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. 2020 Oct;69(10):1989-1999.
doi: 10.1007/s00262-020-02593-w. Epub 2020 May 11.

Expression of immune checkpoints and T cell exhaustion markers in early and advanced stages of colorectal cancer

Reem Saleh  1 , Rowaida Z Taha  1 , Salman M Toor  1 , Varun Sasidharan Nair  1 , Khaled Murshed  2 , Mahwish Khawar  3 , Mahmood Al-Dhaheri  3 , Mahir Abdulla Petkar  2 , Mohamed Abu Nada  3 , Eyad Elkord  4
Affiliations

Expression of immune checkpoints and T cell exhaustion markers in early and advanced stages of colorectal cancer

Reem Saleh et al. Cancer Immunol Immunother. 2020 Oct.

Abstract

Despite recent advances in colorectal cancer (CRC) treatment, a large proportion of patients show limited responses to therapies, especially in advanced stages. There is an urgent need to identify prognostic biomarkers and/or therapeutic targets in advanced stages, aiming to improve the efficacy of current treatments. We aimed to determine prognostic biomarkers in tumor tissue and circulation of CRC patients, with a special focus on T cell exhaustion markers. We found that mRNA levels of PD-1, TIM-3, CTLA-4, TIGIT, CD160, CD244, KLRG1, TOX2, TOX3, Ki-67, and PRDM1 were elevated in CRC tumor tissues. We also investigated differences in gene expression between early and advanced disease stages. We found that TOX and potentially TIM-3, CTLA-4, VISTA, TIGIT, KLRG1, TOX2, SIRT1, Ki-67, and Helios mRNA levels in tumor tissue were elevated in advanced disease stages, suggesting their potential roles in CRC progression. In contrast, PD-1 and CD160 levels in tumor tissue were downregulated in advanced stages. In the circulation of CRC patients, mRNA levels of PD-1, VISTA and LAG-3 were higher than those of healthy individuals. Moreover, in circulation, PD-1, CTLA-4 and TIGIT mRNA levels were reduced in advanced stages. Interestingly, levels of PD-1 in both tumor tissue and circulation were reduced in advanced stages, suggesting that targeting PD-1 in patients with advanced stages could be less effective. Altogether, these findings suggest some potential T cell exhaustion markers that could be utilized as prognostic biomarkers and/or therapeutic targets for CRC. However, further investigations and validations in larger cohorts are required to confirm these findings.

Keywords: Colorectal cancer; Immune checkpoints; Prognostic biomarker; T cell exhaustion; Therapeutic target.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Gene expression of immune checkpoints, T cell exhaustion-related markers and cancer progression-related markers in colorectal cancer tissues. RNA was isolated from normal and tumor tissues of 70 patients, and reverse transcribed to cDNA. Quantitative RT-PCR was performed to assess the expression levels of immune checkpoints (PD-1, CTLA-4, TIM-3, VISTA, CTLA-4, LAG-3, TIGIT, CD160, CD244, and KLRG1) (a), TOX family members (TOX, TOX2, TOX3, and TOX4) (b), and T cell exhaustion-related markers (SIRT1, Ki-67, Helios and PRDM1) (c). Scatter plots show expression levels in TT normalized to NT. The relative expression of each gene was calculated by normalization with β-actin. Each dot represents the relative expression value of a particular gene of an individual patient. Mean ± standard error of the mean (SEM) is shown in red
Fig. 2
Fig. 2
Gene expression of immune checkpoints, and T cell exhaustion and cancer progression-related markers in colorectal cancer tissues from patients at early and advanced disease stages. Scatter plots show the expression of markers in early (I & II) and advanced (III & IV) disease stages (CRC patients n = 70, stage I, n = 5; stage II, n = 24; stage III, n = 30; stage IV, n = 11). Scatter plots show the expression levels of immune checkpoints (PD-1, TIM-3, CTLA-4, VISTA, LAG-3, TIGIT, CD160, CD244 and KLRG1) (a); TOX family members (TOX, TOX2, TOX3 and TOX4) (b); other T cell exhaustion markers (SIRT1, Ki-67, Helios and PRDM1) (c) in tumor tissues of CRC patients. Results are presented as fold change (FC) of gene expression in TT versus NT. Means ± standard error of the means (SEM) are depicted on the scatter plots
Fig. 3
Fig. 3
Gene expression of immune checkpoints, T cell exhaustion and cancer progression-related markers in the circulation of colorectal cancer patients at early and advanced disease stages. RNA was isolated from peripheral blood mononuclear cells (PBMCs) of 30 healthy donors and 68 CRC patients, and reverse transcribed to cDNA. Quantitative RT-PCR was performed to assess the expression levels of immune checkpoints (PD-1, TIM-3, VISTA, CTLA-4, LAG-3, and TIGIT) and T cell exhaustion markers (TOX, TOX2 and SIRT1). Bar plot shows relative expression of each gene, normalized to average expression level recorded for healthy donors (a). Scatter plots show the expression levels of PD-1, TIM-3, VISTA, CTLA-4, LAG-3, TIGIT, TOX, TOX2, and SIRT1 between early (I & II) and advanced (III & IV) disease stages (stage I, n = 10; stage II, n = 23; stage, III n = 24; stage IV, n = 11) (b). Results are presented as fold change (FC) of gene expression in CRC versus HD. Results are presented as mean ± standard error of the mean (SEM)
Fig. 4
Fig. 4
Paired comparison of gene expression levels in the circulation and tumor tissues of CRC patients. Scatter plots show the paired comparison between the circulation and TT of 56 CRC patients based on the expression level of PD-1, TIM-3, VISTA, CTLA-4, LAG-3, TIGIT, TOX, TOX2, and SIRT1. Results are presented as fold change (FC) of gene expression in TT versus NT for tissue and CRC versus HD for circulation
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