Hypotonic stress enhances colon cancer cell death induced by platinum derivatives and immunologically improves antitumor efficacy of intraperitoneal chemotherapy.

Colorectal cancer is a highly metastatic disease that could invade various distal organs and also the peritoneal cavity leading to peritoneal carcinomatosis. This is a terminal condition with poor prognosis and only palliative treatments such as cytoreductive surgery and intraperitoneal chemotherapy are proposed to some patients. However, clinicians use different parameters of treatments without any consensus. Here we decided to evaluate the effect of osmolarity in the efficacy of this procedure to kill colon cancer cells. We first show that a short exposure of platinum derivatives in hypotonic conditions is more efficient to decrease cell viability of human and murine colon cancer cells in vitro as compared to isotonic conditions. This is related to more important incorporation of platinum and the capacity of hypotonic stress to induce the copper transporter CTR1 oligomerization. Oxaliplatin in hypotonic conditions induces caspase-dependent cell death of colon cancer cells. Moreover, hypotonic conditions also modulate the capacity of oxaliplatin and cisplatin (but not carboplatin) to induce immunogenic cell death (ICD). In vivo, oxaliplatin in hypotonic conditions increases CD8+ T cell tumor infiltration and activation. Finally, in a murine peritoneal carcinomatosis model, oxaliplatin in hypotonic conditions is the only tested protocol which is able to slow down the appearance of tumor nodules and increase mice survival, while showing no effect in CD8+ T cells depleted mice or in immunodeficient mice. Altogether, our study provides new information both in vitro and in a preclinical model of peritoneal carcinomatosis, which highlights the importance of hypoosmolarity in intraperitoneal chemotherapy.

Heat shock and HSP70 regulate 5-FU-mediated caspase-1 activation in myeloid-derived suppressor cells and tumor growth in mice.

BACKGROUND: We have previously shown that 5-fluorouracil (5-FU) selectively kills myeloid-derived suppressor cells (MDSCs) and activates NLRP3 (NOD-leucine rich repeat and pyrin containing protein 3) inflammasome. NLRP3 activation leads to caspase-1 activation and production of IL-1ß, which in turn favors secondary tumor growth. We decided to explore the effects of either a heat shock (HS) or the deficiency in heat shock protein (HSP) 70, previously shown to respectively inhibit or increase NLRP3 inflammasome activation in macrophages. METHODS: Caspase-1 activation was detected in vitro in MSC-2 cells by western blot and in vivo or ex vivo in tumor and/or splenic MDSCs by flow cytometry. The effects of HS, HSP70 deficiency and anakinra (an IL-1 inhibitor) on tumor growth and mice survival were studied in C57BL/6 WT or Hsp70-/- tumor-bearing mice. Finally, Th17 polarization was evaluated by qPCR (Il17a, Rorc) and angiogenic markers by qPCR (Pecam1, Eng) and immunohistochemistry (ERG). RESULTS: HS inhibits 5-FU-mediated caspase-1 activation in vitro and in vivo without affecting its cytotoxicity on MDSCs. Moreover, it enhances the antitumor effect of 5-FU treatment and favors mice survival. Interestingly, it is associated to a decreased Th17 and angiogenesis markers in tumors. IL-1ß injection is able to bypass HS+5-FU antitumor effects. In contrast, in Hsp70-/- MDSCs, 5-FU-mediated caspase-1 activation is increased in vivo and in vitro without effect on 5-FU cytotoxicity. In Hsp70-/- mice, the antitumor effect of 5-FU was impeded, with an increased Th17 and angiogenesis markers in tumors. Finally, the effects of 5-FU on tumor growth can be restored by inhibiting IL-1ß, using anakinra. CONCLUSION: This study provides evidence on the role of HSP70 in tuning 5-FU antitumor effect and suggests that HS can be used to improve 5-FU anticancer effect.