Tumour Cell Apoptosis Modulates the Colorectal Cancer Immune Microenvironment Via Interleukin-8-dependent Neutrophil Recruitment

Sporadic apoptosis of tumour cells is a commonly observed feature of colorectal cancer (CRC) and strongly correlates with adverse patient prognosis. The uptake of apoptotic cell debris by neutrophils induces a non-inflammatory, pro-regenerative, and hence potentially pro-tumorigenic phenotype. In this study, we therefore sought to investigate the impact of apoptotic CRC cells on neutrophils and its consequence on other immune cells of the tumour microenvironment. Apoptosis induced by combined TNFα-treatment and UV-C irradiation, as well as various chemotherapeutic agents, led to a substantial release of neutrophil-attracting chemokines, most importantly interleukin-8 (IL-8), in both primary patient-derived and established CRC cells. Accordingly, conditioned media of apoptotic tumour cells selectively stimulated chemotaxis of neutrophils, but not T cells or monocytes. Notably, caspase-inhibition partially reduced IL-8 secretion, suggesting that caspase activity might be required for apoptosis-induced IL-8 release. Moreover, apoptotic tumour cell-conditioned media considerably prolonged neutrophil lifespan and induced an activated CD66bhighCD11bhighCD62Llow phenotype, comparable to that of tumour-associated neutrophils in CRC patients, as assessed by flow cytometry of dissociated CRC tissues. Immunohistochemical analyses of 35 CRC patients further revealed a preferential accumulation of neutrophils at sites of apoptotic tumour cells defined by the expression of epithelial cell-specific caspase-cleaved cytokeratin-18. The same areas were also highly infiltrated by macrophages, while T cells were virtually absent. Notably, neutrophils induced an M2-like CD86lowCD163+CD206+ phenotype in co-cultured monocyte-derived macrophages and suppressed LPS-induced pro-inflammatory cytokine release. In an in vitro transwell model, IL-8 blockade efficiently prevented neutrophil-induced anti-inflammatory macrophage polarisation by inhibiting neutrophil migration towards IL-8 gradients generated by apoptotic CRC cells. To conclude, our data suggest that apoptotic cancer cells release chemotactic factors that attract neutrophils into the tumour, where their interaction with neighbouring macrophages might promote an immunologically unfavourable tumour microenvironment. This effect may contribute to tumour recurrence after chemotherapy-induced apoptosis.