Fig 1: ITGB6 promotes tumor growth by inhibiting the T-cell antitumor immune response. (A) Western blot for ITGB6 in MC38 and CT26 cells that overexpress ITGB6. (B) 3-day proliferation assay with ITGB6 overexpressing and control cells. (C) CT26-ITGB6 and CT26-Ctrl cecum tumors at day 13 after injection into Balb/c mice. (D) Weight of CT26-ITGB6 and CT26-Ctrl cecum tumors at day 13 after injection. (E) Flow cytometry analysis of CD8+ T cells isolated from tumor. (F) Flow cytometry analysis of CD4+ T cells isolated from tumor. (G) Flow cytometry analysis of CD8+ and CD4+ T cells isolated from spleen and lymph node (LN). (H) CT26-ITGB6 and CT26-Ctrl cecum tumors at day 13 after injection into Balb/c nude mice. (I) Weight of CT26-ITGB6 and CT26-Ctrl cecum tumors at day 13 after injection into Balb/c nude mice. Means and SDs are shown (n=5 mice). Unpaired two-tailed t-test was used to calculate statistical significance. ns=not significant (p≥0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 2: Integrin αvβ6 blockade overcomes resistance to checkpoint blockade therapy in tumors without artificial ITGB6 overexpression. (A) RT-qPCR analysis of Itgb6 expression in murine carcinoma cell lines. (B) RT-qPCR analysis of Itgb6 expression in tumors grown from 4T1, CT26-ITGB6 and MC38-ITGB6 cells. (C) Experimental design of αITGB6 (6.8G6) and αPD-1 antibody administration. (D) Tumor volume development of subcutaneous 4T1 tumors treated with αITGB6, αPD-1, αITGB6 and αPD-1 or IgG control. (E) Tumor weight of subcutaneous 4T1 tumors treated with αITGB6, αPD-1, αITGB6 and αPD-1 or IgG control. Means and SDs are shown (n=5 mice, 2 tumors per mouse). One-way analysis of variance (ANOVA) (E) and two way ANOVA (D) with Tukey’s post-hoc test were used to calculate statistical significance. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 3: ITGB6 expression leads to local, but not systemic T-cell inhibition. (A) Experimental design of injection scheme. Subcutaneous injection of CT26-Ctrl tumors or CT26-ITGB6 tumors in both flanks or CT26-Ctrl tumors in one flank and CT26-ITGB6 tumors in the other flank of the mice (Mix). (B) Weight of tumors from mice bearing only CT26-ITGB6 or CT26-Ctrl tumors or mice bearing both tumors (Mix). (C) Tumor volume development of tumors from mice bearing only CT26-ITGB6 or CT26-Ctrl tumors or mice bearing both tumors (Mix). (D) Flow cytometry analysis of T-cells isolated from tumors of mice bearing only CT26-ITGB6 or CT26-Ctrl tumors or mice bearing both tumors (Mix). (E) Immunofluorescent stainings for CD8 and pSmad3 in CT26-ITGB6 tumors from mice bearing only CT26-ITGB6 tumors or mice bearing both tumors (Mix). Means and SDs are shown (n=5 mice). One-way analysis of variance (ANOVA) (B and D) and two-way ANOVA (C) with Tukey’s post-hoc test were used to calculate statistical significance. ns=not significant (p≥0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 4: Integrin αvβ6 blockade sparks T-cell antitumor response and overcomes resistance to CBT. (A) Experimental design of αITGB6 antibody (6.8G6) administration. (B) Subcutaneous CT26-ITGB6 tumors treated with αITGB6 or IgG control. (C) Tumor weight of subcutaneous CT26-ITGB6 tumors treated with αITGB6 or IgG control. (D) Tumor volume development of subcutaneous CT26-ITGB6 tumors treated with αITGB6 or IgG control. (E) Flow cytometry analysis of CD8+ T cells in CT26-ITGB6 tumors treated with αITGB6 or IgG control. (F) Experimental design of αITGB6 (6.8G6) and αPD-1 antibody administration. (G) Representative image of subcutaneous CT26-ITGB6 tumors treated with αPD-1 or αITGB6 and αPD-1. (H) Tumor weight of subcutaneous CT26-ITGB6 tumors treated with αITGB6, αPD-1, αITGB6 and αPD-1 or IgG control. (I) Tumor volume development of subcutaneous CT26-ITGB6 tumors treated with αITGB6, αPD-1, αITGB6 and αPD-1 or IgG control. (J) Flow cytometry analysis of CD8+ T cells in CT26-ITGB6 tumors treated with αITGB6, αPD-1, αITGB6 and αPD-1 or IgG control antibody. Means and SDs are shown (n=5 mice, 2 tumors per mouse). Unpaired two-tailed t-test (C, E) one-way analysis of variance (ANOVA) (H, J) and two way ANOVA (D and I) with Tukey’s post-hoc test were used to calculate statistical significance. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 5: Cytotoxic immune response regulation is the main function of ITGB6 within the tumor. (A) Heatmap of differentially expressed genes (DEGs) (p<0.001) in MC38-ITGB6 and MC38-Ctrl tumors, generated by unsupervised hierarchical clustering (n=5 mice). (B) Volcano plot displaying DEGs from MC38-ITGB6 versus MC38-Ctrl tumors. Y-axis corresponds to p value of –log 10. X-axis displays log 2-fold change value. Indicated limits represent DEGs with p<0.05 and log 2 fold change above +2 or below −2. (C) Gene ontology enrichment analysis of DEGs. Bars in red or blue indicate the number of genes involved in upregulation or downregulation of the respective biological process in ITGB6-expressing tumors.
Supplier Page from Sino Biological, Inc. for Mouse Integrin beta 6/ITGB6 Gene ORF cDNA clone in cloning vector