Triple-Therapy of Peritoneal Metastasis-Partial-Dehydration under Hyperthermic Condition Combined with Chemotherapy: The First Preliminary In-Vitro Results

Abstract

A newly introduced combination of intraperitoneal dehydration and hyperthermia has recently been shown to be feasible and cytotoxic for colon cancer cells in vivo. For the first time, our study now aims to evaluate dehydration under hyperthermic conditions combined with chemotherapy for potential use in the clinical setting. In this study, in vitro colon cancer cells (HT-29) were subjected to single or several cycles of partial dehydration under hyperthermic conditions (45 °C), followed by chemotherapy (triple exposure) with oxaliplatin or doxorubicin in various configurations. The viability, cytotoxicity, and proliferation of cells after the proposed protocols were assessed. Intracellular doxorubicin uptake was measured via flow cytometry. After one cycle of triple exposure, the viability of HT-29 cells was significantly reduced versus the untreated control (65.11 ± 5%, p < 0.0001) and versus only chemotherapy (61.2 ± 7%, p < 0.0001). An increased chemotherapeutic inflow into the cells after triple exposure was detected (53.4 ± 11%) when compared to cells treated with chemotherapy alone (34.23 ± 10%) (p < 0.001). Partial dehydration in a hyperthermic condition combined with chemotherapy increases the overall cytotoxicity of colon cancer cells significantly compared to chemotherapy alone. This could possibly be related to enhanced intracellular uptake of chemotherapeutic agents after partial dehydration. Further studies are required for the further evaluation of this new concept.

Keywords: colorectal cancer; dehydration; doxorubicin; hyperthermia; intraperitoneal chemotherapy; oxaliplatin; peritoneal metastasis.

Figure 1

Model of laparoscopically induced peritoneal surface-dehydration under hyperthermic conditions followed by intraperitoneal chemotherapy, based on Diakun A. et al. (2022). From A to B: Model of physiological peritoneal tissue in (A) followed by laparoscopically induced partial dehydration of the surface under hyperthermic conditions (B). The dehydration process combined with hyperthermia induces partial clotting of the most superficial capillaries and a shrinking/thinning of the peritoneal surface (B). After this part of the procedure, intraperitoneal chemotherapy can be applied to target peritoneal metastasis (C).

Figure 2

Viability (A,C,D) and cytotoxic levels (B) of HT-29 cells in different treatment protocols. (A) Viability and cytotoxicity compared to control (B) after one cycle of dehydration plus hyperthermia only (1), oxaliplatin only (2), dehydration plus hyperthermia followed by oxaliplatin (3), oxaliplatin followed by dehydration plus hyperthermia (4). (C) Development of viability after three cycles of dehydration plus hyperthermia only (red), oxaliplatin only (pink), dehydration plus hyperthermia followed by oxaliplatin (blue). (D) The area under the curve of proliferation was calculated, and appropriate statistical analysis was performed for each group (three cycles). Columns include the standard deviation; significance levels are indicated by: * p < 0.05, ** p < 0.01, **** p < 0.0001; ns—not significant.

Figure 3

Intracellular doxorubicin uptake from the medium under various protocols. Concentration protocol (A,B): In A. HT-29 cells, doxorubicin was incubated at concentrations of 1 μM, 2.5 µM, and 5 µM. Intracellular doxorubicin penetration was then analyzed by flow cytometry. Figure B shows the total amount (in percent) of doxorubicin-marked cells for each group. A statistical analysis was performed to compare the number of doxorubicin-marked cells in each group. Treatment protocol (C,D): flow cytometry (C) and histograms (D) of HT-29 cells treated with 2.5 µM doxorubicin only, dehydration under hyperthermic conditions followed by doxorubicin, and doxorubicin followed by dehydration under hyperthermic conditions. Untreated cells were used as a control. Results obtained are presented as means ± SD of three independent biological repeats. Significance levels were indicated with: ** p <0.01; *** p <0.005; **** p <0.0001; ns—not significant.

Figure 4

Phase contrast microscopy of HT-29 cells showing the morphology changes and disruption of intercellular connections after one treatment cycle of dehydration in hyperthermic conditions followed by oxaliplatin treatment (C). Control cells (A) oxaliplatin-treated cells (B). 20× magnification level.