Additional file 3 of SIRPα antibody combined with oncolytic virus OH2 protects against tumours by activating innate immunity and reprogramming the tumour immune microenvironment

Additional file 3: Figure S1. Cell proliferation assay results. Cell proliferation assay results of Raw264.7 cell lines treated with OH2 MOI=1 (red), OH2 MOI=0.5 (blue) and untreated (black) group by CCK8 assay in 72 hours. Figure S2. OH2 lysates induce RAW264.7 polarization and phagocytosis in vitro. A. Demonstration of the analysis of the phagocytosis by flow cytometry. B. Cell proliferation assay results of CT26, MC38 and 4T-1 cell lines treated with lysate (red), CFS (blue), Cell frozen lysate (purple) and untreated (black) group by CCK8 assay in 24 hours. **, p<0.01. Figure S3. The ratio of M1 (F4/80+CD86+) and M2 (F4/80+CD206+) macrophages in RAW264.7 without any treatment by flow cytometry. Figure S4. The ratio of M1 (F4/80+CD86+) and M2 (F4/80+CD206+) macrophages in RAW264.7 treated with lysate in the blocked SIRPα group and the non-blocked SIRPα group. A. Demonstration of the analysis of the polarization of macrophages by flow cytometry. B. Display of isotype control results for different antibodies. C. The ratio of M1 (F4/80+CD86+) and M2 (F4/80+CD206+) macrophages in the blocked SIRPα group and the non-blocked SIRPα group. D. The ratio of M1 (F4/80+CD86+) and M2 (F4/80+CD206+) macrophages in the blocked SIRPα group and the non-blocked SIRPα group. An unpaired Student’s t test was used to analyze the significance of the difference between two groups. Figure S5. Cell frozen lysate and CFS induce RAW264.7 polarization in vitro. A. Flow cytometric analysis results of one representative sample from each cell line. B. The ratio of M1 (F4/80+CD86+) subtype in the Cell frozen lysate and CFS groups of CT26, MC38 and 4T-1 cells detected by flow cytometry. An unpaired Student’s t test was used to analyze the significance of the difference between two groups. Figure S6. OH2 lysates induce primary macrophages polarization in vitro. A. Demonstration of the analysis of the polarization of macrophages by flow cytometry. B. The percentage of M1 (F4/80+CD86+) subtype and M2 (F4/80+CD206+) subtype in the lysate, CFS, untreated and cell frozen lysate groups detected by flow cytometry. The data are averages from three samples per treatment group. Statistical analysis was performed using ANOVA with multiple comparisons. ns, no significant differences, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. Figure S7. The clear efficiency of CL detected by flow cytometry. Statistical analysis was performed using ANOVA with multiple comparisons. ***, p=0.0003; ****, p<0.0001. Figure S8. Tumor-rechallenge with CT26 of the OH2 or OH2+ CL cured animals. A. The tumor growth curve of CT26 cells rechallenge after the tumors of the CL model had completely regressed. B. The tumorigenic rate of tumor-rechallenge mice in OH2 combined control liposomes group and OH2 combined CL group. Statistical differences were calculated using the Chi-square test. p=0.0291. Figure S9. M-IHC staning of OH2 (left) and OH2 combined CL group (right) including each single staining marker. Figure S10. Expression level of SIRPα on macrophages in spleen from mice detected by flow cytometry. A. Demonstration of the expression level of SIRPα on macrophages in spleen from mice detected by flow cytometry. B. Histogram showing Expression level of SIRPα on macrophages in spleen from mice (n = 3). Figure S11. Combined anti-SIRPα therapy with OH2 enhances anticancer immune responses in a CT26 cancer model. A. The treatment process and experimental timeline for the combined treatment. B. The tumor growth curve of different treatment groups of tumor-bearing mice with smaller tumor volumes at the initial treatment. The red box zoomed in to show the OH2 combined anti-SIRPα antibody group, OH2 combined isotype and OH2 group. Two-way analysis of variance (ANOVA) was performed on the experimental data. n=10 mice/group. ns, no significant differences. C. The survival curve of different treatment groups of tumor-bearing mice with smaller tumor volumes at the initial treatment. The Kaplan-Meier method and log-rank test were used for the survival curve data. ns, no significant differences. Figure S12. M-IHC staining results of anti-SIRPα model. M-IHC stanning of control group (top left), OH2 group (top right), OH2 combined isotype group (bottom left) and OH2 combined anti-SIRPα antibody group (bottom right) including each single staining marker. Figure S13. IHC staining and quantitative results of the anti-SIRPα antibody treatment model. A. Representative IHC staining for CD8, F4/80, CD86 and CD206 of the OH2 combined anti-SIRPα antibody group, control group and anti-SIRPα antibody group with larger tumor volumes at the initial treatment. B. Quantitative results of CD8, F4/80, CD86 and CD206 staining in the the OH2 combined anti-SIRPα antibody group, control group and anti-SIRPα antibody group. Original magnification, ×200. n=5 samples/group. Statistical analysis was performed using ANOVA with multiple comparisons. ns, no significant differences, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. Figure S14. KEGG results of tumor tissues from anti-SIRPα model. A. KEGG results of differentially expressed genes between OH2 combined anti-SIRPα antibody group and control group. B. KEGG results of differentially expressed genes between OH2 combined anti-SIRPα antibody group and OH2 combined Isotype antibody group. Figure S15. GSEA results of tumor tissues from anti-SIRPα model. A. GSEA results of differentially expressed genes between OH2 combined anti-SIRPα antibody group and control group. B. GSEA results of differentially expressed genes between OH2 combined anti-SIRPα antibody group and oHSV-2 combined Isotype antibody group. Figure S16. scRNA-seq data analysis. A. UMAP plots represent the clusters (left) and groups (right) of tumor cells. B. A heatmap showing the scaled average expression of each cell subcluster. C. UMAP plots showing the expression values of canonical marker genes of each subcluster. D. A heatmap showing the scaled average expression of each immune cell subcluster. C. UMAP plots showing the expression values of canonical marker genes of each immune subcluster. Figure S17. CTL Assay. A. The killing functions of lymphocyte co-cultured with different cancer cell lysates detected by CFSE/PI. Three effectors to target ratios were set for each cell line (Lymphocyte: tumour cells=25:1, 50:1, and 100:1). The data are averages from three samples per treatment group. Statistical analysis was performed using ANOVA with multiple comparisons. ns, no significant differences, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. B. Supernatant from the CTL assay was tested for TNF-α, Granzyme B and IFN-γ by ELISA. Statistical analysis was performed using ANOVA with multiple comparisons. ns, no significant differences, ****, p<0.0001.