Objective Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers. Design Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing. Results CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival. Conclusions Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues.

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.

Increasing evidence that cancers originate from small populations of so-called cancer stem cells (CSCs), capable of surviving conventional chemotherapies and regenerating the original tumor, urges the development of novel CSC-targeted treatments. Screening of new anticancer compounds is conventionally conducted on established tumor cell lines, providing sufficient material for high-throughput studies. Whether tumor cell lines might comprise CSC populations resembling those of primary tumors, however, remains highly debated. We have analyzed the expression of defined phenotypic profiles, including CD133+, CD166+CD44+, and CD24+CD44+, reported as CSC-specific in human primary colorectal cancer (CRC), on a panel of 10 established CRC cell lines and evaluated their correlation with CSC properties. None of the putative CSC phenotypes consistently correlated with stem cell-like features, including spheroid formation ability, clonogenicity, aldehyde dehydrogenase-1 activity, and side population phenotype. Importantly, CRC cells expressing putative CSC markers did not exhibit increased survival when treated with chemotherapeutic drugs in vitro or display higher tumorigenicity in vivo. Thus, the expression of CD133 or the coexpression of CD166/CD44 or CD24/CD44 did not appear to reliably identify CSC populations in established CRC cell lines. Our findings question the suitability of cell lines for the screening of CSC-specific therapies and underline the urgency of developing novel platforms for anticancer drug discovery.

OX40 expression enhances the prognostic significance of CD8 positive lymphocyte infiltration in colorectal cancer