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

Decreased Fas expression level is correlated with the CD133⁺CD24^lo 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⁺CD24^lo and CD133⁺CD24^hi subsets of cells. Fas expression levels (MFI) of CD133⁺CD24^lo and CD133⁺CD24^hi cells were then determined and shown as overlays at the right. Shown are gating strategy and representative plots of one of three experiments. B, Fas MFI of CD133⁺CD24^lo and CD133⁺CD24^hi cells as shown in A were quantified and presented. Column, mean; bar, SD. Statistical differences were determined by Student t test.

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⁺CD24^lo and CD133⁺CD24^hi 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.

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 24 hours. 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 24 hours. 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⁺CD24^loFas^lo cell subsets were then quantified and presented at the right. D, The parent and FasL-resistant cell lines were cultured in ultra-low attachment tissue culture plates with serum-free DMEM 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).

FasL treatment increases CSC-like cells in MSS colon cancer cells. A and B, MSI (HCT116 and RKO; A) and MSS (SW480 and HT29; B) human colon carcinoma cell lines were cultured in the presence of FasL (50 ng/mL), Z-VAD (20 μmol/L), or both FasL and Z-VAD for 24 hours. Cells were then stained 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 and 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⁺CD24^lo cells were quantified. Top, representative flow cytometric dot plots. CD133⁺CD24^lo cells were quantified and presented at the bottom.

Fas^lo 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⁺CD24^loFas^lo and CD133⁺CD24^hiFas^hi cells. Showing is the gating strategy for sorting. B, The sorted CD133⁺CD24^loFas^lo and CD133⁺CD24^hiFas^hi cells were injected intravenously into BALB/c mice (1.5 × 10⁵ 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⁺CD24^loFas^lo and CD133⁺CD24^hiFas^hi cells. Showing is the gating strategy for sorting. D, The sorted CD133⁺CD24^loFas^lo and CD133⁺CD24^hiFas^hi cells were injected intravenously into C57BL/6 mice (3 × 10⁵ 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⁺CD24^loFas^lo (n = 6) and CD133⁺CD24^hiFas^hi (n = 7) MC38.met cells were injected into FasL-deficient fasl^gld mice (3 × 10⁵ cells/mouse) intravenously. 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⁺CD24^loFas^lo and CD133⁺CD24^hiFas^hi CT26 cells were injected intravenously into BALB/c mice (2 × 10⁵ cells/mouse, n = 5). Four days later, mice with treated with saline control or perforin-deficient pk03 CTLs (3 × 10⁵ cells/mouse). Mice were sacrificed on day 14 and analyzed as in B. G, CD133⁺CD24^loFas^lo and CD133⁺CD24^hiFas^hi CT26 cells were injected intravenously into BALB/c mice (2 × 10⁵ 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⁺CD24^loFas^lo and CD133⁺CD24^hiFas^hi CT26 cells were injected subcutaneously into BALB/c mice (2 × 10⁵ cells/mouse, n = 5). Ten days later, CD133⁺CD24^loFas^lo and CD133⁺CD24^hiFas^hi 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 2 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.