Loss of Fas Expression and Function Is Coupled with Colon Cancer Resistance to Immune Checkpoint Inhibitor Immunotherapy

Author Manuscript Published OnlineFirst on November 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0455 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Loss of Fas Expression and Function is Coupled with Colon Cancer Resistance to Immune Checkpoint Inhibitor Immunotherapy

Wei Xiao1#, Mohammed L Ibrahim1,2#, Priscilla S. Redd1,2,3, John D. Klement1,2,3, Chunwan Lu1,2,3, Dafeng Yang1,3, Natasha M. Savage4, and Kebin Liu1,2,3*

1Department of Biochemistry and Molecular Biology, and 2Georgia Cancer Center, Medical College of Georgia, Augusta, GA 30912, USA. 3Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. 4Department of Pathology, Medical College of Georgia, Augusta, GA 30912, USA.

# Equal contribution

Running title: Fas expression and colon cancer immune evasion

Key words: Fas, FasL, T cell immunotherapy, Colon cancer stem cell, MSI colon cancer, MSS colon cancer.

Conflict of interest: The authors have declared that no conflict of interest exists Grant support: NIH/NCI CA182518 and CA133085 (K.L.)

Word counts: 5,012

*Correspondence to: Kebin Liu, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA. Tel 706-721-9483, e-mail: [email protected].

Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0455 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Despite the remarkable efficacy of immune checkpoint inhibitor (ICI) immunotherapy in

various types of human cancers, colon cancer, except for the approximately 4% microsatellite-

instable (MSI) colon cancer, does not respond to ICI immunotherapy. ICI acts through activating

cytotoxic T lymphocytes (CTLs) that use the Fas-FasL pathway as one of the two effector

mechanisms to suppress tumor. Cancer stem cells are often associated with resistance to therapy

including immunotherapy, but the functions of Fas in colon cancer apoptosis and colon cancer

stem cells are currently conflicting and highly debated. We report here that decreased Fas

expression is coupled with a subset of CD133+CD24lo colon cancer cells in vitro and in vivo.

Consistent of the lower Fas expression level, this subset of CD133+CD24loFaslo colon cancer

cells exhibit decreased sensitivity to FasL-induced apoptosis. Furthermore, FasL selectively

enriches CD133+CD24loFaslo colon cancer cells. CD133+CD24loFaslo colon cancer cells exhibit

increased lung colonization potential in experimental metastatic mouse models, and decreased

sensitivity to tumor-specific CTL adoptive transfer and ICI immunotherapies. Interesting, FasL challenge selectively enriched this subset of colon cancer cells in microsatellite-stable (MSS) but

not in the MSI human colon cancer cell lines. Consistent with the down-regulation of Fas

expression in CD133+CD24lo cells, lower Fas expression level is significantly correlated with

decreased survival in human colon cancer patients.

Implications

Our data determine that CD133+CD24loFaslo colon cancer cells are capable to evade Fas-

FasL cytotoxicity of tumor-reactive CTLs and targeting this subset of colon cancer cells is

potentially an effective approach to suppress colon cancer immune evasion.

Introduction

CD8+ Cytotoxic T lymphocytes (CTLs) are the primary immune cells that kill target tumor cells in cancer immunosurveillance (1). CTLs use the Fas-FasL and perforin-granzyme

pathways as major effector mechanisms of cytotoxicity (2-4) and for the execution of tumor

rejection (5, 6). The perforin-granzyme pathway is essential for CTL suppression of established

tumors (7). Compelling experimental data have shown that the Fas-FasL apoptosis pathway also

plays an essential role in host cancer immunosurveillance (8-10) and in eradication of the

established tumors (5, 11-14).

Fas (also termed CD95 or APO-1) is a cell surface receptor of the tumor necrosis factor

receptor superfamily and is expressed in hematopoietic and non-hematopoietic cells including

tumor cells. Binding of Fas by its physiological ligand FasL induces Fas receptor trimerization,

followed by formation of the death-inducing signaling complex (DISC) at the cytoplasmic

domain of the Fas receptor, and subsequent cleavage of procaspase-8 at the DISC, leading to the

induction of Fas-mediated apoptosis (15-17). Fas receptor also mediates non-apoptotic signaling

pathways, and paradoxically Fas has been shown to promote tumor growth and progression (18,

19) and protect colon cancer stem cells (CSC) in certain tumor models (20). FasL is a member of

the tumor necrosis factor superfamily and is selectively expressed in activated T cells and NK

cells (21, 22). FasL can be either membrane-bound on the surface of activated T cells and NK

cells (mFasL) (23), or cleaved by metalloproteinases to exist as soluble protein (sFasL) (24). The

two different forms of FasL exhibit opposite functions. mFasL induces Fas-mediated apoptosis

and is essential for its cytotoxicity in cancer surveillance, whereas excess sFasL appears to

promote tumorigenesis through non-apoptotic activities (23). The mechanisms underlying the

contrasting functions of the Fas-FasL pathway in apoptosis and colon CSC differentiation is

currently unknown.

Immune checkpoint inhibitor (ICI) cancer immunotherapy has produced durable efficacy

in various human hematological malignancies and solid tumors. ICIs suppress the interaction of

T cell inhibitory receptors with their cognate ligands on tumor or stromal cells to unleash CTL-

mediated cytotoxicity to kill tumor cells (25). However, human colorectal cancer, except for the

small subset of microsatellite instable (MSI) tumor, does not respond to ICI immunotherapy

(26). The mechanism underlying colorectal cancer non-response to ICI immunotherapy is

currently highly debated (27, 28). CSCs, including colon CSCs, are often the major cause of

resistance to therapies (29, 30). Considering the essential role of the Fas-FasL in CTL-mediated anti-tumor cytotoxicity (1-5, 8-10, 12-14, 31), determining the functions of Fas in colon CSC-

like cells is thus of significance for colorectal cancer immunotherapy (31). Here, we made use of

a recombinant FasL trimer protein that mimics mFasL, and mice with only Fas or FasL

deficiency to determine the relationship between the Fas-FasL pathway and colon CSC

phenotypes. Using these defined and physiologically relevant systems, we determined that Fas is

essential for tumor cells apoptosis and that mFasL selectively enriches CD133+CD24loFaslo

subset of colon cancer cells that are potentially CSC-like cells. This subset of

CD133+CD24loFaslo colon cancer cells exhibit decreased sensitivity to Fas-mediated apoptosis,

increased growth potential, and increased resistance to CTL adoptive transfer and ICI

immunotherapies.

Materials and Methods

Cell lines: The murine CT26, human HCT116, HT29, RKO, SW480, and SW620 colon carcinoma cell lines were obtained from American Type Culture Collection (Manassas, VA) in

2013 and stored in aliquots in liquid nitrogen. Cells were used within 30 passages. Murine colon carcinoma MC38, MC38.met, and MC32a cell lines were provided by Dr. Jeffrey Schlom

(National Cancer Institute, Bethesda, MD) and were characterized previously (32, 33). Sarcoma

MC78 and MC693 cell lines were generated from tumor-bearing C57BL/6 mice, and sarcoma

MC68 and MC69 cell lines were generated from tumor-bearing faslpr mice as previously described (34). All cell lines are tested for mycoplasma every 2 months and all cells used in this study were mycoplasma-negative.

Mouse tumor models: BALB/c and C57BL/6 mice were obtained from Charles River Frederick

Facility (Frederick, MD). Faslgld (B6Smn.C3-Faslgld/J) and faslpr (B6.MRL-Faslpr/J) mice were obtained from Jackson Laboratory (Bar Harbor, ME). To induce spontaneous sarcoma, C57BL/6, faslpr mice were injected subcutaneously (s.c.) with 3-methylcholanthrene (MCA, 100 g/mouse) in peanut oil. Tumors were dissected from the mice and digested with collagenase solution (1 mg/ml collagenase, 0.1 mg/ml hyaluronidase, and 30 U/ml DNase I) to make single cell suspension. Cells were cultured to establish stable cell lines. The cultured cells were pelleted, fixed in formalin, embedded in paraffin, and analyzed histologically by a board certified

Pathologist (N.M.S.). To establish s.c. tumors, BALB/c (for CT26 cells) and C57BL/6 (for

MC32a and MC38 cells) were inoculated in the right unilateral flank with 2.5×105 tumor cells in

Hanks's Buffered Saline Solution. Tumor-bearing mice were sacrificed when the tumor reaches

approximately 150 mm3 in size. Tumor tissues were excised and digested with collagenase solution. For the experimental lung metastasis model, sorted subsets of CT26 (1.5 x105 cells/mouse) and MC38.met (3 x105 cells/mouse) cells were injected i.v. into BALB/c (CT26 cells), and C57BL/6 and faslgld (MC38.met cells) mice, respectively. Fourteen days later, mice were sacrificed and injected with ink to inflate the tumor-bearing lungs as described (35). All animal studies were performed in compliance with a protocol (2008-0162) approved by Augusta

University Institutional Animal Care and Use Committee.

CTL adoptive transfer and anti-PD-1 mAb immunotherapy. For adoptive transfer immunotherapy, tumor-bearing mice were injected i.v. with the tumor-specific perforin-deficient pk03 CTLs (14). For anti-PD-1 immunotherapy, tumor-bearing mice were treated with IgG (200

g/mouse) or anti-PD-1 mAb (clone; 29F.1A12, 200 g/mouse) every 2 days for 5 times.

Cell sorting: Cell sorting was performed as previously described (36). Briefly, cells were stained with CD133-, CD24-, and Fas-specific mAbs (BioLegend). Stained cells were sorted using a BD

FACSAria II SORP or a Beckman Coulter MoFlo XDP cell sorter to isolate cell subsets.

Recombinant FasL protein. Mega-Fas Ligand (kindly provided by Dr. Peter Buhl Jensen at

Oncology Venture A/S, Denmark) is a recombinant fusion protein that consists of three human

FasL extracellular domains linked to a protein backbone comprising the dimer-forming collagen domain of human adiponectin. The Mega-Fas Ligand was produced as a glycoprotein in

mammalian cells using Good Manufacturing Practice compliant process in Topotarget A/S

(Copenhagen, Denmark).

Selection of Fas-resistant cell line: Tumor cells were cultured in the presence of increasing

concentrations of FasL (5, 10, 25, 50, and 200 ng/ml). Cells that survived 200 ng/ml FasL are

maintained as FasL-resistant cell lines.

Fas overexpression. SW480-FasL-R cells were transfected with pLNCX2 or Fas-coding

sequence-containing pLNCX2 (provided by Dr. Richard Siegel, National Institutes of Health,

Bethesda, MD), and selected for stable cell lines SW480-FasLR-Vector SW480-FasLR-Fas.

Tumor cell apoptosis assay: Cells (1×105 cells/well) were seed in 24-well plates in complete

RPMI-1640 media with 10% fetal bovine serum. Recombinant FasL was added into cell culture

and incubated for 24 to 72 hours. Both attached and non-attached cells were harvested, washed in

phosphate-buffered saline (PBS), suspended in Annexin V binding buffer (10 mM Hepes, pH

7.4, 140 mM NaCl, 2.5 mM CaCl2) and incubated with APC-conjugated Annexin V for 30 min.

Propidium iodide (PI) was then added and incubated for another 5 min. Stained cells were

analyzed by flow cytometry. Apoptosis is expressed as % Annexin V+ PI- cells, and apoptotic

cell death is expressed as %Annexin V+ PI+ cells. Genomic DNA was isolated from cells and

analyzed in 1.5% agarose gels.

3H-Thymidine incorporation assay: Cells were cultured in 96-well plates in the absence or

presence of recombinant FasL for 72 hours. 3H- thymidine (1 μCi/well, Amersham Corporation,

USA) was then added to the culture and cultured for another 5 hours. Cells were detached with

10 mM EDTA and then were transferred onto the nitrocellulose membrane. 3H incorporation was quantified in a Microplate Scintillation and Luminescence Counter (PerKinElmer 1450

LSC).

Analysis of subsets of cells by flow cytometry: Cell surface marker staining was performed as

previously described (36). Briefly, cells were incubated with fluorescence-conjugated antibodies

diluted in FACS buffer (2% BSA in PBS buffer) on ice for 15 min. After washing, cells were

acquired using BD LSR II or BD AccriTM C6 Flow Cytometers (BD Biosciences). Monoclonal

antibodies used for cell surface staining were: PE-anti-human CD133 (293C3), PECy7-anti-

human CD24 (ML5), APC-anti-human Fas (DX2), and PE-mouse IgG2b (27-35) and PE/Cy7-

Mouse IgG2a (MOPC-173) isotype controls, which were purchased from Miltenyi Biotec or

BioLegend. Data files were analyzed using FlowJo.V10 software. Dead cells were excluded by

7AAD or DAPI staining. All flow cytometric analyses are done on live singlets cells only.

Tumor cell sphere formation assay. Sphere formation assay was performed as previously

described (37). Briefly, cells were cultured in serum-free DMEM medium plus 20 ng/ml EGF

and 10 ng/ml basic FGF (Pepro Tech) in ultra-low attachment surface 96-well tissue culture flat

bottom plates (Cat# 3474, Costar, Kennebunk, ME).

Transwell and scratch wound healing assay. Cell Migration was assessed using a 24 well plate

with transwell insert (Falcon, Corning, NY). The insert and the plate surface were coated with

0.1% gelatin. Cells were suspended in 200 µl 1% FBS-containing medium and then added to the

upper chamber at a density of 6×104 cells/insert and 700 µl 1% FBS-medium was added to the

lower chamber. Cells were cultured for 16 h, fixed in 4% PFA and the number of migrant cells to

the lower side of the insert was counted under a microscope after staining with crystal violet. For scratch wound healing assay, cells were cultured in 6-well plate to 90-100% confluence. Cell

monolayer was scratched with a pipet tip, washed with PBS and supplied with new medium.

Scratched areas were measured at 0 and 24 h. Scratch closure rate was calculated with the

formula: scratch closure rate (µm/h) = initial scratch width-final scratch width/time.

Statistical analysis: Data are expressed as mean ± SD. Statistical analysis was performed using

ANOVA and paired Student’s t-test.

Results

Fas expression level is decreased in CD133+CD24lo subset of colon cancer cells.

Fas expression level in subsets of colon cancers was compared in murine colon

carcinoma CT26, MC32a and MC38 cell lines and human colon carcinoma HCT116 and SW480

cell lines. CD133+ and CD24lo were used to define the phenotype of colon CSC-like cells (20,

37, 38). Fas protein level, as measured by cell surface Fas protein staining mean fluorescence intensity (MFI), is significantly lower in the CD133+CD24lo subset of colon cancer cells as

compared to the CD133+CD24hi cells in all three murine colon carcinoma cell lines in vitro (Fig.

1A and B). Similarly, Fas protein level is also significantly lower in the CD133+CD24lo subset of

colon cancer cells as compared to the CD133+CD24hi cells in both human colon carcinoma cell

lines in vitro (Fig. 1A & B).

To determine whether the above observations can be extended to colon cancer in vivo,

CT26 cells were injected to BALB/c mice, and MC32a and MC38 cells were injected to

C57BL/6 mice, respectively. Tumor tissues were dissected from the tumor-bearing mice and

analyzed for Fas expression in subsets of colon cancer cells. The level of CD133+CD24lo subset

of tumor cell population in vivo is similar to that in vitro in all three cell lines (Fig. 2A). As in

the in vitro cultured cells, Fas expression level in CD133+CD24lo subset of cells is significantly

lower as compared to the CD133+CD24hi subset of cells in the colon tumor tissues in vivo (Fig.

2B).

Fas receptor has been shown to mediate both apoptosis and survival signaling pathways

(39, 40). We next made use of a physiologically relevant FasL, the MegaFasL, to determine

whether Fas mediates apoptosis in colon carcinoma cells. CT26 cells are resistant to FasL (Fig.

S1A & B). However, MC32a, MC38, HCT116 and SW480 cells are all sensitive to FasL and

exhibited a dose-dependent response in terms of apoptosis (Fig. S1A & B).

To validate the specificity of FasL-induced apoptosis, we next used WT and Fas-deficient

tumor cell lines. These cells are high grade sarcoma cells with active mitotic activity (Fig. S2A).

As expected, the WT tumor cell lines MC78 and MC693 are sensitive to FasL-induced apoptosis

in a dose-dependent manner. In contrast, Fas-deficient MC68 and MC69 cell lines are resistant to

FasL-induced apoptosis (Fig. S2B & C). FasL also suppressed proliferation in MC78 and

MC693 cell lines but not in MC68 and MC69 cell lines (Fig. S2D). Taken together, these

observations indicate that FasL induce tumor cell apoptosis through the Fas-mediate apoptosis

pathway and Fas expression is down-regulated in the CD133+CD24lo colon CSC-like cells.

Colon CSC-like cells exhibit reduced sensitivity to FasL-induced apoptosis in vitro.

The decreased Fas expression level in the subset of CD133+CD24lo cells suggests a

decreased sensitivity to Fas-mediated apoptosis in the colon CSC-like cells. To test this

hypothesis, the murine MC32a and human HCT116 cells were treated with FasL and analyzed

for apoptosis. CD133+ cells were gated into CD24hi and CD24lo populations and analyzed

apoptotic cells (Fig. 3A and C). Apoptosis is significantly lower in the CD133+CD24lo subset of

cells than in the CD133+CD24hi subset of cells in both MC32a and HCT116 cell lines (Fig. 3B &

D). These observations indicate that colon CSC-like cells have reduced sensitivity to apoptosis

induction by FasL.

FasL selectively enriches colon CSC-like cells

The above observation suggests that the CD133+CD24loFaslo subset of colon cancer cell

might be a consequence of immune selection of FasL+ CTLs during tumorigenesis (41). To test

this hypothesis, we used a defined in vitro culture system to culture SW480, RKO and HCT116

cells with increasing concentrations of FasL and generated a FasL-resistant tumor cell sublines.

All three FasL-selected cell lines exhibited decreased sensitivity to FasL-induced apoptosis as

determined by analyzing early apoptosis (Annexin V+ PI-) and apoptotic cell death (Annexin V+

PI+) (Fig. 4A), as well as genomic DNA fragmentation (Fig. 4B). Flow cytometry analysis

revealed that FasL selection significantly enriches CD133+CD24loFaslo cells (Fig. 4C).

Furthermore, all three FasL-selected tumor cell lines exhibited acquired sphere formation

capability (Fig. 4D). These data thus determine that FasL selectively enriches colon CSC-like

cells with a CD133+CD24loFaslo phenotype.

MSS colon carcinoma cells respond to FasL to differentiate to CSC-like cells

MSI but not MSS colon carcinoma cells respond to ICI immunotherapy (26). FasL-

mediated cytotoxicity is a major effector mechanism of ICI-activated CTL anti-tumor

cytotoxicity. Our above data indicate that decreased Fas expression level and sensitivity to FasL-

induced apoptosis is potentially a phenotype of colon CSC-like cells. We therefore reasoned that

the CSC-like cell population may contribute to different MSI and MSS response to FasL. To test this hypothesis, we analyzed responses of MSI (HCT116 and RKO) and MSS (SW480 and

HT29) human colon carcinoma cell lines (42) to FasL. FasL induced apoptosis in all four cell

lines regardless of MSI and MSS phenotypes (Fig. 5A and B). The pan-caspase activation

blocker Z-VAD inhibited FasL-induced apoptosis, suggesting a caspase-dependent apoptosis

induction by FasL (Fig. 5A & B). Analysis of CD133+CD24lo cell population revealed that FasL

treatment did not significantly enrich the CSC-like cell level in the two MSI cell lines. However,

FasL treatment significantly increased the CD133+CD24lo subset of cells in the two MSS colon

carcinoma cell lines (Fig. 5C & D). Furthermore, inhibition of caspase activation blocked both

apoptosis and FasL-induced enrichment of CD133+CD24lo colon CSC-like cells (Fig. 5B and

5D). Our data thus suggest that FasL enriches CD133+CD24lo colon CSC-like cells and doing so

in a caspase-dependent manner.

IFN and TNF both regulate Fas expression and enhance tumor cell response to Fas-

mediated apoptosis. To determine whether IFN and TNF regulates FasL selection and

enrichment of colon CSC-like cells, HCT116 and SW480 cells were pre-treated with IFN and

TNF, followed by FasL treatment. As observed above, FasL increased the level of

CD133+CD24lo cell population in the MSS SW480 but not in the MSI HCT116 cells (Fig. S3A).

IFN and TNF treatment did not significantly change FasL-induced enrichment of

CD133+CD24lo in both HCT116 and SW480 cells (Fig. S3).

CD133+CD24loFaslo colon cancer cells exhibit increased resistance to T cell

immunotherapy.

To determine tumor invasiveness in vivo, we sorted CD133+CD24loFaslo and

CD133+CD24hiFashi from both CT26 (Fig. 6A) and MC38.met (Fig. 6C) cells and injected these

cells to mice and compared their colonization efficacy in lungs in an experimental metastasis

system. CD133+CD24loFaslo cells exhibited significantly higher lung colonization potential in

both the CT26 (Fig. 6B) and MC38.met (Fig. 6D) mouse models. This increased invasiveness is

validated by in vitro tumor cell migration and scratch wound healing assays. CD133+CD24loFaslo

CT26 cells exhibited significantly higher migration and scratch wound healing capability than the CD133+CD24hiFashi CT26 cells (Fig. S4).

To determine the contribution of the Faslo phenotype in the colonization potential,

CD133+CD24loFaslo and CD133+CD24hiFashi MC38.met cells were injected to FasL-deficient mice. The rationale is that if Fas-FasL interaction mediates tumor cell colonization potential,

then these two subsets of tumor cells should colonize in lungs of FasL-deficient mice at similar

rate. Indeed, no significant difference was observed in the tumor nodule numbers between the

two groups of mice that received CD133+CD24loFaslo and CD133+CD24hiFashi cells (Fig. 6E).

To determine the response of CD133+CD24loFaslo and CD133+CD24hiFashi tumor cells to

immunotherapy, we treated tumor-bearing mice with a tumor-specific and perforin-deficient

CTL line. The rationale is that the CD133+CD24loFaslo tumor cells are relatively more resistant to

FasL, this subset of cells should be less susceptible to perforin-deficient CTLs. Indeed, the

CD133+CD24loFaslo CT26 tumor exhibited significantly less response to the perforin-deficient

CTLs as compared to CD133+CD24hiFashi CT26 tumor (Fig. 6F). We then sought to determine

the response of these two subsets of cells to ICI immunotherapy. It is clear that

CD133+CD24loFaslo tumor is significantly less responsive to anti-PD-1 mAb immunotherapy

than the CD133+CD24hiFashi CT26 tumor in both the experimental lung metastasis model and the

s.c. tumor model (Fig. 6G-I). These observations indicate that a CD133+CD24loFaslo subset of

colon cancer cells is at least partially responsible for colon cancer resistance to ICI

immunotherapy.

Lower Fas expression level is correlated with decreased survival in human colorectal cancer patients

The Fas-FasL pathway plays an essential role in host cancer immunosurveillance. Our

above observations suggest that decreased Fas expression may be a key phenotype of colon

CSC-like cells and may contribute to patient disease outcome. To test this hypothesis, we

examined Fas expression levels in human colorectal carcinoma. Kaplan-Meier survival analysis

for Fas mRNA level revealed that Fas expression level is positively correlated with increased

survival in human colorectal cancer patients (Fig. 7). Therefore, it is clear that colon CSCs may

use down-regulation of Fas expression to increase resistance to FasL-induced apoptosis to evade host cancer immunosurveillance in human colon cancer patients.

Discussion

The major and best known function of Fas is apoptotic cell death (39). However, Fas also

mediates non-apoptotic signaling pathways and has been shown to promote tumor growth in

certain tumor models (18, 19, 43). Under physiological conditions, Fas is expressed virtually in

all types of cells, whereas FasL is selectively expressed on the surface of activated T cells and

NK cells (4, 21). Under pathological conditions such as cancer, FasL is also abundantly

expressed in tumor cells in the cytoplasm (20) and are often secreted as sFasL by tumor cells (21,

44), but not on tumor cell surface as membrane-bound form. It has been well demonstrated that

membrane-bound FasL induces apoptosis whereas excess sFasL induce non-apoptotic activities

and promote cellular proliferation (23). Therefore, it is not surprising that Fas may promote

tumor growth and progression under certain pathological conditions (18, 19). Using a

recombinant FasL protein that structurally mimics the mFasL trimer, we observed that two of the

three murine and three of the three human colon cancer cell lines are sensitive to FasL-induced

apoptosis. Fas is weakly expressed on CT26 cells. CT26 cells, as expected, do not respond to

FasL to undergo apoptosis. This phenomenon is validated in the Faslpr tumor cell lines. Although

the Faslpr mice lack an increase in spontaneous tumor development (45), Faslpr mice are more

susceptible to carcinogen induction of spontaneous tumorigenesis (34). We demonstrated here

that loss of Fas function in the Faslpr tumor cell line abolishes tumor cell sensitivity to FasL-

induced apoptosis. In contrast to promotion of cellular proliferation by sFasL, we also observed that FasL suppresses tumor cell proliferation through a Fas-dependent mechanism. These data thereby validate the role of Fas in apoptotic cell death of tumor cells.

In this study, we also observed that a decreased Fas expression level is linked to the

CD133+CD24lo colon CSC-like cell phenotype in mouse and human colon carcinoma cell lines

in vitro and in mouse colon carcinoma in vivo. We observed that CD133+CD24lo colon CSC-like

cells are less sensitive to FasL-induced apoptosis as compared to the CD133+CD24hi cells. This

observation is consistent with the report that CSC have decreased sensitivity to Fas-mediated

apoptosis (20). However, it is unlikely that Fas plays a direct role in colon CSC-like maintenance

since overexpression of Fas in FasL-resistant tumor cells did not alter colon tumor cell sphere

formation (Fig. S5). It has been shown that Fas and FasL, especially tumor cell produced FasL,

promote and protect CSCs (20, 46). Interestingly, we observed that although FasL induces

apoptosis in both MSI and MSS colon carcinoma cells, FasL increases colon CSC-like cells only

in MSS human colon carcinoma cell lines but not in the MSI human colon carcinoma cell lines.

Furthermore, we observed that FasL also selectively enrich the Fas resistant cell subsets that

have a colon CSC-like phenotype. Therefore, it appears that FasL may promote colon CSC both

through inducing colon CSC-like cell differentiation and through eliminating the Fas-sensitive

cells to selectively enrich the Fas-resistant cells with a colon CSC-like phenotype. The

expression of FasL, the physiological ligand for Fas, is primarily restricted to activated T cells

(2, 4, 8, 23). We observed that the CD133+CD24loFaslo and CD133+CD24loFashi colon cancer

cells exhibit significant difference in lung colonization efficiency in the immune competent mice

(Fig. 6D) but not in the FasL-deficient mice (Fig. 6E)., which suggest that host immune cells

modulate these two subsets of colon CSC-like cells. One limitation of this study is thus that the

stemness of the CD133+CD24loFaslo colon cancer cells and the function of Fas in stemness

cannot be determined by the conventional limited dilution assay in immune-deficient mice.

Therefore, whether Faslo phenotype is a colon cancer stem cell marker remains to be determined.

Nevertheless, the CD133+CD24loFaslo subset of cells may represent colon CSC-like cells.

MSI human colorectal cancer but not MSS human colorectal cancer responds to ICI

immunotherapy (26). It is known that CSCs are often the source of resistance to therapy

including chemotherapy and radiotherapy (29, 47). We determined here that the

CD133+CD24loFaslo colon CSC-like cells are more resistant to CTL adoptive transfer immunotherapy and to ICI immunotherapy. The link between colon CSCs and MSS colon cancer is not clear. FasL is one of the two lytic mechanisms that CTLs use to induce tumor cell apoptosis to suppress tumor growth and progression (1-5, 8-10, 12-14, 31). Our observation that

FasL treatment induces colon CSC-like cells in human colon cancer cell lines suggests that MSS

human colon carcinoma cells might use an increase in CSCs to respond to FasL to acquire a Fas-

resistant phenotype. Therefore, in addition to its potent tumor suppression activity, tumor-

reactive CTL FasL may also induce colon CSC differentiation and selectively enrich Faslo colon

CSCs through eliminating Fas-sensitive non-stem cells, resulting in immunoselection (41) and

accumulation of Faslo colon CSCs which underlies MSS colon cancer resistance to ICI

immunotherapy. Considering the significance of MSS/MSI phenotype in ICI immunotherapy and

colon cancer stem cell in resistance to therapies, further studies are warranted to further

determine the role of Fas in colon cancer stem cell pathogenesis and resistance to ICI

immunotherapy in human colorectal cancer patients (31).

Acknowledgement

We thank Dr. Jeanene Pihkala at the Medical College of Georgia Flow Cytometry Core Facility and Dr. Ningchun Xu at Georgia Cancer Center Flow Cytometry Core Facility for assistance in cell sorting.

Author contributions W.X, J.D.K., C.L., M.L.I, D.Y., and P.S.R.: performed experiments and developed methods

concept development and overall study designs. N.M.S.: data analysis. W.X., and K.L., wrote

then manuscript.

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Figure legends

Figure 1. Decreased Fas expression is linked to a CD133+CD24lo colon CSC-like cell

phenotype in vitro. A. Murine (CT26, MC32a, and MC38) and human (HCT116 and SW480)

colon carcinoma cells were stained with DAPI, and with CD133-, CD24- and Fas-specific mAbs,

respectively. The DAPI- live cells were gated and analyzed for CD133+CD24lo and

CD133+CD24hi subsets of cells. Fas expression levels (MFI) of CD133+CD24lo and

CD133+CD24hi cells were then determined and shown as overlays at the right panel. Shown are

gating strategy and representative plots of one of three experiments. B. Fas MFI of

CD133+CD24lo and CD133+CD24hi cells as shown in A were quantified and presented. Column:

mean; Bar: SD. Statistical differences were determined by student t test.

Figure 2. Decreased Fas expression level is correlated with the CD133+CD24lo colon CSC-

like cell phenotype in vivo. A. Murine (CT26, MC32a, and MC38) colon carcinoma cells were

injected subcutaneously to BALB/c (CT26) and C57BL/6 (MC32a and MC38) mice,

respectively. Tumors were dissected from the tumor-bearing mice and digested with collagenase

solution to prepare single cell mixtures. The cell mixtures were stained with 7AAD, and with

CD45.2-, CD133-, CD24-, and Fas-specific mAbs, respectively. The 7AAD- live cells and

CD45.2- tumor cells were then gated out and analyzed for CD133+CD24lo and CD133+CD24hi subsets of cells. Fas expression levels (MFI) of CD133+CD24lo and CD133+CD24hi cells were

then determined and shown as overlays at the right panel. Shown are gating strategy and representative plots of one of three experiments. B. Fas MFI of CD133+CD24lo and

CD133+CD24hi cells as shown in A were quantified and presented. Column: mean; Bar: SD.

Statistical differences were determined by student t test.

Figure 3. Colon CSC-like cells exhibit decreased sensitivity to Fas-mediated apoptosis.

Murine MC32a (A) and human HCT116 (C) colon carcinoma cells were treated with Fas ligand

(50 ng/ml) for 24 hours. Shown are representative flow cytometric dot-plots of the gating

strategy. Apoptosis (Annexin V+DAPI-) of CD133+CD24lo and CD133+CD24hi cells were

determined by flow cytometry. Apoptosis (Annexin V+DAPI-) of the two subsets of cells in

MC32a (B) and HCT116 (D) cells were then quantified. Data are representatives of three independent experiments. Column: mean; Bar: SD. Statistical differences were determined by

student t test.

Figure 4. FasL selection enriches colon CSC-like cells. A. SW480, RKO, HCT116, and the

respective FasL-resistant cell lines as indicated were cultured in the presence of FasL at the

indicated concentrations for 24h. Cells were stained with PI and annexin V. Early apoptosis

(Annexin V+PI-) and apoptotic cell death (Annexin V+PI+) were quantified. B. The three pairs of

parent and FasL-resistant cell lines were either untreated or treated with FasL (200 ng/ml) for

24h. Genomic DNA was isolated from the cells and analyzed by 1.5% agarose gel

electrophoresis. C. The three pairs of parent and FasL-resistant cell lines were stained with

CD133-, CD24-, and Fas specific mAbs and analyzed by flow cytometry. Shown are

representative plots of CD133 and CD24 phenotypes (left two panels). CD133+CD24loFaslo cell

subsets were then quantified and presented at the right panel. D. The parent and FasL-resistant

cell lines were cultured in ultra-low attachment tissue culture plates with serum-free DMEM

medium supplemented with EGF (20 ng/ml) and basic FGF (10 ng/ml), respectively, for 10 days.

Shown are representative images of cell morphology of three independent experiments. a1

SW480, a2: SW480-FasLR, b1: RKO, b2: RKO-FasLR, c1: HCT116, C2: HCT116-FasLR.

Figure 5. FasL treatment increases CSC-like cells in MSS colon cancer cells. A & B. MSI

(A. HCT116 and RKO) and MSS (B. SW480 and HT29) human colon carcinoma cell lines were

cultured in the presence of FasL (50 ng/ml), Z-VAD (20 M) or both FasL and Z-VAD for 24h.

Cells were then stained with with PI and annexin V. Top panel shows representative flow cytometry dot plots. Apoptosis (Annexin V+PI-) and apoptotic cell death (Annexin+PI+) were quantified and presented at the bottom panel. Column: mean; Bar: SD. Data are representative result of one of two independent experiments. C & D. The MSI and MSS human colon

carcinoma cell lines were treated as in A and B and then stained with CD133- and CD24-specific

mAbs and analyzed by flow cytometry. CD133+CD24lo cells were quantified. Top panel shows

representative flow cytometric dot plots. CD133+CD24lo cells were quantified and presented at

the bottom panel.

Figure 6. Faslo colon CSC-like cells exhibit a higher lung colonization potential and

resistance to T cell immunotherapy. A. CT26 cells were stained with CD133-, CD24- and Fas-

specific mAbs and sorted into CD133+CD24loFaslo and CD133+CD24hiFashi cells. Showing is the

gating strategy for sorting. B. The sorted CD133+CD24loFaslo and CD133+CD24hiFashi cells were

injected i.v. into BALB/c mice (1.5x105 cells/mouse, n=5). Fourteen days later, mice were sacrificed and india ink was perfused into the lung. The ink-inflated lungs were fixed. Shown are

tumor-bearing lungs. The tumor nodule number was counted and presented at the right.

Statistical significance was determined by student t test. C. MC38.met cells were stained with

CD133-, CD24- and Fas-specific mAbs and sorted into CD133+CD24loFaslo and

CD133+CD24hiFashi cells. Showing is the gating strategy for sorting. D. The sorted

CD133+CD24loFaslo and CD133+CD24hiFashi cells were injected i.v. into C57BL/6 mice (3x105

cells/mouse, n=5). Fourteen days later, mice were sacrificed and india ink was perfused into the

lung. The ink-inflated lungs were fixed. Shown are tumor-bearing lungs. The tumor nodule

number was counted and presented at the right. E. Sorted CD133+CD24loFaslo (n=6) and

CD133+CD24hiFashi (n=7) MC38.met cells were injected into FasL-deficient faslgld mice (3x105

cells/mouse) i.v. Fourteen days later, mice were sacrificed and india ink was perfused into the

lung. The ink-inflated lungs were fixed. Shown are tumor-bearing lungs. The tumor nodule

number was counted and presented at the bottom. F. CD133+CD24loFaslo and

CD133+CD24hiFashi CT26 cells were injected i.v. into BALB/c mice (2x105 cells/mouse, n=5).

Four days later, mice with treated with saline control or perforin-deficient pk03 CTLs (3x105 cells/mouse). Mice were sacrificed on day 14 and analyzed as in B. G. CD133+CD24loFaslo and

CD133+CD24hiFashi CT26 cells were injected i.v. into BALB/c mice (2x105 cells/mouse, n=5).

Four days later, mice were treated with IgG (200 mg/mouse) or anti-PD-1 mAb (200 mg/mouse)

every 2 days for 5 times. Lung tumors were analyzed as in B. H. CD133+CD24loFaslo and

CD133+CD24hiFashi CT26 cells were injected s.c. into BALB/c mice (2x105 cells/mouse, n=5).

Ten days later, CD133+CD24loFaslo and CD133+CD24hiFashi CT26 tumor-bearing mice were

randomly grouped into two groups, respectively, and treated with IgG or anti-PD-1 mAb as in G

every 2 days for 5 times. Mice were sacrificed two days after the last treatment. Shown are tumor

images. I. The sizes and weights of tumors as shown in H were measured and analyzed by

student t test.

Figure 7. Decreased Fas expression is correlated with decreased survival in human

colorectal cancer patients. Fas mRNA levels were extracted from the TCGA database and

plotted against survival in human colorectal patients.

Loss of Fas Expression and Function is Coupled with Colon Cancer Resistance to Immune Checkpoint Inhibitor Immunotherapy

Wei Xiao, Mohammed L. Ibrahim, Priscilla S Redd, et al.

Mol Cancer Res Published OnlineFirst November 14, 2018.

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