New therapeutic strategies for peritoneal carcinomatosis, a preclinical approach

Abstract

About 25-35 % of patients with colorectal cancer (CRC) will develop peritoneal carcinomatosis (PC) at some point in time after initial diagnosis. PC has a very poor prognosis. The gold standard treatment for these patients is cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC). HIPEC enables usage of higher chemotherapy doses, and the heated solution may increase the absorption of the drugs into tumor cells. Consequently, this could remove all microscopic tumor and free cancer cells after CRS. CRS-HIPEC have improved the overall survival (OS) of PC-CRC patients, however most patients experience relapse and treatment associated morbidity and mortality. Relapse is caused by residual cancer cells, which may have developed resistance against chemotherapy/hyperthermia. Thermo-tolerance in cancer is usually based on the induction of heat-shock proteins (HSPs). In this master thesis, we aimed to investigate the sensitizing effect of hyperthermia on the chemotherapy drugs, Mitomycin C and Oxaliplatin, which are commonly used in the clinic HIPEC procedure to treat PC-CRC, was studied in our newly developed in vitro model, which closely mimic the clinical HIPEC condition. An ex vivo model on CRC-PC tumor tissues samples, was also tested in attempts to bring our results even closer to the clinic. Methods: For this experiment, we used two CRC cell lines HCT116 and HT29 with different mutation profiles, and fresh PC-tumor tissues from patient-derived xenograft (PDX)-mice and one patient. Hyperthermic chemotherapy experiments were performed in temperature controlled water baths, 37°C and 42°C. HSP90 inhibitor (17-AAG) and HSP70 inhibitor (HS-72) were included in the experiments, in effort to enhance the cytotoxicity effect of the treatments. In addition, HSF1 was silenced using a short interfering RNA (siRNA). Cell viability assay was performed by MTS-assay, either 24, 48 or 72 hours post treatment. Furthermore, we investigated the effect of hyperthermia to induces immunogenic cell death (ICD) by measuring extracellular HMGB1. HSPs, HSF1 and HMAGB1 expression was measured using Western blot. Results: Treatment response including hyperthermia was observed to be cell line and chemotherapy drug selective. We found HT29 to be less sensitive than HCT116 cells to both drugs at 37°C. HSPs inhibition did not provide additional effect on the treatment. 17AAG itself did contribute to significant cell viability reduction, but addition of hyperthermia did not further decrease in cell viability. Silencing HSF1 did not increase the effect of hyperthermic chemotherapy treatment, which was our hypothesis. Interestingly, we found hyperthermic chemotherapy to increase a much higher HMGB1 release compared to cells treated at 37℃, indicating immunogenic cell death. Conclusion: We conclude that hyperthermia has some form of beneficial effect, depending on the drug and/or cancer cell populations examined. Hyperthermia induces high expression of HSPs, however, inhibition of HSPs by HSP inhibitors and silencing HSF1, did not enhance the cytotoxic effect of hyperthermic chemotherapy. Interestingly, hyperthermic chemotherapy increases HMGB1 release, which indicates immunogenic cell death. This could perhaps verify the beneficial effect of hyperthermia in HIPEC treatment. However, further studies, using other cytotoxicity assay are required to validate our result.