MFSD2A suppresses CD8+ T cell exhaustion in colorectal cancer by inhibiting ERK/p38 MAPK-PD-L1 signaling

Figure 1 Major facilitator superfamily domain containing 2A inhibits the malignant progression of colorectal cancer cells in vitro. A: Western blotting analysis of major facilitator superfamily domain containing 2A (MFSD2A) expression in normal colonic epithelial cells, Caco-2, SW480, and MC38 cells; B: Validation of MFSD2A overexpression and knockdown by western blotting; C: The relative MFSD2A expression level in cells was determined by quantitative polymerase chain reaction; D: Cell proliferation was determined using the cell counting kit-8 assay; E and F: Apoptosis detected using allophycocyanin Annexin V/propidium iodide flow cytometry; G and H: Wound healing assays were performed to evaluate the migration capability of MFSD2A-overexpressing MC38 and MFSD2A-knockdown Caco-2 cells; I and J: Transwell assays were performed to evaluate the invasion capability of MFSD2A-overexpressing MC38 and MFSD2A-knockdown Caco-2 cells. ^aP < 0.0001 vs overexpression negative control, ^bP < 0.01 vs overexpression negative control, ^cP < 0.0001 vs siRNA negative control, and ^dP < 0.01 vs siRNA negative control. MFSD2A: Major facilitator superfamily domain containing 2A; NCM460: Normal colonic epithelial cells; oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; oeNC: Overexpression negative control; siMFSD2A: Major facilitator superfamily domain containing 2A-knockdown; siNC: SiRNA negative control.

Figure 2 Major facilitator superfamily domain containing 2A modulates the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase-programmed death-ligand 1 axis and alleviates CD8+ T cell exhaustion. A: Western blotting analysis of extracellular signal-regulated kinase/p38 mitogen-activated protein kinase pathway and programmed death-ligand 1 expression in MC38 [overexpression negative control (oeNC), major facilitator superfamily domain containing 2A-overexpressing (oeMFSD2A)] and Caco-2 [siRNA negative control (siNC), major facilitator superfamily domain containing 2A-knockdown (siMFSD2A)] cells; B: The expression of programmed cell death protein 1 and cytotoxic T lymphocyte-associated antigen 4 in CD8+ T cells following co-culture of MC38 (control, oeNC, oeMFSD2A) cells or Caco-2 (control, siNC, siMFSD2A) cells with CD8+ T cells; C: Killing assay (24 hours) showing the lysis proportion of MC38 (control, oeNC, oeMFSD2A) cells and Caco-2 (control, siNC, siMFSD2A) cells co-cultured with CD8+ T cells, respectively; D: Following co-culture of MC38 cells (control group, oeNC group, oeMFSD2A group) or Caco-2 cells (control group, siNC group, siMFSD2A group) with CD8+ T cells, enzyme-linked immunosorbent assay assays were performed to detect the interleukin-2, interferon-γ, and tumor necrosis factor-α secreted by CD8+ T cells. ^aP < 0.0001 vs overexpression negative control, ^bP < 0.0001 vs siRNA negative control. oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; oeNC: Overexpression negative control; siMFSD2A: Major facilitator superfamily domain containing 2A-knockdown; siNC: SiRNA negative control; ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase; PD-L1: Programmed death-ligand 1; CTLA4: Cytotoxic T lymphocyte-associated antigen 4; IL-2: Interleukin-2; IFN-γ: Interferon-γ; TNF-α: Tumor necrosis factor-α.

Figure 3 Major facilitator superfamily domain containing 2A suppresses malignant phenotypes and programmed death-ligand 1 expression in colorectal cancer cells via the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase pathway. A: Western blotting analysis of major facilitator superfamily domain containing 2A (MFSD2A), p-extracellular signal-regulated kinase 1/2, extracellular signal-regulated kinase 1/2, p-p38 mitogen-activated protein kinase, and p38 mitogen-activated protein kinase expression in MFSD2A-overexpressing MC38 cells treated with or without epidermal growth factor (EGF); B: Cell counting kit-8 assay was performed on MC38 cells with or without EGF (50 ng/mL) treatment; C: Apoptosis assay was performed on MC38 cells with or without EGF (50 ng/mL) treatment; D: Wound healing assay was used to assess the effects of EGF on MC38 cell migration; E: Transwell assay was used to assess the effects of EGF on MC38 cell invasion; F: Western blotting analysis of programmed death-ligand 1 expression in MFSD2A-overexpressing MC38 cells treated with or without EGF (50 ng/mL). ^aP < 0.0001 vs control, ^bP < 0.0001 vs major facilitator superfamily domain containing 2A-overexpressing, ^cP < 0.01 vs major facilitator superfamily domain containing 2A-overexpressing, and ^dP < 0.001 vs Major facilitator superfamily domain containing 2A-overexpressing. oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; MFSD2A: Major facilitator superfamily domain containing 2A; ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase; EGF: Epidermal growth factor; PD-L1: Programmed death-ligand 1.

Figure 4 Major facilitator superfamily domain containing 2A suppresses CD8+ T cell exhaustion via the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase signaling pathway. A: Western blotting analysis of programmed cell death protein 1 and cytotoxic T lymphocyte-associated antigen 4 expression in CD8+ T cells following co-culture with MC38 cells treated with or without epidermal growth factor (EGF); B: Killing assay (24 hours) showing lysis rates of major facilitator superfamily domain containing 2A-overexpressing MC38 cells co-cultured with CD8+ T cells in the presence or absence of EGF; C: Enzyme-linked immunosorbent assay quantification of interleukin-2, interferon-γ, and tumor necrosis factor-α secretion by CD8+ T cells incubated with major facilitator superfamily domain containing 2A-overexpressing MC38 cells under EGF-treated or untreated conditions. ^aP < 0.0001 vs major facilitator superfamily domain containing 2A-overexpressing. oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; EGF: Epidermal growth factor; PD1: Programmed cell death protein 1; CTLA4: Cytotoxic T lymphocyte-associated antigen 4; IL-2: Interleukin-2; IFN-γ: Interferon-γ; TNF-α: Tumor necrosis factor-α.

Figure 5 Major facilitator superfamily domain containing 2A overexpression inhibits tumor growth and promotes CD8+ T cell infiltration, concurrent with suppressed mitogen-activated protein kinase signaling in vivo. A: Subcutaneous tumor growth following injection of MC38, overexpression negative control MC38, or major facilitator superfamily domain containing 2A-overexpressing MC38 cells in C57BL/6 mice; B: Hematoxylin and eosin staining; C: Ki67 immunohistochemistry of tumor tissue; D: Western blotting analysis of p-extracellular signal-regulated kinase 1/2, extracellular signal-regulated kinase 1/2, p-p38 mitogen-activated protein kinase, and p38 mitogen-activated protein kinase expression in tumor tissue; E: Immunofluorescence staining of CD8+ T cell infiltration in tumor tissue. oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; oeNC: Overexpression negative control; HE: Hematoxylin and eosin; ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase.