Cytotoxic T-Lymphocyte Antigen-4 in Colorectal Cancer: Another Therapeutic Side of Capecitabine

Abstract

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory immune checkpoint that can be expressed in tumor-infiltrating lymphocytes and colorectal cancer (CRC) cells. This immune checkpoint can attenuate anti-tumoral immune responses and facilitate tumor growth and metastasis. Although capecitabine is an effective chemotherapeutic agent for treating CRC, its effect on the tumoral CTLA-4 expression remains unclear. In the current research, we applied the GSE110224 and GSE25070 datasets to characterize CTLA-4 expression in CRC patients. Then, we analyzed CTLA-4 expression in CRC samples, HT-29, HCT-166, and SW480 cell lines using real-time PCR. Our bioinformatic results have highlighted the overexpression of CTLA-4 in the CRC tissues compared to the adjacent non-tumoral tissues. Our in vitro studies have indicated that SW480 cells can substantially overexpress CTLA-4 compared to HT-29 and HCT 116 cells. In addition, capecitabine can remarkably downregulate the expression of CTLA-4 in SW480 cells. Collectively, capecitabine can inhibit the expression of CTLA-4 in CRC cells and might bridge the immunotherapy approaches with chemotherapy.

Keywords: CTLA-4; capecitabine; chemotherapy; colorectal cancer; immune checkpoint; immune checkpoint inhibitors; immunotherapy.

Figure 1

Data preprocessing, heatmaps of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in GSE110224, and GSE25070 datasets and CTLA-4 expression in cancer cells and adjacent. (A) The flowchart of data preparation, processing, and analysis in this study. (B) Sample dendrogram and trait heatmap. (I) GSE25070 and (II) GSE110224 dataset; the color is proportional to the pathological stage (red = primary CRC and white = normal adjacent sample). (C) The expression analysis of CTLA-4 in different groups has shown that this gene is highly expressed in colorectal cancer (CRC) cells compared to the adjacent cells. (D) CTLA-4 expression in both datasets has been higher in the cancer samples than in the controls, C: control, and T: tumor.

Figure 2

Obtained data from the cancer genome atlas (TCGA) and cancer cell line encyclopedia (CCLE). (A) Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression profiles and clinicopathological data of patients with CRC. (B) The CCLE showed a high CTLA-4 expression level in SW480 cells. (C) Overall survival of colorectal cancer (CRC) patients stratified by CTLA-4 expression levels (the cut-off criterion is lower than 3.867 and higher than 5.701).

Figure 3

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression was higher in tumor tissues than in the adjacent non-tumoral tissues; however, this change was not statistically significant; N.S: non-significant, Adj: adjacent.

Figure 4

The gene expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in HT-29, HCT 116, and SW480 cells. The real-time quantitative reverse transcription PCR (qRT-PCR) assay has indicated that CTLA-4 expression in the SW480 cells is substantially higher than the HT-29 and HCT 116 cells. N.S: non-significant, *** p < 0.001, and **** p < 0.0001.

Figure 5

The dose–response and cell viability curve of SW480 cells. The results indicated that the half-maximal inhibitory concentration (IC50) of capecitabine in the SW480 cells was 289.7 ng/mL.

Figure 6

The mRNA expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in the SW480 cells. (A): The CTLA-4 expression has been significantly lower in the control group compared to the capecitabine-treated group. Error bars display the standard deviation. (B): Gel electrophoresis of CTLA-4 (92 bp) and GAPDH (102 bp). From right to left, lines show DNA ladder (50–1500 bp), CTLA-4 expression in the SW480 cell line (control), CTLA-4 expression after treatment with capecitabine. The other lanes are related to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (control and treated, respectively); **** p-value < 0.0001.