Inhibition of integrin αvβ6 sparks T-cell antitumor response and enhances immune checkpoint blockade therapy in colorectal cancer

Abstract

Background: Integrin αvβ6 is a heterodimeric cell surface protein whose cellular expression is determined by the availability of the integrin β6 subunit (ITGB6). It is expressed at very low levels in most organs during tissue homeostasis but shows highly upregulated expression during the process of tumorigenesis in many cancers of epithelial origin. Notably, enhanced expression of integrin αvβ6 is associated with aggressive disease and poor prognosis in numerous carcinoma entities. Integrin αvβ6 is one of the major physiological activators of transforming growth factor-β (TGF-β), which has been shown to inhibit the antitumor T-cell response and cause resistance to immunotherapy in mouse models of colorectal and mammary cancer. In this study, we investigated the effect of ITGB6 expression and antibody-mediated integrin αvβ6 inhibition on the tumor immune response in colorectal cancer.

Methods: Using orthotopic and heterotopic tumor cell injection, we assessed the effect of ITGB6 on tumor growth and tumor immune response in wild type mice, mice with defective TGF-β signaling, and mice treated with anti-integrin αvβ6 antibodies. To examine the effect of ITGB6 in human colorectal cancer, we analyzed RNAseq data from the colon adenocarcinoma dataset of The Cancer Genome Atlas (TCGA-COAD).

Results: We demonstrate that expression of ITGB6 is an immune evasion strategy in colorectal cancer, causing inhibition of the antitumor immune response and resistance to immune checkpoint blockade therapy by activating latent TGF-β. Antibody-mediated inhibition of integrin αvβ6 sparked a potent cytotoxic T-cell response and overcame resistance to programmed cell death protein 1 (PD-1) blockade therapy in ITGB6 expressing tumors, provoking a drastic increase in anti-PD-1 treatment efficacy. Further, we show that the majority of tumors in patients with colorectal cancer express sufficient ITGB6 to provoke inhibition of the cytotoxic T-cell response, indicating that most patients could benefit from integrin αvβ6 blockade therapy.

Conclusions: These findings propose inhibition of integrin αvβ6 as a promising new therapy for colorectal cancer, which blocks tumor-promoting TGF-β activation, prevents tumor exclusion of cytotoxic T-cells and enhances the efficacy of immune checkpoint blockade therapy.

Keywords: CD8-positive T-lymphocytes; cytotoxicity; drug evaluation; immunologic; immunotherapy; preclinical; tumor microenvironment.

Figure 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.

Figure 2

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.

Figure 3

T-cell inhibiting effect of ITGB6 is mediated through TGF-β activation. (A) Immunohistochemical (IHC) stainings for pSmad2, pSmad3 and SOX4 in subcutaneous CT26-ITGB6 and CT26-Ctrl tumors. Representative images of IHC stainings (top) and quantification of the number of stained cells (below). Scale bar=50 µm. (B) Representative image of subcutaneous CT26-ITGB6 and CT26-Ctrl tumors grown in CD4-dnTGFBR2 mice or C57BL/6 WT mice at day 14 after injection. (C) Tumor volume development of CT26-ITGB6 and CT26-Ctrl tumors grown in CD4-dnTGFBR2 mice or C57BL/6 WT mice. (D) Weight of CT26-ITGB6 and CT26-Ctrl tumors grown in CD4-dnTGFBR2 mice or C57BL/6 WT mice at day 14 after injection. Means and SDs are shown. Unpaired two-tailed t-test (A) (n=5 mice), one-way analysis of variance (ANOVA). (D) and two-way ANOVA (C) with Tukey’s post-hoc test (n=5 mice, 2 tumors per mouse) were used to calculate statistical significance. ns=not significant (p≥0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4

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.

Figure 5

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.

Figure 6

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.

Figure 7

Integrin αvβ6 inhibits T-cell immune response in the majority of patients with colorectal cancer. (A) Kaplan-Meier curves of disease free survival by ITGB6 gene expression status. Minimum p value approach was used to obtain an optimal discrimination of the total patient group into two subgroups with different disease-free survival depending on the level of ITGB6. Differences in survival were compared by logrank test. ITGB6 low: n=109, ITGB6 high: n=234. (B) Relative ITGB6 mRNA expression of all 521 patients in the TCGA-COAD dataset. (C) Relative mRNA expression in the 50 patients with lowest ITGB6 expression compared with the remaining 471 patients of the TCGA-COAD dataset. Means and SDs are shown. Mann-Whitney test was used to calculate statistical significance. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001.