Inhibition of TFF3 improves the infiltration and function of CD8+ T cells by downregulating the expression of PD-L1 in colorectal cancer

Abstract

Background: Colorectal cancer is a common malignant tumor of the digestive tract with a high mortality rate. TFF3 is a secreted protein expressed in various cancers. The aim of the study is to report that inhibiting TFF3 increases the function and infiltration of CD8⁺ T cells in colorectal tumor tissues.

Methods: The proteomics analysis confirmed the high expression of secreted TFF3 in the supernatant of colorectal cancer cells, and databases were used to analyze the immune infiltration in tumor tissues. qPCR test was used to demonstrate the high levels of secreted TFF3 affecting the function of CD8⁺ T cells. In vivo experiments were used to observe the effects of TFF3 knockdown on tumor growth and immune cell infiltration. Tumor-infiltrating T lymphocytes were sorted for RNA sequencing and mechanism explanation.

Results: Inhibiting the expression of TFF3 in tumor tissues improved immune cell infiltration and enhanced anti-tumor effects. TFF3 knockdown downregulated the expression of PD-L1 in tumor tissues through the AKT/mTOR pathway, which enhanced the function of CD8⁺ T cells.

Conclusion: Knockdown of TFF3 improved the infiltration of CD8⁺ T cells and anti-tumor capabilities.

Keywords: Immune infiltration; PD-L1; TFF3.

Fig. 1

TFF3 has a high secretion level in the colorectal tumor conditional medium. A Gene expression level related to the function of CD8⁺ T cells (mean ± SD, n = 4). B The differentially expressed proteins between the CM and TCM groups. C Subcellular localization of differentially expressed proteins. D The most differentially expressed extracellular secreted proteins. E. The secretion level of TFF3 (mean ± SD, n = 6)

Fig. 2

TFF3 affects the infiltration and function of CD8⁺ T cells into colorectal tumor. A The immune infiltration in tumor tissues of all patients from the TCGA-COAD database. B The correlation of immune cells from the TCGA-COAD database. C Analysis of immune cell infiltration in tumor samples with varying levels of TFF3 expression. D The level of GZMB and PRF1 based on the different expression of TFF3. E The secretory level of TFF3 after knockdown (mean ± SD, n = 4). F The expression level of GZMB, TNF, and IFNG after the knockdown of TFF3 (mean ± SD, n = 3)

Fig. 3

Knockdown of TFF3 restricted tumor growth. A Tumor growth curve of CT-26 inoculated with control (shNC) or shTFF3 tumor cells (mean ± SEM, n = 6). B The tumor was excised from the mice at the time of euthanasia. C The concentration of TFF3 from tumor interstitial fluid (n = 3). D The infiltration of CD8⁺ and CD4⁺ T cells in the mice tumor. E Flow cytometry analysis of the immune infiltration (n = 5). F The secretory level of TNF and GZMB in mouse serum (mean ± SD, n = 4)

Fig. 4

Knockdown of TFF3 increases the infiltration of CD8⁺ T cells in vivo. A Schematic diagram for RNA-seq of tumor cells and T cells sorted from CT-26 tumors formed by shNC and shTFF3 tumor cells. B Detection of CD8⁺ T cells in tumors with immunofluorescence. Scale bars represent 50 µm (mean ± SD, n = 3). C Volcano plot of differentially expressed genes for TIL-T. D KEGG enrichment analysis of differentially expressed genes for TIL-T

Fig. 5

Knockdown of TFF3 reduces the expression level of PD-L1 on tumor. A The KEGG enrichment analysis on immune infiltration-related genes was performed by the Cytoscape software. B The expression level of PD-L1 in tumor tissues (mean ± SD, n = 3). C Genes participated in the PD-L1 expression and PD-1 checkpoint pathway in cancer

Fig. 6

Knocking down TFF3 inhibits the AKT/mTOR pathway. A The expression level of p-AKT/AKT (mean ± SD, n = 3). B The expression level of p-mTOR/mTOR (mean ± SD, n = 3). C Validation of the interaction between TFF3 and AKT by Co-IP. D The construction of CEA-CAR. E The apoptosis rates of DLD1 shTFF3 and DLD1 shNC cancer cell lines (mean ± SD, n = 7)