Gene Expression Analysis of Immune Regulatory Genes in Circulating Tumour Cells and Peripheral Blood Mononuclear Cells in Patients with Colorectal Carcinoma

Abstract

Information regarding genetic alterations of driver cancer genes in circulating tumour cells (CTCs) and their surrounding immune microenvironment nowadays can be employed as a real-time monitoring platform for translational applications such as patient response to therapeutic targets, including immunotherapy. This study aimed to investigate the expression profiling of these genes along with immunotherapeutic target molecules in CTCs and peripheral blood mononuclear cells (PBMCs) in patients with colorectal carcinoma (CRC). Expression of p53, APC, KRAS, c-Myc, and immunotherapeutic target molecules PD-L1, CTLA-4, and CD47 in CTCs and PBMCs were analysed by qPCR. Their expression in high versus low CTC-positive patients with CRC was compared and clinicopathological correlations between these patient groups were analysed. CTCs were detected in 61% (38 of 62) of patients with CRC. The presence of higher numbers of CTCs was significantly correlated with advanced cancer stages (p = 0.045) and the subtypes of adenocarcinoma (conventional vs. mucinous, p = 0.019), while being weakly correlated with tumour size (p = 0.051). Patients with lower numbers of CTCs had higher expression of KRAS. Higher KRAS expression in CTCs was negatively correlated with tumour perforation (p = 0.029), lymph node status (p = 0.037), distant metastasis (p = 0.046) and overall staging (p = 0.004). CTLA-4 was highly expressed in both CTCs and PBMCs. In addition, CTLA-4 expression was positively correlated with KRAS (r = 0.6878, p = 0.002) in the enriched CTC fraction. Dysregulation of KRAS in CTCs might evade the immune system by altering the expression of CTLA-4, providing new insights into the selection of therapeutic targets at the onset of the disease. Monitoring CTCs counts, as well as gene expression profiling of PBMCs, can be helpful in predicting tumour progression, patient outcome and treatment.

Keywords: CTLA-4; KRAS; circulating tumour cells; immune checkpoint molecules; immune escape mechanism; molecular characterisation.

Figure 1

Enumeration of circulating tumour cells (CTCs) and numbers of different subpopulation of CTCs in patients with colorectal carcinoma (CRC). The figure depicts: (A) representative images of CTCs detected from patients with CRC captured using an Olympus Fluoview FV1000 Confocal Microscope (scale bar: 20 µm); (B) a comparison of the number of different subpopulations of CTCs detected in patients with CRC; and (C) the number of populations recruited in different groups based on the range of CTC counts, along with healthy donors. (HD, healthy donor; N, No CTC; L, low CTC-positive group; H, high CTC-positive group).

Figure 2

Relative fold change values (2^–ΔΔCt) of p53, APC, KRAS, c-Myc and CD47, CTLA-4 in CTC-enriched fraction from patients with CRC and from healthy donors (HDs) (n = 6). All the values are plotted as a scatter plot with the median. Line indicates the normal fold change value. ** p < 0.005, * p < 0.05.

Figure 3

Comparison of the gene expression of oncogenes (KRAS, c-Myc), tumour suppressor genes (p53, APC) and immune checkpoint molecules (CTLA-4, CD47) between high versus low CTC-positive groups in CTCs and PBMCs from patients with colorectal carcinoma (CRC). Data are depicted as scatter plots interleaved with bar plots, indicating min. to max. value. All the values are plotted as mean ± SEM. The dashed line indicates the normal fold change value. The PCR data were shown on a log2 scale and analysed by unpaired two-way ANOVA (Bonferroni’s multiple comparison test). Comparisons were considered significant at p ≤ 0.05.

Figure 4

Correlation between KRAS gene and CTLA-4 expression in CTCs. (A) The mRNA expression level of CTLA-4 correlated positively with KRAS (r = 0.6878, p = 0.0002). r; coefficient correlation value (Spearman’s rank test). (B) The mRNA expression levels of CTLA-4 in CTCs and PBMCs from patients with CRC according to the KRAS mutation status of the primary tumour.

Figure 5

Relationship of the gene expressions (P53, APC, KRAS, c-Myc, CD47 and CTLA-4) between high and low CTC groups in CTCs and PBMCs, in patients with CRC.

Figure 6

Heat map depicting the mRNA expression level and percentage of positive expression of the tumour suppressor genes p53 and APC; the oncogenes, KRAS and c-Myc; and the immune-regulatory molecules CD47 and CTLA-4 in CTCs and PBMCs among individual patients. The values were calculated from the log2 value of the relative quantification of each gene. The colour indicates the expression level for each gene. Red fields represent downregulated genes; blue fields represent upregulated genes; crossed-out fields represent no expression.