The immune landscape in colorectal cancer: identifying exhaustion markers on tumor infiltrating CD8 using multiplex immunohistochemistry

Abstract

Patients with colorectal cancer (CRC) have a significant proportion of exhausted tumor infiltrating CD8, and that would reduce the efficacy of the immune response. Exhausted T cells characterized by impaired cytokines secretion, decreased cytotoxicity and upregulation of inhibitory receptors such as T cell immunoglobulin and mucin-3 (TIM-3), cytotoxic lymphocyte-activation gene - (LAG-3) and programmed cell death-1 (PD-1). Accordingly, this study aimed to identify the expression of different exhaustion markers on tumor infiltrating CD8 in CRC tissue samples. Chromogenic immunohistochemistry (IHC) staining was used to optimize the concentrations of anti-LAG-3 and anti-TIM-3 using a whole CRC tissue and tissue microarrays (TMAs). Consequently, optimized concentrations were used in multiplex immunohistochemistry (IHC) staining to identify the expression of different exhaustion markers on the surface of tumor infiltrating CD8. We found that concentrations of 1:100 of anti-LAG-3 and 1:400 of anti- TIM-3 were the ideal ratios to provide positive expression with limited background staining in chromogenic IHC. Additionally, our study revealed that multiplex IHC staining had successfully demonstrated the presence of double positive cells including CD8+LAG-3+, CD8+TIM-3+, and CD8+PD- 1+. The presence of triple positive cells was detected in multiplex IHC including CD8+TIM-3+PD-1+ and CD8+TIM-3+LAG-3+. In conclusion, after a successful optimization, this study was effectively recognized the expression of different exhaustion markers on tumor infiltrating CD8, and the expression of triple positive cells considered to be more exhausted than double positive cells. Hence, we suggest further research should be conducted to investigate more exhaustion markers.