Fig 2: WEE1 inhibitor MK1775 enhances the anticancer effect of immune checkpoint blockade (ICB) therapy in MUS81 deficient gastric cancer cells. a Schematic of isotype control IgG, MK1775, anti–PD-L1 antibody and combination treatment. Treatments were started on day 7 after inoculation and stopped on day 29. b Tumor volume of the indicated groups on day 29. Data of tumor volume are represented as mean ± SD (n = 6 for each group). c Tumor growth curve of indicated groups. Data of tumor volume are represented as mean ± SD in indicated groups (n = 6 for each group). d Images of tumor of MFC gastric cancer mice model. e multiplexed immunofluorescence (mIF) staining of CD8 (red), perforin (green), and PD-L1 (pink) in parental and MUS81 knockdown MFC tumors. Scale bar: 50 μm. f Quantitative analysis of CD8 and PD-L1 positive cells and the fluorescence intensity of perforin. Top, proportion of CD8 positive cells in MFC shScramble (left) or shMUS81 (right) tumors. Middle, proportion of PD-L1 positive cells in MFC shScramble (left) or shMUS81 (right) tumors; Bottom, the fluorescence intensity of perforin in MFC shScramble (left) or shMUS81 (right) tumors. The data are presented as mean ± SD (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns., not significant

Fig 3: WEE1 inhibition enhances the CD8+ T cell activation induced by MUS81 knockdown. a Representative images of CD8+ T cells activation after co-cultures. CD69+ cells were gated in CD3+/CD8+ T cells. MFC shScramble or shMUS81 cells were treated with dimethyl sulfoxide (DMSO) or MK1775 (1.0 μM). b Statistics analysis of the proportion of CD69+ cells to CD3+/CD8+ T cells in the indicated four groups. The data are presented as mean ± SD (n = 3). c–e ELISA analysis of TNF-α (left), IL-1β (middle), and IL-6 (right) in the supernatants of the co-cultures. The data are presented as mean ± SD (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns., not significant

Fig 4: The binding of PD‐L1 to PD‐1 promotes the immune escape in NPC cells. (A) Flow cytometric sorting of CD8+ T cells from human peripheral blood mononuclear cells. (B) Confocal microscopic observation of CFSE‐labeled C666‐1 cell‐derived exosomes binding to PD‐1 on the CD8+ T‐cell surface. In this panel, unstimulated + EXO indicates that the CD8+ T cells unstimulated with anti‐CD3/CD28 antibodies were co‐cultured with CFSE‐labeled C666‐1 cell‐derived exosomes. CD3/CD28 stimulated + EXO indicates that the CD8+ T cells stimulated with anti‐CD3/CD28 antibodies were co‐cultured with CFSE‐labeled C666‐1 cell‐derived exosomes. CFSE‐EXO indicates the CFSE‐labeled exosomes. (C) Flow cytometric analysis of the binding ratio between C666‐1 cell‐derived exosomes and activated/inactivated CD8+ T cells (i.e. CD8+ T cells with/without pre‐stimulation by anti‐CD3/CD28 antibodies). The green curve in the figure represents the fluorescence intensity of CSFE in the control group, and the red curve represents the fluorescence intensity of CSFE after activation. (D) Flow cytometric analysis of the binding ratio between exosomes from IFN‐γ‐treated C666‐1 cell and activated CD8+ T cells. The green curve in the figure represents the fluorescence intensity of CSFE in the unstimulated + EXO group, and the red and blue curves represent the fluorescence intensity of CSFE in the CD3/CD28 stimulated + EXO group and the CD3/CD28 stimulated + EXO + IFN‐r group, respectively. (E) Flow cytometric analysis of the proportion of CD8+ T cells in response to co‐culture with the exosomes alone or in combination with treatment by anti‐PD‐L1 antibody. (F) Flow cytometric analysis of the proportion of Ki‐67‐positive cells in CD8+ T cells in response to co‐culture with the exosomes alone or in combination with treatment by anti‐PD‐L1 antibody. (G) Levels of IFN‐γ, IL‐2, and TNF‐α in CD8+ T cells in response to co‐culture with the exosomes alone or in combination with treatment by anti‐PD‐L1 antibody measured by ELISA. *P < 0.05. Each cellular experiment was repeated three times

Fig 5: Exosomal PD‐L1 facilitates the tumor growth of NPC cells in mice. (A) The knockdown efficiency of lentivirus‐mediated sh‐PD‐L1 treatment in C666‐1 cells validated by qRT‐PCR. (B) Images of NPC cell‐implanted tumors in mice of each group. (C) The volume of NPC cell‐implanted tumors in mice of each group, measured every 5 days. (D) The weight of NPC cell‐implanted tumors in mice of each group. (E) Immunohistochemical staining of the infiltration of CD8+ TILs in tumor tissues of mice of each group (the y axis represents the number of CD8+ TILs in every five high‐power fields of view). *P < 0.05