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The Role of CD47 and PD-L1 in Tumor Regression upon MYC Inactivation

in MYC-induced Tumors

Rachel Do

May 2016

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The Role of CD47 and PD-L1 in Tumor Regression upon MYC Inactivation

in MYC-induced Tumors

An Honors Thesis Submitted to

the Department of Biology

in partial fulfillment of the Honors Program

STANFORD UNIVERSITY

Rachel Do

May 2016

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Acknowledgements:

I would like to thank the other contributing authors on this project: Stephanie Casey, Ling Tong,

Yulin Li, Susanne Walz, Kelly Fitzgerald, Arvin Gouw, Virginie Baylot, Ines Gutgemann,

Martin Eilers, and Dean Felsher. In particular, I would like to thank my mentor, postdoctoral

researcher Stephanie Casey, for training me in the techniques I have used in this project, for her

assistance and critique throughout the experimental process, and for her work on the project

design and data analysis. I would also like to thank my PI, Dr. Dean Felsher, for his mentorship

and guidance throughout this project, as well as during my four years in the Felsher Lab.

Additionally, I would like to thank Stanford Undergraduate Advising and Research and the

Stanford Bio-X program for providing funding through a Major Grant (2012) and a Summer

Research Grant (2011). Lastly, I would like to thank Dean Felsher for his advice and support as

my Research Sponsor for this thesis and Patricia Jones for her feedback as my Second Reader.

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Table of Contents:

List of figures……………………………………………………………………………………...5

Abstract……………………………………………………………………………………………6

Introduction………………………………………………………………………………………..7

Materials and Methods…………………………………………………………………………….9

Results……………………………………………………………………………………………12

Discussion………………………………………………………………………………………..14

References………………………………………………………………………………………..17

Figures…………………………………………………………………………………………....19

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List of Figures:

Figure 1: Model of MYC expression and inactivation in T-ALL 4188 line; model of enforced

CD47 and PD-L1 expression.

Figure 2: Primer sequences for qPCR analysis of mRNA expression.

Figure 3: Constitutive expression of CD47 and PD-L1 are required for tumor regression and

survival.

Figure 4: MYC, CD47, and PD-L1 mRNA expression upon MYC inactivation.

Figure 5: Sustained expression of CD47 or PD-L1 inhibits the recruitment of macrophages and

activated lymphocytes upon MYC inactivation.

Figure 6: Sustained expression of CD47 or PD-L1 inhibits angiogenic shutdown upon MYC

inactivation.

Figure 7: Sustained expression of CD47 or PD-L1 inhibits cellular senescence upon MYC

inactivation.

Figure 8: Sustained expression of CD47 or PD-L1 does not affect apoptosis or proliferation upon

MYC inactivation.

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Abstract:

The MYC oncogene, which is overexpressed in many human cancers, is a transcription factor

that regulates cell proliferation, growth, differentiation, and apoptosis. Upon MYC inactivation

in MYC-driven tumors, an intact immune system is essential for sustained tumor regression, but

the role of specific immune effectors is unclear. Our lab has recently found that MYC

suppression results in decreased gene and protein expression of the immunoinhibitory molecules

CD47 and PD-L1 in a conditional mouse model system for MYC-induced tumors. CD47 and

PD-L1 are critical to the mechanism by which tumors evade the host immune system.

Specifically, we show that when MYC is inactivated in tumors with enforced CD47 or PD-L1

expression, the host immune response is suppressed, preventing sustained tumor regression and

leading to a faster rate of relapse. Through immunohistochemistry, we further show that enforced

expression of CD47 and PD-L1 prevents angiogenic shutdown, blocks senescence, and inhibits

recruitment of immune cells after MYC inactivation. Therefore, the data presented here suggests

that MYC appears to initiate and maintain tumorigenesis in part through the increased expression

of CD47 and PD-L1.

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Introduction:

Cancer is caused by the activation of proto-oncogenes and/or inactivation of tumor

suppressors [1]. MYC is a transcription factor that regulates cell proliferation, growth,

differentiation, and apoptosis in conjunction with other oncogenes and the loss of tumor

suppressor genes [2]; its overexpression is associated with most types of human cancers,

including T cell acute lymphoblastic leukemia (T-ALL). T-ALL is characterized by the

proliferation and accumulation of cancerous blast cells in the peripheral blood and bone marrow.

On average, it affects thirty out of every million Americans and occurs during childhood.

Prognosis for T-ALL patients who relapse following initial treatment is poor, which necessitates

development of targeted anti-ALL therapies for sustained tumor regression.

Our lab [3, 4] has demonstrated that tumors exhibit “oncogene addiction,” in which

inactivation of the oncogene driving tumor growth, such as MYC, reprograms the physiology of

the tumor cell back to its normal state [5]. Tumor regression is associated with proliferative

arrest, apoptosis, cellular senescence, and angiogenic shutdown. In general, tumors coevolve

with host immune effectors and chemokines through a process described as immune editing;

thus, the host immune system is involved in this reprogramming by both promoting and

suppressing tumorigenesis [6]. Many different components of the immune system, such as

macrophages, CD8+ and CD4

+ T cells, B cells, and NK cells, may play specific roles. It is known

that CD8+ T cells contribute to antigen-dependent and NK cell-mediated tumor elimination [7]

and that CD4+ T cells are required for sustained tumor regression, and that cytokines mediate this

antitumor activity [8]. Our lab has identified TSP-1, a cytokine secreted by CD4+ T cells, as a

significant contributor to CD4+ T cell-mediated tumor regression. TSP-1 interacts with CD47, a

ubiquitous cell surface receptor that, upon activation, regulates angiogenesis and sends a “don’t

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eat me” signal to the innate immune system, allowing tumors to evade phagocytosis [9]. PD-L1

binds to the PD-1 receptor on activated T cells, suppressing T cell proliferation, which sends a

“don’t find me” signal to the adaptive immune system [10]. We have recently found that both

CD47 and PD-L1 are downregulated upon MYC inactivation in MYC-driven tumors [11].

Oncogene addiction is associated with both cell-autonomous mechanisms, including

proliferative arrest and induction of apoptosis, and host-dependent mechanisms, such as

inhibition of angiogenesis and induction of senescence [5]. Two of these hallmarks, shutdown of

angiogenesis in the tumor microenvironment and tumor cell senescence, have been linked to the

T-cell mediated immune response [1]. Cancer cells exhibit the ability to maintain chronic

proliferation through self-production of growth factor ligands, which can be directly responded

to, or which can stimulate normal support cells in the tumor environment [12]. This

independence from exogenous growth factors is linked to mutations that can activate a number of

signaling pathways or disrupt negative feedback mechanisms. In order to sustain such growth,

cancer cells must resist apoptosis, or programmed cell death [123. An apoptotic trigger is

controlled by the balance between pro- and anti-apoptotic factors, which in turn is regulated by

abnormality sensors that flag DNA damage, insufficient survival factor signaling, and

oncoprotein activity levels [14].

Senescence, or cellular aging, is believed to be a protective barrier to neoplastic

expansion, as cancer cells have been understood to have unlimited replicative potential [14]. It is

triggered by proliferation-associated abnormalities including high oncogene signaling and the

subcritical shortening of telomeres, though it has been shown that cancer cells may be capable of

proliferating unimpeded in culture up until the point of crisis, upon the induction of apoptosis

[15]. To support rapid proliferation, angiogenesis, which generates tumor-associated vasculature,

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is induced early in the development of invasive cancers. During tumorigenesis, an angiogenic

switch is almost always activated and remains on, causing normal quiescent vasculature to

continuously branch and sprout new blood vessels [16]. The angiogenic switch is controlled by

the balance between factors that induce and oppose angiogenesis, some of which are signaling

proteins whose pathways are disrupted by oncogenic mutations.

An emerging hallmark, as previously described, is the ability for cancer cells to evade

immune destruction; recently, there has been a recent focus on tumor-host immunological

interactions, which may be critical in understanding how the anti-tumor immune response is a

barrier to tumorigenesis in humans [14]. Thus, our research may have significant therapeutic

implications, in optimizing MYC-targeted or immune specific therapies for the treatment of

leukemia and lymphoma patients. More generally, the results of this project will enhance our

understanding of how oncogene inactivation leads to tumor regression and how tumor relapse, in

which tumor cells overcome oncogene addiction, may occur.

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Materials and Methods:

Cell lines:

All experiments were completed using the MYC-induced T-cell acute lymphoblastic

leukemia (T-ALL) 4188 cell line; expression of MYC is regulated by the tetracycline

transactivating protein (tTA) so that administration of the tetracycline derivative doxycycline

(Dox) will cause MYC inactivation (Fig. 1A) [17]. We engineered MYC T-ALL 4188 cells to

constitutively express CD47 or PD-L1 even upon MYC inactivation. Using the pMSCV-

puromycin system, plasmids containing retroviral expression vectors with either CD47 or PD-L1

cDNA, or empty control MSCV vector, were transfected into 293T cells, and virus-containing

medium supernatants were used to infect tumor cells using polybrene methodology (Fig. 1B).

Infected cells were selected, expanded, forced expression confirmed, and prepared for injection.

Tumor growth assays:

10 x 106

tumor cells were injected subcutaneously in a volume of 100 µL into 4-6 week

old female FVB WT mice. 6 mice were allocated for each group: control (MYC T-ALL 4188),

constitutively-expressing CD47, and constitutively-expressing PD-L1 cell lines. After tumors

reached approximately 1.5 cm3, 3 of the mice in each group were sacrificed, and their tumors

collected, noted as “MYC on”/Day 0 (d0). The remaining mice were treated with Dox in their

drinking water (100 µg/mL concentration) to inactivate MYC, and their tumors were collected 4

days after the start of treatment, noted as “MYC off”/Day 4 (d4).

A separate cohort of FVB RAG1-/-

mice transplanted with control, CD47-expressing, and

PD-L1 expressing MYC T-ALL 4188 tumor cells was followed for survival analysis; mice were

treated with Dox once tumors reached approximately 1.5 cm3, and tumor burden was monitored.

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Animals were identically raised and housed at Stanford University. All experiments were

approved by the Administrative Panel on Laboratory Animal Care (APLAC) at Stanford

University in accordance with institutional and national guidelines.

In vivo bioluminescence imaging:

Bioluminescence imaging was performed as previously described [8]. Cell lines were

infected with pMSCVneo containing fLuc. For fLuc immune labeling, mice were injected

intravenously with 4 x 106 purified CD4

+ T cells that had been magnetically enriched from

CAG-luc-eGFP L2G85 mice (Miltenyi Biotec beads and columns); control, CD47-expressing,

and PD-L1 expressing MYC T-ALL 4188 cells were injected into FVB RAG1-/-

mice 1 week

after reconstitution with fLuc+ CD4

+ T cells.

Mice were anesthetized with inhaled isoflorane/oxygen using the Xenogen XGI-8 5-port

Gas Anesthesia System. d-luciferin (150 mg/kg) was injected intraperitoneally 10 min prior to

imaging. Animals were placed into the light-tight chamber and were imaged with an IVIS-200

cooled CCD camera (Xenogen). Living Image (Xenogen) was used to collect and analyze data

and generate pseudocolor images.

Immunohistochemistry and immunofluorescence:

Immunohistochemistry and immunofluorescence were performed as previously described

[1]. Senescence-associated β-galactosidase staining was performed on tissue sections that had

been embedded in OCT freezing medium (Tissue Tek) and stored at -80°C. Tissue sections were

warmed to room temperature, fixed in 0.5% glutaraldehyde in PBS, and washed in PBS pH 5.5.

Sections were stained for 8 hours in a solution containing 250 mM potassium ferricyanide,

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potassium ferrocyanide, and MgCl2 in PBS (pH 5.5), mounted in 50% glycerol/PBS, and

promptly imaged. CD31 staining was also performed on frozen sections. Slides were thawed,

fixed in acetone, blocked in Dako serum-free blocking reagent, incubated in rat anti-mouse

CD31 (1:50, BD Pharmingen) for 1.5 hours at room temperature, incubated with anti-rat

biotinylated IgG (1:300) and Cy3-streptavidin (1:300) for 30 min at room temperature), and

counterstained with DAPI. CC3, PH3 (Cell Signaling), F4/80 (Invitrogen), and CD69 primary

antibodies were used according to manufacturer recommendations. Tumors were fixed in 4%

paraformaldehyde, embedded in paraffin, and sectioned. Sections were stained as previously

described [18]. CC3 and PH3 stainings were counterstained with DAPI, and CD69 and F4/80

stainings were counterstained with hematoxylin. Pictures were taken with 20-40x objectives on a

Leica DMI6000 microscope with LASAF software. At least 5 random fields per section and at

least 3 tumors per experimental condition were imaged.

RNA isolation, cDNA preparation, and qPCR:

All tissues and cells were flash-frozen in liquid nitrogen and stored at -80°C until RNA

preparation. RNA was isolated using the TRIzol Reagent (Invitrogen) according to manufacturer

instructions. cDNA was synthesized using SuperScriptIII (ThermoFisher). qPCR was performed

using specific primers (Fig. 2) and the SYBR Green qPCR Kit (Roche) in an Applied Biosystems

Real-Time PCR System (Life Technologies) with QuantStudio12K Flex Software. Data was

analyzed using the cycle threshold method (normalized to UBC). A minimum of 3 biological and

3 technical replicates were used for all qPCR experiments. S. Casey provided assistance with the

analysis and graphing of qPCR results.

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Results:

Enforced expression of CD47 or PD-L1 reduces survival and blocks tumor regression following

MYC inactivation.

Expression of CD47 or PD-L1 prevented the sustained tumor regression that was

observed upon MYC inactivation in control tumors (Fig. 3A); mice with CD47 or PD-L1

expressing tumors survived a maximum of 30 days post MYC inactivation, compared to a 100%

survival rate for controls after 60 days. Bioluminescence imaging showed a significant decrease

in tumor size in control tumors for 20 days post MYC inactivation, compared to a minor,

temporary decrease for CD47 and PD-L1 expressing tumors (Fig. 3B). qPCR data confirms that

MYC expression in control, CD47 expressing, and PD-L1 expressing tumors is at the same level

before MYC inactivation and decreases equally afterwards (Fig. 4A). 4 days after MYC

inactivation, CD47 mRNA levels are significantly higher in CD47 expressing tumors than in

control tumors, and PD-L1 mRNA levels are significantly higher in PD-L1 expressing tumors

than in control tumors (Fig. 4B).

Enforced expression of CD47 or PD-L1 inhibits the recruitment of immune cells following MYC

inactivation.

Recruitment of macrophages (F4/80) and activated lymphocytes (CD69) was assessed

through immunohistochemistry of control MSCV, CD47-expressing, and PD-L1-expressing

tumors following MYC inactivation in WT FVB hosts (Fig. 5A). The density of F4/80 and CD69

markers was significantly reduced when CD47 and PD-L1 were constitutively expressed by the

tumor cells, compared to the control (Fig. 5B). 4 days post-MYC inactivation, the density of both

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markers significantly increased in the control tumors, but remained low in the CD47 and PD-L1

expressing conditions.

Enforced expression of CD47 or PD-L1 prevents angiogenic shutdown and blocks senescence

following MYC inactivation, but does not affect cellular proliferation or apoptosis.

Angiogenesis (CD31) was assessed through immunofluorescence of control MSCV,

CD47 expressing, and PD-L1- expressing tumors (Fig. 6A). The mean vessel density (MVD), as

measured by the presence of CD31+ microvessels, was significantly lower 4 days post-MYC

inactivation in control tumors; there were insignificant differences in CD47 and PD-L1

expressing tumors (Fig. 6B). In control tumors, 4 days after MYC inactivation, RNA levels of

Ang2, which promotes vascular regression, are increased, and RNA levels of Tie2, which

promotes vascularization, are decreased. CD47 and PD-L1 expressing tumors exhibit opposing,

or insignificant effects (Fig. 6C).

Senescence was assessed through SA-β-gal staining, measured by β-galactosidase density

(Fig. 7A). In control tumors, β-galactosidase density increased over 20-fold after 4 days of

MYC-inactivation, and there were insignificant changes in density in CD47 and PD-L1

expressing tumors (Fig. 7B). On the mRNA level, there was decreased expression of p15Ink4b

and p19ARF, both implicated in mediating senescence, after 4 days of MYC inactivation in

CD47 and PD-L1 expressing tumors (Fig. 7C).

There were no significant differences in apoptosis or proliferation, as assessed through

immunofluorescence for the quantification of cleaved caspase 3, an apoptosis marker, (Fig. 8, A

and C) and phospho-histone H3, a proliferation marker, (Fig. 8, B and D), in control, CD47

expressing, and PD-L1 expressing tumors.

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Discussion:

We have demonstrated that the immunoinhibitory molecules CD47 and PD-L1 play a

critical role in the initiation and maintenance of MYC-induced tumors. CD47 and PD-L1 are

upregulated in MYC-induced tumors and downregulated upon MYC inactivation. Here, we have

shown that constitutive expression of CD47 or PD-L1 prevents sustained tumor regression,

which is generally observed when MYC is inactivated, and this significantly increases the

likelihood for tumor relapse. In vivo, this is associated with a drastically reduced lifespan and

chance of survival in WT FVB mice.

Specifically, MYC inactivation induces tumor regression through oncogene addiction,

mediated through proliferative arrest, apoptosis, cellular senescence, and angiogenic shutdown,

so we investigated the effect of enforced CD47 and PD-L1 expression on these mechanisms. We

found that upon MYC inactivation in MYC-driven tumors, CD47 or PD-L1 expression prevented

angiogenic shutdown, measured by the presence of CD31+ microvessels, decreased Ang2

expression, and unaffected Tie2 expression. Similarly, CD47 or PD-L1 expression prevented

cellular senescence, measured through a β-galactosidase assay and decreased p15Ink4b and

p19ARF expression. We discovered that enforced CD47 and PD-L1 expression had no effect on

apoptosis and proliferation, measured by immunofluorescence for cleaved caspase 3 and

phosphor-histone H3, respectively. In addition, we found that CD47 and PD-L1 expression

inhibited the recruitment of immune cells to the tumor microenvironment, as measured by

immunohistochemistry for F4/80, a macrophage marker, and CD69, a marker of activated

lymphocytes.

Enforced expression of CD47 and PD-L1 prevents the shutdown of angiogenesis, blocks

cellular senescence, and inhibits the recruitment of immune cells upon MYC inactivation.

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Therefore, the downregulation of CD47 and PD-L1 observed when MYC is inactivated is

required for angiogenic shutdown, senescence induction, and the host immune response, which is

necessary for sustained tumor regression. This suggests that, in MYC-induced tumors, MYC

overexpression leads to CD47 and PD-L1 overexpression, masking tumor cells from immune

surveillance, suppressing the host immune response, inducing angiogenesis, and preventing

senescence to promote tumorigenesis. MYC regulation of CD47 and PD-L1 may have a direct

role in MYC-driven tumorigenesis.

Our lab has also found that MYC inactivation rapidly downregulates CD47 and PD-L1

mRNA and protein expression, but expression of other cell surface immune molecules was not

affected. This has been found in mouse and human solid tumors and in multiple human cancer

types. MYC binds to the promoters of Cd47 and Pd-l1 genes in a dose-dependent manner, and

there is a lower, nonspecific binding to the promoters of other surface immune molecules [11].

This may provide evidence of a transcriptional regulatory mechanism by which MYC regulates

CD47 and PD-L1 expression.

In mice, enforced expression of CD47 and PD-L1 prevents sustained tumor regression,

leading to relapse. Additional findings from our lab show that shRNA knockdown of CD47 and

PD-L1 prevents the growth of MYC T-ALL cells and the initiation of tumorigenesis [11]. The

inability for tumor engraftment to occur when CD47 and PD-L1 expression is inhibited,

combined with the lack of complete tumor cell clearance upon enforced CD47 and PD-L1

expression, suggests that CD47 and PD-L1 directly affects the initiation and maintenance of

MYC-induced tumorigenesis.

The results of this project have set the stage for investigation into the mechanisms

through which CD47 and PD-L1 are involved in specifically regulating angiogenesis and

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senescence, and how these immunoinhibitory molecules directly affect the host immune response

to tumorigenesis. More broadly, our findings have implicated MYC with having a role in

impairing anti-tumor immune responses and therefore a critical target for MYC-driven human

cancers. Though many therapies have been developed to inhibit MYC function or stability, in

vivo application has not been successful; targeting CD47 and PD-L1, which has been shown to

be effective in our mouse model, should be explored. It would also be important to investigate

how MYC may work with other oncogenic pathways [19], as this interaction may also contribute

to the aforementioned impairment of the anti-tumor immune response.

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References:

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57-70.

2) Felsher, Dean W., and J. Michael Bishop. "Reversible tumorigenesis by MYC in

hematopoietic lineages." Molecular Cell 4.2 (1999): 199-207.

3) Bellovin, David I., Bikul Das, and Dean W. Felsher. "Tumor dormancy, oncogene addiction,

cellular senescence, and self-renewal programs." Systems Biology of Tumor Dormancy.

Springer New York, 2013. 91-107.

4) Felsher, Dean W. "Reversing cancer from inside and out: oncogene addiction, cellular

senescence, and the angiogenic switch." Lymphatic research and biology 6.3-4 (2008): 149-

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Immunology 179.10 (2007): 6651-6662.

8) Rakhra, Kavya, et al. "CD4+ T cells contribute to the remodeling of the microenvironment

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485-498.

9) Parsa, Andrew T., et al. "Loss of tumor suppressor PTEN function increases B7-H1

expression and immunoresistance in glioma." Nature medicine 13.1 (2007): 84-88.

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10) Jaiswal, Siddhartha, et al. "CD47 is upregulated on circulating hematopoietic stem cells and

leukemia cells to avoid phagocytosis." Cell 138.2 (2009): 271-285.

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and PD-L1." Science 352.6282 (2016): 227-231.

12) Lane, D.P. (1992). Cancer. p53, guardian of the genome. Nature 358, 15–16. Lemmon, M.A.,

and Schlessinger, J. (2010). Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134.

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Figures:

Fig. 1: (A) Expression of MYC in our MYC-driven T-ALL cell line is regulated by tTA, and

administering doxycycline inactivates MYC. (B) Retroviral mediated expression of CD47 (blue)

and PD-L1 (green), or an empty control vector (gray), in MYC T-ALL 4188 cells for constitutive

CD47 or PD-L1 expression upon MYC inactivation.

A B

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Fig. 2:

Primer sequences used for qPCR analysis of mRNA expression.

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Fig. 3:

Constitutive expression of CD47 and PD-L1 are required for tumor regression and survival.

(A) Survival curve and (B) tumor size measurements for FVB WT mice with control MSCV

(gray), CD47 expressing (blue), or PD-L1 expressing MYC T-ALL 4188 tumor cells. MYC was

inactivated when tumors reached 1.5 cm3 (d0). For (B), bioluminescence imaging data for 3

representative animals per group are shown.

A B

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Fig. 4:

MYC, CD47, and PD-L1 mRNA expression upon MYC inactivation. (A) MYC expression

before (d0) and after MYC inactivation (d4) in control MSCV (gray), CD47 expressing (blue),

and PD-L1 expressing (green) tumors. (B) CD47 and PD-L1 expression before (d0) and after

MYC inactivation (d4).

A B

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Fig. 5:

Sustained expression of CD47 or PD-L1 inhibits the recruitment of macrophages and activated

lymphocytes upon MYC inactivation. (A) Immunohistochemistry of macrophages (F4/80) and

activated lymphocytes (CD69) in control MSCV, CD47-expressing, and PD-L1-expressing

tumors before (d0) and after MYC inactivation (d4). (B, C) Quantification of F4/80+ or CD69+

cells in control MSCV (gray), CD47 expressing (blue), and PD-L1 expressing (green) tumors

before or 4 days after MYC inactivation, by immunohistochemistry using markers for

macrophages (F4/80) and activated T cells (CD69).

A

B C

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Fig 6:

Sustained expression of CD47 or PD-L1 inhibits angiogenic shutdown upon MYC inactivation.

(A) Immunofluorescence for CD31, an angiogenic marker, in control MSCV, CD47-expressing,

and PD-L1- expressing tumors 0 or 4 days following MYC inactivation. (B) Quantification of

mean vessel density, measured by the presence of CD31+ microvessels in control MSCV (gray),

CD47 expressing (blue), and PD-L1 expressing (green) tumors before (d0) or 4 days after MYC

inactivation. (C, D) Relative mRNA expression, measured by qPCR, of Ang2 and Tie2 in control

MSCV (gray), CD47-expressing (blue), and PD-L1-expressing (green) tumors before (d0) and

after MYC inactivation (d4).

A

B Mean Vessel Density in Tumors

C D

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Fig 7:

Sustained expression of CD47 or PD-L1 inhibits cellular senescence upon MYC inactivation.

(A) Senescence-associated beta-gal assay in control MSCV, CD47-expressing, and PD-L1-

expressing tumors 0 or 4 days following MYC inactivation; cells stained blue are senescent.

(B) Quantification of senescent cells in control MSCV (gray), CD47 expressing (blue), and PD-

L1 expressing (green) tumors before (d0) or 4 days after MYC inactivation. (C, D) Relative

mRNA expression, measured by qPCR, of p15Ink4b and p19ARF in control MSCV (gray),

CD47-expressing (blue), and PD-L1-expressing (green) tumors before (d0) and after MYC

inactivation (d4).

C

A

Cellular Senescence in Tumors A B

D

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Fig 8:

Sustained expression of CD47 or PD-L1 does not affect apoptosis or proliferation upon MYC

inactivation. (A-B) Immunofluorescence in control MSCV, CD47 expressing, and PD-L1

expressing tumors 0 or 4 days following MYC inactivation for CC3, an apoptosis marker, (A),

and PH3, a proliferation marker detecting cells in metaphase (B). (C, D) Quantification of CC3+

(C) and PH3+ (D) cells in control MSCV (gray), CD47-expressing (blue), and PD-L1-expressing

(green) tumors before (d0) and after MYC inactivation (d4).

A

B C Apoptosis in Tumors Proliferation in Tumors