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Molecular Cancer Research
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Cell Death and Survival

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, John D. Klement, Chunwan Lu, Dafeng Yang, Natasha M. Savage and Kebin Liu
Wei Xiao
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.
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Mohammed L. Ibrahim
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.
2Georgia Cancer Center, Medical College of Georgia, Augusta, Georgia.
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Priscilla S. Redd
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.
2Georgia Cancer Center, Medical College of Georgia, Augusta, Georgia.
3Charlie Norwood VA Medical Center, Augusta, Georgia.
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John D. Klement
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.
2Georgia Cancer Center, Medical College of Georgia, Augusta, Georgia.
3Charlie Norwood VA Medical Center, Augusta, Georgia.
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Chunwan Lu
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.
2Georgia Cancer Center, Medical College of Georgia, Augusta, Georgia.
3Charlie Norwood VA Medical Center, Augusta, Georgia.
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Dafeng Yang
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.
3Charlie Norwood VA Medical Center, Augusta, Georgia.
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Natasha M. Savage
4Department of Pathology, Medical College of Georgia, Augusta, Georgia.
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Kebin Liu
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.
2Georgia Cancer Center, Medical College of Georgia, Augusta, Georgia.
3Charlie Norwood VA Medical Center, Augusta, Georgia.
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  • ORCID record for Kebin Liu
  • For correspondence: Kliu@augusta.edu
DOI: 10.1158/1541-7786.MCR-18-0455 Published February 2019
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  • Figure 1.
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    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.

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    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. 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.

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    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.

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    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 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+CD24loFaslo 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).

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    Figure 5.

    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+CD24lo cells were quantified. Top, representative flow cytometric dot plots. CD133+CD24lo cells were quantified and presented at the bottom.

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    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 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+CD24loFaslo and CD133+CD24hiFashi cells. Showing is the gating strategy for sorting. D, The sorted CD133+CD24loFaslo and CD133+CD24hiFashi 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+CD24loFaslo (n = 6) and CD133+CD24hiFashi (n = 7) MC38.met cells were injected into FasL-deficient faslgld 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+CD24loFaslo and CD133+CD24hiFashi 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+CD24loFaslo and CD133+CD24hiFashi 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+CD24loFaslo and CD133+CD24hiFashi CT26 cells were injected subcutaneously into BALB/c mice (2 × 105 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 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.

  • Figure 7.
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    Figure 7.

    Decreased Fas expression is correlated with decreased survival in patients with human colorectal cancer. Fas mRNA levels were extracted from the TCGA database and plotted against survival in human patients with colorectal cancer.

Additional Files

  • Figures
  • Supplementary Data

    • Supplementary Figure Legend - Supplemental Figure Legends 1-5
    • Figure S1 - Figure S1. Sensitivity of colon carcinoma cell lines to FasL-induced apoptosis in vitro.
    • Figure S2 - Loss of Fas function leads to resistance to FasL-induced apoptosis.
    • Figure S3 - FasL induces apoptosis to increase the frequency of colon cancer stem cell-like cells in MSS colon cancer cells.
    • Figure S4 - CD133+CD24loFaslo colon cancer stem cell-like cells exhibit increased invasiveness in vitro.
    • Figure S5 - Overexpression of Fas does not alter sphere formation.
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Molecular Cancer Research: 17 (2)
February 2019
Volume 17, Issue 2
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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, John D. Klement, Chunwan Lu, Dafeng Yang, Natasha M. Savage and Kebin Liu
Mol Cancer Res February 1 2019 (17) (2) 420-430; DOI: 10.1158/1541-7786.MCR-18-0455

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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, John D. Klement, Chunwan Lu, Dafeng Yang, Natasha M. Savage and Kebin Liu
Mol Cancer Res February 1 2019 (17) (2) 420-430; DOI: 10.1158/1541-7786.MCR-18-0455
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