytes to tumor cells would
enhance tumor growth in vivo and increase TGF-beta activity. We used an orthotopic human
xenograft model developed in the lab in which human NB cells are injected in the adrenal gland
of immunodeficient NOD SCID Gamma (NSG) mice and set up four experimental groups
including Group 1: implantation of NB cells alone, Group 2: implantation of NB cells
+Monocytes, Group 3: implantation of NB+CAF-MSCs, Group 4: implantation of
10

NB+Monocytes+CAF-MSCs. To show the enhancing effect of TGF-beta activity we use the
detection of p-SMAD2 by immunohistochemistry.
3. Experimental design
Both the male and female 6-8 weeks old mice were divided in a group of 3-4 mice. Each mouse
was orthotopically implanted in the adrenal gland fat pad with NB cells (CHLA-255, CHLA-
136, SK-N-BE (2)), either alone or mixed with monocytes and/or CAF-MSCs. The number of
cells: NB cells (1x 10
6
), monocytes (1x 10
6
), CAF-MSCs (2.5x 10
5
). Tumor growth was
accessed by bioluminescence imaging every week. Mice were sacrificed after four weeks, and
tumors were resected. Paraffin-embedded sections were cut at 5μm thickness, stained with
hematoxylin and eosin to assess the morphology of the tumor, or used for
immunohistochemistry. (Figure 4)
4. Materials and Methods
Cell lines
Luciferase expressing human NB cell lines: CHLA-255-luc, CHLA-136-luc and SK-N-BE (2)-
luc were obtained from Dr. R Seeger (Children’s Hospital Los Angeles, Los Angeles, CA),
which CHLA-255-luc is a MYCN-non-amplified (MYCN-NA) cell line, CHLA-136-luc and SK-
N-BE (2)-luc are MYCN- amplified (MYCN-A) cell lines.
MSCs were obtained from the bone marrow of healthy donors or purchased from American Type
Culture Collection (ATCC, PCS-500-OR, Bethesda, MD, USA).
Cultures were periodically assessed for absence mycoplasma contamination. MSCs were injected
with tumor cells and became educated as CAF-MSCs.
11

Fresh monocytes were isolated from healthy donors’ peripheral blood at Children’s Hospital Los
Angeles (CHLA)
47
.
Immunohistochemistry (IHC)
Tumor tissues were fixed in paraformaldehyde (4% w/v in PBS) overnight at 4℃, then
dehydrated in several changes of ethanol solutions at increased concentrations from 70% to
100% and dried in several changes of xylene at increased concentrations from 50% to 95%.
Tissues were then incubated in 60℃ melted paraffin/xylene (50/50 v/v) for 1 hour and after then
immersed in 2 changes of paraffin, one hour and overnight respectively. The next day,
embedding the tissues in paraffin blocks with fresh paraffin. Paraffin-embedded sections (5μm)
were deparaffinized in xylene for two times and rehydrated in several ethanol solutions at a
decreasing concentration from 100% to 50%. After washing in phosphate-buffered saline (PBS,
composition: 8.0 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4), sections were
circled carefully with a PAP pen. Antigen recovery was performed by treating sections with the
proteinase K (20μg/ml) for 10 minutes at room temperature. Slides were rinsed with 0.1% (v/v)
Tween-20 in PBS (PBST) and incubated with “Bloxall” blocking buffer for 10 minutes to block
endogenous peroxidase activity. Then we blocked tissue sections in 5% (v/v) goat serum in PBS
at room temperature for 1 hour. Slides were then incubated with the primary antibody solution:
Rabbit anti-human p-SMAD2 (Ser465/467) antibody at the concentration of 1: 500 in 5%(v/v)
biotinylated goat serum/ PBS overnight at 4℃. For example, if we need 1ml primary antibody
solution, we need 2μl primary antibody, 50μl goat serum and 948μl PBS mixed. After washing in
PBST, we applied 1:1000 biotinylated goat anti-rabbit IgG antibody (H+L) for one-hour
incubation at room temperature. After washing washed two times with PBST, slides were
incubated with ABC reagent for half an hour at room temperature. In our immunohistochemistry
12

experiments, we used VECTASTAIN ELITE® ABC Kits to increase the sensitivity of detection,
as these kits offer a uniform avidin-biotin complex for greater accessibility to biotinylated targets
within tissue samples. After washing with PBST, slides were stained with DAB chromogen. We
used ImmPACT DAB in this experiment to increase the visualization of the target protein
antigen and show a relative expression level. Finally, slides were counterstained with
hematoxylin and mounted in the permanent mounting medium. Negative controls included
incubation with the PBS solution instead of the primary antibody. (Figure 5, Table 1)

Figure 4. Experimental design. Setting up four groups of NGS mice, each group has equally 3-4 mice.
Injecting each group in the fat of the adrenal gland with NB alone, NB+monocytes, NB+ CAF-MSCs,
NB+ monocytes + CAF-MSCs, respectively. Follow tumor growth, bioluminescence every week,
dissection mice and resection of tumors. Then embedding tumors in paraffin, cutting the blocks to 5μm
sections. Finally, perform immunohistochemistry and quantification. (Cartoon resource:
https://smart.servier.com/)

13

Figure 5. Immunohistochemistry method. Heating slides for half an hour at 60℃, then deparaffinized
in xylene and rehydrated in ethanol. Antigen recovery was performed by proteinase K for 10 min. Slides
are incubated with “Bloxall” blocking buffer for 10 min and blocked in 5% (v/v) goat serum in PBS for 1
hour. Slides are incubated with the primary antibody at 4°C overnight followed by 1-hour incubation with
the secondary antibody. Then apply ABC reagent for half an hour and stained with DAB chromogen.
Finally, sections were counterstained with hematoxylin and applied with the mounting medium. (Cartoon
resource: https://smart.servier.com/, https://www.abcam.com/)

Table 1. List of reagents and concentrations used in immunohistochemistry

Reagent Concentration Company & Cat.
PAP pen ready to use Vector Laboratories # H-4000
Proteinase K dilute to 20ug/ml Roche # 03 115 828 001
Bloxall block solution ready to use Vector Laboratories # SP-6000
normal goat serum dilute to 5% (v/v) goat serum in PBS Vector Laboratories # S-1000
VECTASTAIN Elite ABC kit,
Peroxidase 1 drop A+ 1 drop B + 2.5ml PBS Vector Laboratories # PK-6100
ImmPACT DAB substrate kit,
Peroxidase 1 drop DAB reagent + 1ml DAB diluent Vector Laboratories # SK-4105
Hematoxylin QS ready to use Vector Laboratories # H-3404
VectaMount permanent mounting
medium ready to use Vector Laboratories # H-5000
primary antibody: Rabbit Anti-Human p-
SMAD2 Antibody(Ser465/467) dilute to 1:500 Millipore # AB3849-I
secondary antibody: biotinylated Goat
Anti-Rabbit IgG Antibody (H+L) dilute to 1:1000 Vector Laboratories # BA-1000
14

Quantification
Quantification of p-SMAD2 expression was performed using the QuPath software (version
0.2.3). We created a new project profile and imported the immunohistochemistry images with
setting the image type: H-DAB, then reset the pixel size with the matched image size. Creating
an annotation object with the rectangle tool, which was the area needed to be analyzed, avoiding
areas of tissue necrosis and marginal areas. Next, we ran the Analyze-Cell analysis-Cell
detection command and adjusted the list of parameters to adapt detection, including the setup
parameter, the nucleus parameters, the intensity parameters and the cell parameters, especially
the threshold in the intensity parameters. Then we would get the image result of the detection
object. By choosing the Cells- Nuclei only, the result showed the cells with red and blue circles.
The red circle identifies the dark brown nucleus, and the blue circle identifies the light blue
nucleus in the image A in Figure 6. From the IHC result, the dark brown nucleus means the
presence of p-SMAD2 (p-SMAD2-positive), and the light blue nucleus means the absence of p-
SMAD2 (p-SMAD2-negative). The QuPath software created a table with the number of
detections, the intensity values for all areas of positive nuclei and negative nuclei in the
annotation object, and the positive cell percentage for each IHC image. The data of all IHC
images were analyzed in GraphPad. (Figure 6)
Statistical Analysis
ANOVA one-way was used for statistical analysis for the overall condition with GraphPad Prism
9.0 software. All data are presented in scatter diagram as mean ± SEM of one independent
experiment with 2 different mice and different sections. The symbol related to a P-valve:
****P<0.0001, **P<0.01 and ns: not significant. (Figure 8B, 9B,10B)
15

Figure 6. Quantification of immunohistochemistry images with QuPath software.
A. Original immunohistochemistry image. B. Immunohistochemistry image with cell detection result.
C&D. Color deconvolution of immunohistochemistry image with cell detection result.

16

5. Results
CAF-MSCs and monocytes enhance NB cell growth
In vitro experiment, bioluminescence of the tumor in each mouse has been taken every week,
totally four weeks. Figure 7A shows the images of bioluminescence of each mouse. Figure 7B
shows the bioluminescence intensity of different groups over the weeks. By comparing four
different groups at the same time point, we can conclude that the groups with CAF-MSCs or
monocytes or together have more active tumors than the group injected with NB alone (Group
1). Besides, the Group 4-NB together with CAF-MSCs and monocytes had the largest tumor. By
comparing four time points in each group, we can conclude that the rate of tumor growth
increased over time, for example, in the last week, tumors grown fastest. From the results above,
we can clearly get the conclusion that CAF-MSCs and monocytes can enhance the growth of NB
cell and their joint presence resulted in maximal effect.
CAF-MSCs and monocytes enhance TGF-beta activity
To understand the mechanism underlying the tumor promoting of CAF-MSCs and monocytes,
we did H&E and IHC-p-SMAD2 experiments on mouse tumor samples of each group, and the
results shown in Figure 8A, 9A and 10A. H&E results show on the top row. The medium row is
negative control without the primary antibody, which all show negative. The bottom row shows
the expression of rabbit anti-human p-SMAD2 antibody. The dark brown color indicates the
nuclear presence of p-SMAD2. The transduction of TGF-β signal requires the phosphorylation
of Smad2/3. Thus, the expression of p-SMAD2 is the result of TGF-beta/Smad2/3 pathway
intensification and the p-SMAD2 is the marker of TGF-beta activity.
48

17

In Figure 8B, the expression of p-SMAD2 in NB alone group (Group 1) is at a relatively low
level (6.39±4.25%) compared to other groups, and the p-value is lower than 0.0001 between
Group 1 and the other three groups in CHLA-255 cell lines. NB with monocytes or CAF-MSCs
or together all show a high expression of p-SMAD2, especially the joint presence of monocytes
and CAF-MSCs in Group 4 (71.57±2.81%). And in Figure 9B and 10B, the expression of p-
SMAD2 in four groups all at a high level (all above 40%), the groups with monocytes or/and
CAF-MSCs have the higher expression of p-SMAD2 compared to NB alone Group. These
results suggest that the joint presence of CAF-MSCs and monocytes resulted in a higher
expression of p-SMAD2, reflecting an increase in TGF-beta activity.
Difference between MYCN-A and MYCN-NA tumors (Different results between CHLA-
255, CHLA-136 and SK-N-BE (2) cell lines)
We hypothesized that the addition of human CAF-MSCs and monocytes to NB tumor cells
would enhance tumor growth in vivo and increase TGF-beta activity, thus, NB alone group
which without CAF-MSCs and monocytes was expected to have a very low expression of p-
SMAD2. From the results in Figure 8B, 9B and 10B, it is easy to find that the expression of p-
SMAD2 in NB alone group (Group 1) of the CHLA-255 cell line differs from the other two cell
lines. The expression of p-SMAD2 in NB alone group is 6.39±4.25% in CHLA-255 cell line.
However, the expression of p-SMAD2 in NB alone group is 76.36±3.63% in CHLA-136 cell line
and is 42.3±3.9% in SK-N-BE (2) cell line. In our experiment, CHLA-255 is a MYCN-NA cell
line, CHLA-136 and SK-N-BE (2) are MYCN-A cell lines. From the data above, the expression
of p-SMAD2 in MYCN-A and MYCN-NA NB tumors is different. We suspect that the
difference is related to the status of MYCN in these cells.
18

19

20

21

22

Chapter IV. Discussion
Through in vitro experiment and the images of bioluminescence of each mouse in Figure 7, we
observed that CAF-MSCs and monocytes can enhance the growth of NB cell and their joint
presence results in higher stimulatory effect than in their individual presence. Moreover, the joint
presence of CAF-MSCs and monocytes leads to the higher expression of p-SMAD2 showing the
increase of TGF-beta activity from the H&E and IHC results on mouse tumor samples of each
group in Figure 8, 9 and 10. p-SMAD2 is a marker of TGF-beta activity and the expression of p-
SMAD2 is the result of TGF-beta/Smad2/3 pathway intensification. In our experiment, we used
anti-human p-SMAD2 antibody to show primarily (but not exclusively) tumor cells. In groups
together with CAF-MSCs and Monocytes, the tumor grown fastest and shown the highest
expression of p-SMAD2, which means highly active TGF-beta in TME. However, in this study,
we did not block TGF-beta to determine if it would lose the growth promotion activity. From Dr.
Seeger’s research, Galunisertib, a type of inhibitor of TGF-beta receptor type I that suppress
phosphorylation of SMAD2 and SMAD3 in human NB cells, reduced the growth of tumor cells
implanted subcutaneously in immunodeficient mice
49
. Thus, we can only assume that CAF-
MSCs and monocytes enhance the growth of NB in a TGF-beta-dependent manner.
The second important observation reported in this manuscript is the difference between MYCN-
A and MYCN-NA NB tumors regarding to the result of IHC- p-SMAD2. Since the identification
of the MYCN oncogene to NB in 1983 by Kohl et al
50
, people were beginning to understand the
contribution of genetic changes to the progression of NB. MYCN gene plays a significant role in
NB tumor cell growth, proliferation and apoptosis. Recent studies suggest a significant
difference in the TME of NB between MYCN-A and MYCN-NA tumors in the content of
stromal and immune cells
51
. Genomic amplification of MYCN is most associated with poor
23

prognosis and treatment failure, and nearly 25% of all NB tumor cases detected MYCN
amplification
52
. An analysis of the TARGET dataset and a series of IHC on NB tumors indicated
that MYCN-A tumors are more vascularized and have fewer immune cells, myeloid and
lymphoid cells in the TME landscape. In contrast, MYCN-NA tumors are less vascularized and
have more stromal cells in TME landscape
51
.
From our hypothesis, the addition of human CAF-MSCs and monocytes to NB tumor cells
would enhance tumor growth in vivo and increase TGF-beta activity. We expected lower TGF-
beta activity in MYCN-A tumors (CHLA-136 and SK-N-BE (2) cell lines) in Figure 9 and
Figure 10. However, without adding CAF-MSCs and monocytes, the expression of p-SMAD2 is
76.36±3.63% in CHLA-136 cell line and is 42.3±3.9% in SK-N-BE (2) cell line in NB alone
group. Taken together, we are considering two hypotheses. The first hypothesis is MYCN-A NB
cells have the ability to secrete their own TGF-beta and thus being less dependent on the TME to
activate TGF-beta, whereas MYCN-NA NB cells are more dependent on the TME to make TGF-
beta. A recent study suggested there are several crosslinks between MYCN gene expression and
the TGF-beta pathway at various levels, such as regulating TGF-beta 1 mRNA expression and
TGF-beta 1 target genes
53
. Another hypothesis is that they are differences in the type or number
of receptor molecules between MYCN-A NB cells and MYCN-NA NB cells. There are three
TGF  receptors (1, 2 and 3) and they respond differently to TGF -1, TGF R1 and 2 stimulating
proliferation and TGF R3 promoting differentiation
54
.
The status of MYCN gene in the NB tumor cell has an important biological effect on the activity
of TGF-beta. Further research can be done regarding to the relationship between the expression
of MYCN gene and the TGF-beta activity. For example, setting control experiments of MYCN-
A cell lines, MYCN-A cell lines with downregulating MYCN gene amplification and MYCN-
24

NA cell lines. PIK-75 is an effective PI3K inhibitors which can downregulate MYCN Protein
55
.
If downregulating the MYCN amplification in MYCN-A NB cell lines will lead to less TGF-beta
activity, we could indicate that MYCN-A NB cells have the ability to secrete TGF-beta by itself
and is less dependent on the TME to activate TGF-beta.
In conclusion, the data above indicates that increasing the activity of TGF-beta can promote the
growth of NB tumor cells, thus it raises the question of targeting TGF-beta in tumor therapy. In
recent clinical trials, several therapies targeting TGF-beta signaling have been tested blocking
the TGF-beta pathway. Fresolimumab (GC1008), Galunisertib (LY2157299), Vactosertib (TEW-
7197) and Bintrafusp alfa (M7824) are the TGF-beta targeting drugs that have been extensively
studied recently. Fresolimumab is a TGF-beta neutralizing antibody; Galunisertib is a small
molecule inhibitor of the TGF-beta RI kinase, and Vactosertib is a TGF-beta receptor
ALK4/ALK5 inhibitor
56,57
. Bintrafusp alfa is a bifunctional agent that targeting programmed
death ligand 1 (PD-L1) and TGF-beta receptor II molecules
58
.
Besides, a study found that Bintrafusp alfa shows more effectively suppression of tumor growth
and metastasis compared with applying the anti-PD-L1 antibody or anti-TGF-beta reagent alone
in vivo
59
. More clinical trials are active to verify this, such as NCT03840902 in Table 2, where
the durvalumab is a kind of PD-L1 inhibitors. The Table 2 summarizes several still active
clinical trials and completed clinical trials with the conditions, study arms and status. Although
some are only testing the effect of single drugs, other more study teams start testing the effect of
the combination of drugs targeting TGF-beta with other immunotherapeutic agents,
chemotherapy and radiation therapy, avenues that the combination may be more promising.

25

Table 2. Clinical trials of TGF-beta targeting drugs. (Resource: https://www.clinicaltrials.gov)

Drug Mechanism Conditions
Study
Phase
Enrollment Study Arms Status
ClinicalTrials.gov
Identifier
Metastatic
Breast Cancer Phase 2 23 participants
Two arms. Fresolimumab is administered
intravenously (i.v.) at a dose of 1 mg/kg or
10 mg/kg on day 1 of weeks 0, 3, 6, 9 & 12.
Radiation Therapy.
Completed.
Results First
Posted on April
6, 2017. NCT01401062
Pleural
Malignant
Mesothelioma Phase 2 14 participants
Single-Arm. All subjects will receive the
investigational agent, GC1008 in 3 week
cycles of treatment.
Completed.
Results First
Posted on April
10, 2020. NCT01112293
Early Stage
Non-small Cell
Lung Cancer
Phase 1
Phase 2 24 participants
Phase 1: Evaluate the safe dose of
fresolimumab in combination with
stereotactic ablative radiotherapy (SABR) in
patients.
Phase 2. Evaluate the rate of radiation
induced pulmonary fibrosis after SABR plus
fresolimumab. Completed. NCT02581787
Advanced
Hepatocellular
Carcinoma
(HCC) Phase 1 15 participants
Single-Arm. Galunisertib 150mg by mouth
twice a day. Stereotactic Body Radiotherapy
(SBRT). Completed. NCT02906397
Glioblastoma
(GB) Phase 2 180 participants
Study of LY2157299 Monohydrate
Monotherapy or LY2157299 Monohydrate
Plus Lomustine Therapy Compared to
Lomustine Monotherapy in Patients.
Active, not
recruiting. NCT01582269
Neoplasm
Metastasis
Pancreatic
Cancer
Phase 1
Phase 2 170 participants
Phase 1b: To determine the safe and tolerable
dose of galunisertib in combination with
gemcitabine.
Phase 2a: To compare the overall survival
(OS) of patients with Stage II to IV
unresectable pancreatic cancer when treated
with a combination of galunisertib and
gemcitabine with that of gemcitabine plus
placebo.
Completed.
Results First
Posted on May
16, 2018. NCT01373164
Metastatic
Colorectal or
Gastric Cancer
Phase 1
Phase 2 67 participants
Single-Arm. TEW-7197 will be administered
orally for 5 days per week (5D/W) and
Pembrolizumab will be administered as a
dose of 200 mg every 3weeks.
Active, not
recruiting. NCT03724851
Advanced Non-
Small Cell
Lung Cancer
Phase 1
Phase 2 60 participants
Single-Arm. Dose Escalation of TEW-7197.
TEW-7197 will be administered orally for 5
days per week (5D/W) and Durvalumab
administration.
Active, not
recruiting. NCT03732274
Metastatic
Gastric Cancer
Phase 1
Phase 2 62 participants
Study of TEW-7197 (Vactosertib) Plus
Weekly Paclitaxel as Second-line Treatment.
Dose Escalation of TEW-7197. TEW-7191
will be given twice daily (BID) for 5 days
followed by 2 days off with a cycle of 4
weeks.
Active, not
recruiting. NCT03698825
Non-small Cell
Lung Cancer Phase 2 168 participants
Two arms. Arm 1: cCRT plus M7824
followed by M7824. Arm 2: cCRT plus
placebo followed by durvalumab.
Active, not
recruiting NCT03840902
Neoplasms Phase 1 25 participants
Single-Arm. Participants receiving
Bintrafusp alfa. A Phase Ib Trial to Evaluate
the Efficacy and Safety of Bintrafusp Alfa
Monotherapy in Metastatic or Locally
Advanced/Unresectable Urothelial Cancer.
Active, not
recruiting NCT04349280
Mesothelioma;
Lung Phase 2 47 participants
Single-Arm. Bintrafusp alfa (M7824):
1200mg, over 60 minutes IV infusion. The
treatment will be administered at day 1 of 14-
day intervals. Treatment will be administered
until unacceptable toxicity, loss of clinical
benefit, disease progression or completion of
2 years of therapy. Recruiting NCT05005429
Fresolimumab
(GC1008)
TGFβ
neutralizing
antibody
Galunisertib
(LY2157299)
small molecule
inhibitor of the
TGFβRI kinase
Vactosertib
(TEW-7197)
TGF-β receptor
ALK4/ALK5
inhibitor
Bintrafusp alfa
(M7824)
targeting PD-
L1 and
TGFβRII
26

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Abstract (if available)

Abstract Cancer associated fibroblasts (CAFs) and their precursor cells mesenchymal stromal cells (MSC) are one of the most abundant cell types that exist in the tumor microenvironment (TME) and have an important role to cancer progress. The heterogeneous nature of CAFs and their contribution to Transforming Growth Factor beta (TGF-beta) activity in neuroblastoma (NB) is discussed in this thesis. We analyzed in silico the mRNA expression of several CAF markers in different types of human cancers and provide data showing that CAF-mesenchymal stromal cells (CAF-MSCs) and monocytes can increase TGF-beta activity and NB tumor growth.

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Creator Li, Rongrong (author)

Core Title Contribution of cancer associated fibroblasts to cancer progression

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2022-08

Publication Date 07/26/2022

Defense Date 06/23/2022

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

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