The AGS and Caco-2 cell lines were purchased from American Type Cell Culture (ATCC Manassas, VA, United States). AGS cell line is derived from a gastric adenocarcinoma of the stomach of a 54 year-old Caucasian female with no prior anti-cancer treatment. Caco-2 cells were isolated from a primary colonic tumor in a 72-year-old Caucasian male using the explant culture technique. Forms moderately well differentiated adenocarcinomas consistent with colonic primary grade II, in nude mice. T3M4 cell line was obtained as a gift from the European Pancreas Center (Heidelberg, Germany). This cell line was derived from a lymph node metastasis of the Japanese male patient, diagnosed with pancreatic ductal adenocarcinoma. It is characterized as pancreatic adenocarcinoma producing CEA, K-ras activated, and with slow cell growth. Cells were grown in RPMI medium (Gibco/Invitrogen, Carlsbad, CA, United States) with the addition of 10% fetal bovine serum (Gibco/Invitrogen) and 1% penicillin/streptomycin solution (Gibco/Invitrogen). Flasks with cells were cultured in a humid incubator with a CO₂ level of 5% and temperature of 37 °C.

Cancer cells were cultivated for 24 h in the conditions described above. Afterwards, cells were treated by one of two separate factors: temperature (37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C) or IC₅₀ dose of cisplatin, which was specifically determined for each cell line. Moreover, the combination of hyperthermia and cisplatin treatment was applied (Figure 1). The duration of treatment was 1 h as it reflected the treatment time under the clinical conditions of HIPEC. Cells were heated in humid incubators at particular temperature regimens. When temperature of the media reached desired level, we started the countdown of one hour exposure. Medium temperature was controlled by the electronic thermometer. Following the treatment, the medium was changed, and cells were cultivated for 48 h under the conditions as previously described. Afterwards, MTT and flow cytometry were performed (see “MTT assay” and “Cell apoptosis”). All experiments were repeated at least three times.

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Figure 1 Design of experiment.

Cytotoxicity of cisplatin or/and hyperthermia was determined by MTT [3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide] (Gibco/Invitrogen) assay. The plate was incubated for 4 h at 37 °C after 5 mg/mL of the MTT reagent was added to the wells. Then the supernatant was removed, and DMSO (dimethyl sulfoxide) (Carl Roth GmbH, Karlsruhe, Germany) was added to solubilize the resulting formazan crystals. Absorbance measurements were made at 570 nm on the Sunrise spectrophotometer (Tecan GmbH, Grodig, Austria).

The changes in apoptosis were evaluated after treatment of the cells by hyperthermia (43 °C) and the previously determined IC₅₀ of cisplatin (specific for particular cell line). Untreated cisplatin cells and normothermia (37 °C) cultivated cells were defined as controls. Forty-eight hours after the experiment (Figure 1), the rate of apoptosis was determined by flow cytometer Guava Nexin Annexin V Assay (Merck, Millipore United States and Canada). All the procedures were performed according to the manufacturer’s instructions. Samples were measured using a Guava Personal Cell Analysis (PCA) Flow Cytometer (Merck, Millipore) and CytoSoft software.

Immediately after the experiment, the cell lysate samples were diluted ten times with high purity deionized water. Concentration of total intracellular cisplatin was analyzed by inductively coupled plasma mass spectrometer (ICP-MS) NexION™ 300D (PerkinElmer, United States) equipped with nickel cones and a quartz cyclonic spray chamber as a sample introduction system and using collision mode kinetic energy discrimination (KED) with helium gas (purity ≥ 99.999%) to remove polyatomic interferences. The calibration graphs for determination of Platinum by ICP-MS were prepared in the range of 0-50 ng/mL using Pure Plus 10 mg/L Multi-Element calibration standard 4 (PerkinElmer, United States). The working conditions of the spectrometer were optimized daily in order to obtain the maximal sensitivity and stability.

To identify the confidence limits for the additive action of hyperthermia and cisplatin, isobologram analysis was applied. Data for analysis was obtained from the MTT test. Temperature data points of 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, and 45 °C, and cisplatin concentration data points of 25 μmol/L, 50 μmol/L, 100 μmol/L, 200 μmol/L, and 400 μmol/L were used to define the dose effect curve for temperature and cisplatin, respectively. For combined treatment analysis, the following combinations of factors were used: 39 °C + 50 μmol/L; 39 °C + 100 μmol/L; 39 °C + 200 μmol/L; 40 °C + 50 μmol/L; 40 °C + 100 μmol/L; 40 °C + 200 µmol/L; 41 °C + 50 μmol/L; 41 °C + 100 μmol/L; 41 °C + 200 μmol/L; 42 °C + 50 μmol/L; 42 °C + 100 μmol/L; 42 °C + 200 μmol/L; 43 °C + 50 μmol/L; 43 °C + 100 μmol/L; 43 °C + 200 μmol/L; 44 °C + 50 μmol/L; 44 °C + 100 μmol/L; 44 °C + 200 μmol/L. The Chou median effect equation was used[16]. It provides the theoretical basis for the combination index (CI), which enabled quantification of drug interaction affects defining synergism (CI < 1), additivity (CI = 1), or antagonism (CI > 1). The CompuSyn software (CompuSyn, Inc., Paramus, NJ, United States) was used for calculations.

To evaluate the effect of hyperthermia per se on different cancer cells in vitro, we examined the response of the AGS, Caco-2, and T3M4 cell lines to temperature using a stepwise increase of one degree ranging from 37 °C to 45 °C. Cell viability was determined by MTT assay. Every cell line demonstrated a different pattern of response to hyperthermia (Figure 2). AGS cells were the most sensitive one to hyperthermia, where a temperature rise from 41 °C to 45 °C gradually decreased its viability by 30%. In the 37 °C to 41 °C interval, the viability stayed constant. Cells affected by 43 °C, 44 °C, and 45 °C dropped their viability rate by 16%, 22 °C, and 30%, respectively, as compared to the control (37 °C). A temperature increase from 37 °C to 42 °C had no significant effect on Caco-2 cells, but at 43 °C and 44 °C, its viability dropped by 14% and 20%, respectively, but stayed constant at higher temperatures. The response of T3M4 cells to the changes of temperature was different. Temperatures from 37 °C to 42 °C decreased its viability by 30%. At 43 °C, the viability of T3M4 cells sharply increased by 20% and stayed at this level in higher temperatures. Overall, only the AGS cell line responded to hyperthermia as implied, with gradual inhibition of cell viability.

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Figure 2 The different effect of the temperature on cancer cell viability. Following a one-hour exposure of AGS, Caco-2, and T3M4 cells to temperatures ranging from 37 °C to 42 °C, there were no significant viability changes. Starting at 43 °C, viability gradually decreased in AGS and Caco-2 cells. Activation of viability was evident in T3M4 cells at temperatures of 43 °C and 44 °C. All data were compared with a control group (viability of cells at temperature of 37 °C set as 100%). Statistical analysis was performed using the Mann-Whitney U test comparing selected groups to the control. ^aP < 0.05 vs control.

Cisplatin induced a dose-dependent suppressor effect on cancer cell viability, with a similar linear response in the AGS, Caco-2, and T3M4 cell lines. Pancreatic cancer cells (T3M4) were the most sensitive, revealing cisplatin IC₅₀ at 48 μmol/L. The IC₅₀ for Caco-2 was 194 μmol/L, and for AGS, it was 182 μmol/L (Figure 3).

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Figure 3 The influence of different cisplatin doses on cancer cell viability. Exposure to gradually increasing doses of cisplatin resulted in a linear decrease of viability in all cancer cell lines. T3M4 cells were the most sensitive to cisplatin, followed by Caco-2 and AGS cells. All data were compared with the control group (viability of cells at 37 °C set as 100%). Data are presented as the mean ± SE from ≥ 3 replicates.

Previously determined IC₅₀ doses of cisplatin were used for particular cell lines during this experiment. Simultaneous exposure of cells to hyperthermic conditions and cisplatin triggered different response in AGS, Caco-2, and T3M4 cells (Figure 4). Overall, temperatures ranging from 37 °C to 41 °C had no detectable additive effect to cisplatin cytotoxicity. Interestingly, we observed peaks of viability in AGS (42 °C, increase by 33%) and T3M4 (43 °C, increase by 32%) cells. Higher temperatures, in addition to cisplatin exposure, inhibited cell growth dramatically; AGS viability dropped by 70% and T3M4 dropped by 76% at 45 °C. Application of hyperthermia in addition to cisplatin in the Caco-2 cell line gradually improved the cytotoxic effect and decreased the viability of cells by one-fourth from 43 °C to 45 °C. In summary, application of hyperthermia (43 °C and higher) in addition to cisplatin might enhance its cytotoxic effect in the Caco-2 cell line. However, lower (e.g., < 43 °C) temperature regimens may even promote cell proliferation and worsen expected hyperthermal chemotherapy effects in AGS and T3M4 cell lines.

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Figure 4 Effect of temperature and cisplatin (IC₅₀) combinations on cancer cell viability. The increase of temperature had no significant additional effect on cancer cell viability with the exception of extreme hyperthermia (44 °C-45 °C). Peaks of enhanced viability were observed in AGS and T3M4 cells at temperatures of 42 °C and 43 °C. Data are equalized to control (untreated cells) as 100% and shown in percentages. Dashed line shows IC₅₀.

We constructed isobolograms to reveal if the combined application of hyperthermia and cisplatin had synergistic, antagonistic, or additive effects. Combination of temperature and cisplatin was strongly antagonistic for AGS cells in all studied data points. Combined application of hyperthermia and cisplatin was tripled in Caco-2 cells: synergistic (at 40 °C with 50 μmol/L and 100 μmol/L of cisplatin, at 43 °C with 50 μmol/L, 100 μmol/L, and 200 μmol/L cisplatin, at 44 °C with 50 μmol/L , 100 μmol/L, and 200 μmol/L of cisplatin), additive (at 39 °C with 100 μmol/L of cisplatin, at 41 °C with 100 μmol/L cisplatin, and at 42 °C with 50 μmol/L and 100 μmol/L of cisplatin), and antagonistic (at 39 °C with 200 μmol/L of cisplatin, at 40 °C with 200 μmol/L cisplatin, at 41 °C with 50 μmol/L and 200 μmol/L cisplatin, and at 42 °C with 200 μmol/L of cisplatin). Combined treatment of T3M4 at 41 °C with 200 μmol/L cisplatin, at 42 °C with 50 μmol/L cisplatin, and at 44 °C with 100 μmol/L and 200 μmol/L of cisplatin had an additive effect. The remaining combinations were antagonistic (Figure 5).

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Figure 5 Interaction of temperature and cisplatin using the isobologram method for cancer cell viability. Mathematically revealed combined effects of hyperthermia and cisplatin differ in AGS, Caco-2, and T3M4 cell lines. The isobologram represents the interaction between cisplatin and temperature resulting in inhibition of cancer cell growth. The solid line in the diagram (Combination Index = 1) indicates the alignment of theoretical values of an additive interaction between two effectors and represents calculated conditions required to inhibit cell growth by 50%. Mathematically calculated IC₅₀ for AGS cells: temperature 51 °C, cisplatin 159 μmol/L; Caco-2 cells: temperature 66 °C, cisplatin 328 μmol/L; T3M4 cells: temperature 61 °C, cisplatin 0 μmol/L. Treatment combination points falling above the line indicate mathematical antagonism (below synergism, on the line additivity). Combination points that are distant from the line are not shown. Tables on the right represent the CI of selected combinations.

Annexin V-PE flow cytometry analysis was performed to evaluate rates of early apoptosis. Cisplatin induced early apoptosis in Caco-2 and T3M4 cells 1.5-fold and 3.4-fold, respectively. The number of dead/late apoptotic cells was insignificant in the following groups: 0.5% of Caco-2 cells and 2.9% of T3M4 cells. Hyperthermia of 43 °C in addition to cisplatin induced early apoptosis as compared to cells treated in normothermia by 20% in Caco-2 (1% of dead cells) and 19% (3.9% dead cells) in T3M4, respectively. Interestingly, early apoptosis was not significantly induced by isolated cisplatin treatment in AGS cells. Rates of AGS early apoptosis were not significantly induced by an isolated cisplatin treatment. However, application of combined treatment with hyperthermia and cisplatin increased early apoptosis by 61% (0.4% dead cells) (Figure 6).

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Figure 6 Apoptotic effect of temperature and cisplatin in cancer cells. A: Early apoptosis was significantly induced by hyperthermia in cisplatin-treated cells, with the exception of Caco-2 cells, where temperature had no significant role. The dashed line represents control rates of apoptosis in untreated cells. Statistical analysis was performed using the Mann-Whitney U test. ^aP < 0.05. B: Dot plots presenting change of apoptosis in cisplatin untreated/treated cells and cultivated in hyperthermia. “-cis”: Cisplatin untreated cells; “+cis”: Cisplatin treated cells.

Hypothesizing that hyperthermia promotes delivery of cisplatin to cancer cells, we aimed to measure intracellular cisplatin concentrations. Analysis was performed immediately following a one-hour exposure to the IC₅₀ of cisplatin at 37 °C and 43 °C. Overall, 43 °C promoted an increase of intracellular cisplatin concentration. The concentration was significantly increased in AGS, Caco-2, and T3M4 cells by 30%, 20%, and 18%, respectively (Figure 7).

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Figure 7 The change of the intracellular cisplatin concentration. Temperature of 43 °C significantly increased the intracellular concentration of cisplatin in all analyzed cell lines. Data are shown as concentrations of ng/mL. Statistical analysis was performed using the Mann-Whitney U test. ^aP < 0.05. “+cis”: Cisplatin treated cells.

Hyperthermal intraperitoneal chemotherapy is an option to treat peritoneum invading gastrointestinal cancer. Until now the results of hyperthermal intraperitoneal chemotherapy (HIPEC) treatment are controversy, needing unification in selected parameters of the treatment. In different cancer centers, the procedure varies in time setting, hyperthermia level and different chemotherapy drugs are used.

As HIPEC is widely used in clinical practice, still there is a lack of studies, analyzing the impact of hyperthermia and cisplatin to cancer cells. The cellular response to the treatment is still not clear. There is no clear data defining optimal timing and temperature of the procedure.

Our objective was to analyze gastric, pancreatic and colorectal cancer cells response to hyperthermia and cisplatin treatment regarding viability, change of intracellular cisplatin concentration and apoptosis rate.

We used AGS (gastric cancer), T3M4 (pancreatic cancer) and Caco-2 (colorectal cancer) cells. Mimicking HIPEC procedure, cells were treated with specific to each cell line IC₅₀ dose of cisplatin at the temperature regimens ranging from 37 °C to 45 °C. Treatment lasted for one hour. Later cells were harvested in normothermia, changing cisplatin containing media to fresh one. Immediately after experiment, intracellular cisplatin concentration was measured, using mass spectrometer analysis. For other readouts cells were harvested for 48 hours in normal conditions. MTT test was performed for cellular viability evaluation and isobologram analysis. We used flow cytometry to determine apoptosis change of hyperthermia and cisplatin treatment.

Cells responded to hyperthermia (ranging from 38 °C to 45 °C) in a different manner. Viability of AGS cells was the most hyperthermia-dependent, decreasing by 30% (from 41 °C to 45 °C). Caco-2 cell viability had no change in the interval from 38 °C to 42 °C. Higher temperature regimens dropped its viability rate by 14%-20%. T3M4 cells reacted differently. Viability dropped until 42 °C, but at higher temperature regimens, we observed increase of viability. While in simultaneous hyperthermia and cisplatin treatment, we observed no change of viability until 41 °C in all cancer cells. Higher temperatures inhibited cell growth. Interestingly, we observed peaks of viability in AGS (42 °C, increase by 33%) and T3M4 (43 °C, increase by 32%) cells. Putting all MTT data to isobologram analysis, we observed synergistic, antagonistic, or additive effects of combined treatment. Hyperthermia and cisplatin treatment was strongly antagonistic in AGS cells. In Caco-2 cells we observed synergistic, additive and antagonistic effects of simultaneous treatment. Few combined treatment regimens were additive for T3M4 cells, and remaining antagonistic. Cisplatin induced early apoptosis in Caco-2 and T3M4 cells 1.5-fold and 3.4-fold, respectively. Hyperthermia of 43 °C in addition to cisplatin induced early apoptosis as compared to cells treated in normothermia by 20% in Caco-2 and 19% in T3M4, respectively. Hyperthermia strongly decreased intracellular cisplatin concentration in AGS, Caco-2, and T3M4 cells by 30%, 20%, and 18%, respectively.

Our data suggest that HIPEC conditions have to be cancer type dependent and well revised. Particular temperature regimens can do more harm, than benefit, by activating cell division and growth.

To get better knowledge of hyperthermia and cisplatin treatment effects, future studies should include more cancer cell lines per cancer type. Also, in vivo vehicle should be established.