The aim of this study was to elucidate the prognostic impact of putative cancer stem cell markers CD133, CD166, CD44s, EpCAM, and aldehyde dehydrogenase-1 (ALDH1) in colorectal cancer. A tissue microarray of 1420 primary colorectal cancers and 57 normal mucosa samples was immunostained for CD133, CD166, CD44s, EpCAM, and ALDH1 in addition to 101 corresponding whole tissue sections. Invasive potential of three colorectal cancer cell lines was tested. Differences between normal tissue and cancer were observed for all markers (P<0.001). Loss of membranous CD166 and CD44s were linked to higher pT (P=0.002, P=0.014), pN (P=0.004, P=0.002), an infiltrating growth pattern (P<0.001, P=0.002), and worse survival (P=0.015, P=0.019) in univariate analysis only. Loss of membranous EpCAM expression was also linked to higher pN (P=0.023) and infiltrating growth pattern (P=0.005). The CD44s, CD166, and EpCAM expression were lost towards the invasive front. The CD44−/CD166− cells from three colorectal cancer cell lines exhibited significantly higher invasive potential in vitro than their positive counterparts. Loss, rather than overexpression, of membranous CD44s, CD166, and EpCAM is linked to tumour progression. This supports the notion that the membranous evaluation of these proteins assessed by immunohistochemistry may be representative of their cell adhesion rather than their intra-cellular functions.

Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment

Purpose: Tumor infiltration by different T lymphocyte subsets is known to be associated with favorable prognosis in colorectal cancer. Still debated is the role of innate immune system. We investigated clinical relevance, phenotypes, and functional features of colorectal cancer–infiltrating CD66b⁺ neutrophils and their crosstalk with CD8⁺ T cells. Experimental Design: CD66b⁺ and CD8⁺ cell infiltration was analyzed by IHC on a tissue microarray including >650 evaluable colorectal cancer samples. Phenotypic profiles of tissue-infiltrating and peripheral blood CD66b⁺ cells were evaluated by flow cytometry. CD66b⁺/CD8⁺ cells crosstalk was investigated by in vitro experiments. Results: CD66b⁺ cell infiltration in colorectal cancer is significantly associated with increased survival. Interestingly, neutrophils frequently colocalize with CD8⁺ T cells in colorectal cancer. Functional studies indicate that although neutrophils are devoid of direct antitumor potential, coculture with peripheral blood or tumor-associated neutrophils (TAN) enhances CD8⁺ T-cell activation, proliferation, and cytokine release induced by suboptimal concentrations of anti-CD3 mAb. Moreover, under optimal activation conditions, CD8⁺ cell stimulation in the presence of CD66b⁺ cells results in increasing numbers of cells expressing CD45RO/CD62L “central memory” phenotype. Importantly, combined tumor infiltration by CD66b⁺ and CD8⁺ T lymphocytes is associated with significantly better prognosis, as compared with CD8⁺ T-cell infiltration alone. Conclusions: Neutrophils enhance the responsiveness of CD8⁺ T cells to T-cell receptor triggering. Accordingly, infiltration by neutrophils enhances the prognostic significance of colorectal cancer infiltration by CD8⁺ T cells, suggesting that they might effectively promote antitumor immunity. Clin Cancer Res; 23(14); 3847–58. ©2017 AACR.

Background The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. Objective To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. Methods IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. Results IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. Conclusions Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.