Results

Initially, 100 NOD/LtSz-scid IL2Rγnullmice were used for this study. Three weeks after being xenotransplanted with RH-30 cells, tumor growth was observed in 91 mice. No tumor dissemination occurred in 9 mice. Five mice showed extraperitoneal tumor growth after failed intraperitoneal injection. No mouse was prematurely sacrificed due to tumor progression. Sixteen mice died prematurely during treatment and four mice were used for the MRI scans. The remaining 66 mice were finally included into the statistical analysis.
Conventional laparotomy after treatment revealed an extensive peritoneal dissemination of RMS in 91% of the cases. Tumors could easily be distinguished from surrounding tissue by their specific morphology. The histological examination confirmed alveolar rhabdomyosarcoma cells in the observed tumors. The largest tumor masses (> 3 mm size) were typically found in the epigastric region on the greater curvature of the stomach. Smaller tumors (1 – 3 mm size) were detected in the right and left upper region, perihepatic and perisplenic or in the left lower abdomen. Disseminated small tumor nodes (< 1 mm size) occurred at the mesentery of the small and large intestine. Other tumor localizations like the flank, pelvis or peritoneum of the abdominal wall only showed small tumor spots. Mean PCI was 8.03 (Fig 2).
Anesthesia and intraperitoneal lavage were feasible without acute side effects in 87% of cases. 16 mice died during treatment due to respiratory failure. All these 16 mice had additionally lower body weight compared to those mice which tolerated the intraperitoneal lavage well. HIPEC at 37 °C resulted in mild hypothermia (body temperature 34 – 36 °C) only in isolated cases (n=6). Body temperature could be normalized by using a physical heat source. Hyperthermia (body temperature > 39 °C) during HIPEC at 42 °C did not occur due to fast heat losses in anesthetized mice.
MRI with or without contrast agent during HIPEC was technically feasible. One mouse died during MRI scans due to breathing arrest. MRI scans confirmed the observed tumor dissemination and could illustrate distribution of the lavage during HIPEC for the first time (Fig. 3).
By H&E-staining, no cell morphological changes were evident in the tissues of the tumors (Fig. 4A) and the representatively examined control organs (liver, spleen, and peritoneum) (Supplemental Figure S1).
Evaluation of tumor proliferation by Ki-67 immunohistochemistry and digital image analysis revealed early temperature-dependent effects after intraperitoneal lavage on the tumors (Fig. 4B). There was a significant difference in tumor proliferation after having used isotonic saline solution heated up to 42 °C compared to the group, in which 37 °C warm isotonic saline solution was infused. Early concentration- or temperature-dependent effects of cisplatin-based HIPEC on the tumors could be revealed. Significant effects on tumor proliferation were shown in each HIPEC group compared to the control group (saline solution, 37 °C) dependent on either Cisplatin-concentration, temperature of the lavage or the combination of both. Comparing the HIPEC groups with each other there was no significant difference in the proliferation index (Fig. 4C).
Cleaved-Caspase 3 staining revealed no early apoptosis induction. There were no differences in the sub-groups and no temperature- or chemotherapy-dependent effect was seen especially at the outer tumor surface. Only sporadic apoptotic effects scattered all over the tumor without clear focus were seen (Fig. 5A).
Using the TUNEL-assay method early apoptotic effects at the outer tumor surface could be detected. Immunofluorescence microscopy revealed an increase in apoptotic cell layers dependent on the treatment. Five to ten cell layers of the tumor surface have been affected dose and temperature dependent after HIPEC. The penetration depth of the treatment was approximately 30 to 40 µm of the outer tumor surface. Lavage heated up to 42 °C compared to 37 °C warm lavage (saline solution or Cisplatin) tends to result in more apoptotic affected cells visually observed. The most affected and apoptotic cell layers or the deepest penetration of the lavage was seen in the HIPEC sub-group using 60mg/m2 Cisplatin heated up to 42 °C (Fig. 5B)