Fig 2: PD-L1 is a cell cycle dependent protein and regulates sister chromatid cohesion in TNBC.a PD-L1 level fluctuates during the cell cycle. Target proteins were detected by Western blot in MDA-MB-231 cells at indicated time points after double thymidine block. b Immunostaining of PD-L1 in interphase cells. Wildtype (WT) or PD-L1 knockout (KO) MDA-MB-231 cells were permeabilized and fixed using the indicated methods and cells were then stained with anti-PD-L1 antibody. PFA + Triton X-100: cells were fixed with 3% paraformaldehyde followed by 0.5% Triton X-100. Triton X-100 + PFA: cells were treated with 0.5% Triton X-100 first to remove most cytoplasmic/membrane proteins, and followed by fixation with 3% paraformaldehyde. Nucleus was stained with DAPI. Scale bar: 10 µm. c Detection of PD-L1 in different fractions of control and PD-L1 knockout MDA-MB-231 cells. Cytoplasmic/membrane (C/M) and nuclear (N) fraction. d Cytoplasmic/membrane (C/M) and nuclear (N) fractions of PD-L1 from different cancer cell lines. SE short exposure; LE long exposure. e Cytoplasmic/membrane (C/M) and nuclear (N) fractions of MDA-MB-231 cells with or without PNGase F treatment. Arrows indicate PD-L1 bands. f, g Loss of PD-L1 promotes multinucleation. f Representative nuclear staining by DAPI in MDA-MB-231 cells three days post-infection with control shRNA virus or PD-L1 shRNA virus. Scale bar: 10 µm. g Quantification of multinucleated cells was performed. Data are presented as means ± SD, and were independently replicated three times. Statistical significance was calculated using Student’s t test. **P < 0.01, ***P < 0.001. h, i PD-L1 is required for sister chromatid cohesion. h Representative metaphase spreads showing MDA-MB-231 cells with normal, partial loss and total loss of sister chromatid cohesion. Scale bar: 5 µm. i Quantification of different sister chromatid status in control and PD-L1 knockdown cells. Data are presented as means ± SD, independently replicated three times with similar results. j FISH assays were performed in MDA-MB-231 cells expressing control shRNA, PD-L1 shRNA, SCC1 shRNA or Sororin shRNA. myb gene probe was used and the distance between paired FISH signals was measured and quantified. Bar: 5 µm. k Western blots showing the knockdown efficiency for the experiments in j.

Fig 3: Overexpression of miR-326 enhances TNF-α and IFN-γ expression of infiltrated CD8+ T cells and prevents metastasis by targeting PD-L1 and B7-H3 on cancer cells.5 × 105 miR-326 overexpressed LLC cells or empty vector control LLC cells were subcutaneously injected into C57BL/6 mice. MiR-326 antagomir or NC antagomir were intratumor injected when the tumor diameter reached 0.5 cm. The mice were sacrificed 28 days after inoculation. A Tumor volume (n = 7). B Tumor weight (n = 6). C Pulmonary metastasis nodule calculated by hematoxylin–eosin staining. (n = 6) D, E PD-L1, B7-H3 expression, and CD8+ T cells infiltration were determined by IHC staining in tumor tissues. F–I PD-L1, B7-H3, TNF-α, and IFN-γ were detected by qRT-PCR in tumor sites (n = 3). J TNF-α and IFN-γ expression of infiltrated CD8+ T cells by flow cytometry (n = 3). All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way (B, C and F–I) or two-way (A) ANOVA with Tukey’s post hoc test or Student’s t test (J). *P < 0.05, ***P < 0.001 versus LV-NC. #P < 0.05, ##P < 0.01, ###P < 0.001 versus LV-miR-326. Scale bar, 200 µM.

Fig 4: FGD1 regulates OS immune response through PTEN/PD-L1 axis. A, U-2OS, MNNG/HOS and 143B cells were infected with indicated siRNAs. After 48 h, cells were harvested for Western blotting analysis. B, U-2OS, MNNG/HOS and 143B cells were transfected with indicated constructs. 48 h post-transfection, cells were subjected to Western blotting analysis. C, U-2OS, MNNG/HOS and 143B cells were infected with indicated siRNAs. After 48 h, cells were treated with or without LY294002 (40 uM) for other 24 h. WCL of cells collected for Western blotting analysis. D, U-2OS, MNNG/HOS and 143B cells were infected with indicated constructs. After 48 h, cells were treated with or without LY294002 (40 uM) for other 24 h. WCL of cells collected for Western blotting analysis. E-G, K7M2 cells were infected with lentivirus vectors expressing control or Fgd1-specific shRNAs. 72 h after puromycin selection, 5 x 106 cells were injected subcutaneously into C57BL/6 mice. Mice (n=5/group) were treated with anti-PD-L1 (200 µg) or non-specific IgG for 45 days. Growth curves of tumors with different treatments are shown in (E). Kaplan-Meier survival curves for each treatment group demonstrate the improved efficacy of combining PD-L1 mAb with the knockdown of Fgd1. **, P < 0.01; ***P < 0.001. (Gehan-Breslow-Wilcoxo test) (F). At the end of treatment, the numbers of infiltrated CD45+CD8+ T cells, CD45+CD4+ T cells, and CD11b+Gr1+ myeloid cells in tumors with different treatments were analyzed by FACS (G). All data are shown as mean values ± SD. ns, not significant, ** P < 0.01, *** P < 0.001. H, a hypothesis model depicting that FGD1 inhibits PTEN to trigger PI3K/AKT/NK-kB signaling pathway activation and promote tumor progression in osteosarcoma. Meanwhile, FGD1 modulated immune response of osteosarcoma via PTEN/PD-L1 axis.

Fig 5: Apatinib decreased MVD and cell proliferation in liposarcoma PDX. IHC analysis of MVD, TYMS, MVD, RRM2, TYMS, Ki-67, and PD-L1 expression in tumor sections from each group. Compared with the control group, Apatinib could down-regulate MVD and the expression of TYMS and RRM2.The positive Ki-67 ratio decreased in all treatment groups. PD-L1 expression did not significantly differ among groups. ×200 magnification.