Effects Of β-catenin/TCF Inhibition On Tumor Immune Microenvironment In Colorectal Cancer

Colorectal cancer has high incidence and mortality,with most of the patients characterized with mutations in Wnt/β-catenin pathway components,especially in APC andβ-catenin.These mutations may cause abnormal activation of the Wnt/β-catenin pathway,overexpression of the cancer-related pathway target genes,and initiation and progression of colorectal tumor as a result.An analysis of 1211 primary tumor samples indicated that Wnt/β-catenin pathway activity and T cell infiltration is negatively correlated in the TCGA Colorectal Cancer Cohort,but the mechanism behind that remains unclear.This suggests abnormal activation of Wnt/β-catenin may also produce negative impacts on the immune microenvironment of the colorectal cancer.Therefore,targeting Wnt/β-catenin pathway can be vital in the treatment of colorectal cancer.To date,most inhibitors work on the upstream of the Wnt/β-catenin pathway in colorectal cancer,whose effects are subject to mutations of their downstream components such as APC andβ-catenin.This study used a small molecule inhibitor named iCRT14,which mainly works on the downstream ofβ-catenin.iCRT14 can not only impact theβ-catenin/TCF interaction,but also interfere with TCF binding to DNA,thus effectively inhibiting the transcription of Wnt/β-catenin pathway target genes.As a first step,this study compared the Wnt/β-catenin pathway activity of two common mice colorectal cancer cell lines CT26 and MC38,and CT26 with higher Wnt/β-catenin activity was chosen for the subsequent investigations.We found that iCRT14 could significantly inhibit Wnt/β-catenin pathway activity and in-vitro proliferation of CT26.Afterwards,we established CT26 subcutaneous tumor model and examined the effects of iCRT14 on the tumor growth and tumor immune microenvironment.It showed that there was a remarkable deceleration in tumor growth after treating with iCRT14.Results from flow cytometry analysis indicated iCRT14 dramatically facilitated infiltration of CD8~+T,CD4~+T and NK cells into tumor,and granzyme B expression by NK.Afterwards,we investigated the mechanism of the remarkable promotion of iCRT14 in T cell infiltration.Firstly,it was found that iCRT14 did not affect the expression of T cell proliferation marker Ki67,indicating that T cell proliferation was not influenced by iCRT14.By applying Q-PCR,we examined the influence of iCRT14 on the expression of T cell chemokines CXCL9,CXCL10 and CXCL11.The results indicated upregulating effects of iCRT14 in T cell chemokine expression for both human and mice colorectal cancer cells,with impacts on CXCL10and CXCL11 being relatively more remarkable.Based on these findings,we inferred that iCRT14 might boost the expression of T cell chemokine CXCL9,CXCL10 and CXCL11,which further boosted T cell to move to the tumors,and thus contributed to a significant increase in T lymphocytes in tumor.Finally,we combined iCRT14 with FOXP3~+Treg cell depletion therapy and OVA257-264(SIINFEKL)peptide vaccine therapy,respectively,and revealed that the combined therapy more significantly inhibited the growth of colorectal cancer tumor compared to the individual therapy.To summarize,this study unraveled that iCRT14 evidently inhibited the growth of CT26 subcutaneous tumor,and remarkably boosted the T cell infiltration.Moreover,iCRT14 significantly increased the expression of T cell chemokines CXCL9,CXCL10 and CXCL11 in colorectal cancer cell,which is a possible mechanism for the boost of T cell infiltration by iCRT14.In addition,iCRT14 improved the efficacy of FOXP3~+Treg cell depletion therapy and OVA257-264(SIINFEKL)peptide vaccine therapy for colorectal cancer tumor,expected to be a reference for improving colorectal cancer treatment clinically.