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Background Sub-ablative heat induces pleiotropic biological effects in cancer cells, activating programmed cell death or survival processes. These processes decide the fate of the heated cell. This study investigates these and assesses whether heat, in combination with HSP90 inhibition, augments cell death and induces a pro-immune phenotype in these cells. Methods HCT116 and HT29 cells were subjected to thermal doses (TID) of 60 and 120CEM43 using a PCR thermal cycler. HSP90 was inhibited with NVP-AUY922. Viability was assessed using the MTT assay. Cellular ATP and HSP70 release were assessed using ATP and Enzyme-linked Immunosorbent assays, respectively. Flow cytometry and immunoblotting were used to study the regulation of biomarkers associated with the heat shock response, the cell cycle, and immunogenic and programmed cell death. Results Exposure of HCT116 and HT29 cells to TIDs of 60 and 120CEM43 decreased their viability. In addition, treatment with 120CEM43 increased intracellular HSP70 and the percentage of HCT116/HT29 cells in the G2/M cell cycle phase, ATP release and Calreticulin/HSP70/HSP90 exposure in the plasma membrane, while downregulating CD47 compared to sham-exposed cells. When combined with NVP-AUY922, treatment of HCT116/HT29 cells with 120CEM43 resulted in a synergistic decrease of cell viability associated with the induction of apoptosis. Also, the combined treatments increased Calreticulin exposure, CD47 downregulation, and HSP70 release compared to the sham-exposed cells. Conclusion Sub-ablative heating can act synergistically with the clinically relevant HSP90 inhibitor NVP-AUY922 to induce a pro-immunogenic form of cell death in colon cancer cells.

Pulsed high-intensity focused ultrasound (pHIFU) uses acoustic pressure to physically disrupt tumours. The aim of this study was to investigate whether pHIFU can be used in combination with immune checkpoint inhibitors (ICIs) to enhance survival of tumour-bearing animals. Murine orthotopic pancreatic KPC tumours were exposed both to a grid of pHIFU lesions (peak negative pressure = 17 MPa, frequency = 1.5 MHz, duty cycle = 1%, 1 pulse s−1, duration = 25 s) and to anti-CTLA-4/anti-PD-1 antibodies. Acoustic cavitation was detected using a weakly focused passive sensor. Tumour dimensions were measured with B-mode ultrasound before treatment and with callipers post-mortem. Immune cell subtypes were quantified with immunohistochemistry and flow cytometry. pHIFU treatment of pancreatic tumours resulted in detectable acoustic cavitation and increased infiltration of CD8+T cells in the tumours of pHIFU and pHIFU + ICI-treated subjects compared with sham-exposed subjects. Survival of subjects treated with pHIFU + ICI was extended relative to both control untreated subjects and those treated with either pHIFU or ICI alone. Subjects treated with pHIFU + ICI had increased levels of CD8+IFNγ+T cells, increased ratios of CD8+IFNγ+to CD3+CD4+FoxP3+and CD11b+Ly6G+cells, and decreased CD11chighcells in their tumours compared with controls. These results provide evidence that pHIFU combined with ICI may have potential for use in pancreatic cancer therapy.

Aim: Thermal isoeffective dose (TID) has not been convincingly validated for application to predict biological effects from rapid thermal ablation (e.g., using >55 °C). This study compares the classical method of quantifying TID (derived from hyperthermia data) with a temperature-adjusted method based on the Arrhenius model for predicting cell survival in vitro, after either ‘rapid’ ablative or ‘slow’ hyperthermic exposures. Methods: MTT assay viability data was obtained from two human colon cancer cell lines, (HCT116, HT29), subjected to a range of TIDs (120–720 CEM43) using a thermal cycler for hyperthermic (>2 minutes, 55 °C). TID was initially estimated using a constant RCEM>43°C=0.5, and subsequently using RCEM(T), derived from temperature dependent cell survival (injury rate) Arrhenius analysis. Results: ‘Slow’ and ‘rapid’ exposures resulted in cell survival and significant regrowth (both cell lines) 10 days post-treatment for 240 CEM43 (RCEM>43°C=0.5), while 340-550 CEM43 (RCEM>43°C =0.5) delivered using ‘rapid’ exposures showed 12 ± 6% viability and ‘slow’ exposures resulted in undetectable viability. Arrhenius analysis of experimental data (activation energy ΔE = 5.78 ± 0.04 × 105 J mole−1, frequency factor A = 3.27 ± 11 × 1091 sec−1) yielded RCEM=0.42 * e0.0041*T which better-predicted cell survival than using R CEM> 43°C=0.5. Conclusions: TID calculated using an RCEM(T) informed by Arrhenius kinetic parameters provided a more consistent, heating strategy independent, predictor of cell viability, improving dosimetry of ablative thermal exposures. Cell viability was only undetectable above 305 ± 10 CEM43 using this revised measure.

The pleiotropic effects of heat on cancer cells have been well documented. The biological effects seen depend on the temperature applied, and the heating duration. In this study we investigate the cytotoxic effects of heat on colon cancer cells and determine how different cell death processes such as autophagy, apoptosis and necroptosis play a role in cell response.The thermal dose concept was used to provide a parameter that will allow comparison of different thermal treatments. Two human colon cancer cell lines, HCT116 and HT29, were subjected to ablative temperatures using a polymerase chain reaction thermal cycler. Temperature was recorded using thermocouples. Cell viability was assessed using the MTT assay. Induction of apoptosis was estimated using an enzyme-linked immunosorbent assay that detects cleaved cytoplasmic nucleosomes. Protein regulation was determined using immunoblotting. The percentage of cells undergoing apoptosis and autophagy was determined with annexin V/propidium iodide staining and a cationic amphiphilic tracer using fluorescence-activated cell sorting analysis.Exposure of colon cancer cells to ablative thermal doses results in decreased cell viability. The cytotoxic effect of heat is associated with induction of apoptosis and autophagy, the amount depending on both the thermal dose applied and on the time elapsed after treatment. Autophagy induction is mainly seen in live cells. RIPK3 protein levels are increased after exposure of cells to heat. A necroptosis inhibitor does not affect cell viability.Autophagy, apoptosis and necroptosis are associated with the response of these cancer cell lines to supra-normal temperatures.