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

Abstract

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.Additional Supporting Information may be found in the online version of this article.

Platinum Derivatives Effects on Anticancer Immune Response

“…In our recent work, we performed in vitro experiments on CT26 murine colon cancer cells with a brief exposure of cancer cells to chemotherapy to reproduce clinical settings and, in particular, hyperthermic intraperitoneal chemotherapy (HIPEC). Under these conditions (30 min exposure and 24 h resting in medium), oxaliplatin had no effect on ICD, contrarily to what has been previously observed [25]. This discrepancy can be explained by the time of exposure.…”

“…To detect this feature, non-permeabilized cells are stained with a specific anti-CRT antibody and with DAPI (4 ,6-diamidino-2-phenylindole) or propidium iodide. Because anti-CRT can recognize both extra and intra-cellular CRT, it is imperative to remove DAPI + or PI + cells (corresponding to cells with permeabilized membranes) to be sure to detect only externalized CRT [9,25,26]. After staining, cells can also be observed under a microscope.…”

“…ER stress is responsible for CRT translocation from the ER to the cell membrane. So, an indirect way to evaluate this phenomenon is to analyze ER stress response, such as eIF2α phosphorylation by western blot [25], XBP1 (X-box binding protein 1) mRNA splicing by real-time qPCR [29], or ATF6 (activating transcription factor 6) nuclear translocation by fluorescence microscopy [30].…”

“…Hence, the more ATP is present in the supernatant or in cell lysates, the more light is produced. ATP secretion can be determined by an increase in the supernatant, a decrease in cells, or both [25,31]. Quinacrine (a fluorescent probe which can bind ATP) can also be used to detect intracellular ATP levels by fluorescence microscopy [32].…”

“…HMGB1 release in the cell supernatant can be monitored using specific commercialized ELISA kits [25]. Since HMGB1 first translocates from the nucleus to the cytoplasm before release, HMGB1 release can alternatively be assessed by fluorescence microscopy.…”