Abstract
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.
Highlights
Spontaneous apoptosis of tumour cells is frequently observed in colorectal cancer (CRC), presumably as a consequence of genomic instability and enhanced cellular turnover [1]
To confirm the apoptotic tumour cell-mediated impact debris, resulting in a non-inflammatory, pro-regenerative polarisa- on neutrophil viability in a culture model more accurately tion [10, 11], we hypothesised that the accumulation of cell debris reflecting the in vivo CRC microenvironment, neutrophils were as a consequence of spontaneous or therapy-induced apoptosis embedded in a 3D collagen matrix together with CG08 tumour modulates the neutrophil phenotype in the CRC microenviron- cells and cancer-associated fibroblasts
IL-8 blockade almost completely abrogated neutrophil migration towards conditioned media (CM) of apoptotic CG08 (Fig. 3D) and HT29 cells (Fig. S6), while concomitantly, neutrophil viability remained unaffected (Fig. 3E). These findings indicate that IL-8 blockade is capable of effectively preventing neutrophil migration towards apoptotic CRC cells, while factors other than IL-8 are responsible for the observed survival prolongation
Summary
Spontaneous apoptosis of tumour cells is frequently observed in colorectal cancer (CRC), presumably as a consequence of genomic instability and enhanced cellular turnover [1]. Despite being traditionally regarded as a purely pro-inflammatory cell population, numerous studies suggest that neutrophils are functionally and phenotypically heterogeneous [6, 7] This is exemplified by the indispensable functions of neutrophils in the orchestration of inflammation resolution and tissue repair, such as the generation of pro-resolving lipid mediators or induction of a pro-regenerative macrophage polarisation with enhanced efferocytosis capacity [8, 9]. To confirm the apoptotic tumour cell-mediated impact debris, resulting in a non-inflammatory, pro-regenerative polarisa- on neutrophil viability in a culture model more accurately tion [10, 11], we hypothesised that the accumulation of cell debris reflecting the in vivo CRC microenvironment, neutrophils were as a consequence of spontaneous or therapy-induced apoptosis embedded in a 3D collagen matrix together with CG08 tumour modulates the neutrophil phenotype in the CRC microenviron- cells and cancer-associated fibroblasts.
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