Establishment of a new valid animal model for the evaluation of hyperthermic intraperitoneal chemotherapy (HIPEC) in pediatric rhabdomyosarcoma

Abstract

Background: Cytoreductive surgery in combination with hyperthermic intraperitoneal chemotherapy has been established as a novel treatment approach for peritoneal sarcomatosis. Despite promising clinical reports, there is still a lack of knowledge regarding optimal drug usage and local effects. Therefore, we intended to establish a murine animal model for further evaluation. Procedure: Alveolar rhabdomyosarcoma cells were xenotransplanted into NOD/LtSz-scid IL2Rγnullmice (n=100). The mice received a continuous intraperitoneal lavage with isotonic saline solution as control- or with cisplatin (30 or 60 mg/m2) as treatment group for 60 minutes at 37 or 42 °C (6 subgroups, each n=16). Tumor spread was documented by an adapted peritoneal cancer index and MRI (n=4). Tumor and tissue samples, harvested at the end of the perfusion, were evaluated regarding morphology, proliferation and apoptosis (H&E-, Ki-67-, Cleaved Caspase 3-staining, TUNEL-assay). Results: Extensive peritoneal sarcomatosis in over 91% of the cases was observed. HIPEC was feasible without acute side effects. Ki-67 staining revealed concentration- or temperature-dependent effects of cisplatin-based HIPEC on the tumors. While Cleaved Caspase-3 showed only sporadic apoptotic effects. TUNEL-assay detected concentration- or temperature-dependent apoptotic effects at the outer tumor surface. MRI scans confirmed the observed tumor dissemination. Conclusion: This is the first animal model for evaluation of HIPEC in pediatric RMS in mice. Cisplatin-based HIPEC had early effects on the proliferation whereas circumscribed apoptotic effects could be detected at the tumor surface. This model allows further insights on the possible efficiency of HIPEC in RMS. Further studies using other drug combinations and treatment will follow.

Increasing the efficiency of hyperthermic intraperitoneal chemotherapy (HIPEC) by combination with a photosensitive drug in pediatric rhabdomyosarcoma in an animal model

“…The observed penetration depth of PDT after cisplatin‐based HIPEC was 10 μm deeper than after cisplatin‐based HIPEC without PDT (40 μm, Wagner et al. ), 16 but considerably more superficial as those demonstrated by Goodman et al. (1–3 mm) 31 .…”

“…Based on previous studies with HYP and RMS, we used our HIPEC mouse model of intraperitoneal disseminated pediatric RMS to evaluate the feasibility and benefits combining HYP and HIPEC in vivo 9–11,16,19 . Besides reliable intraperitoneal tumor growth of human alveolar RMS and good tolerance of HIPEC and ip HYP‐PVP25, we could demonstrate a selective uptake and accumulation of HYP in RMS even after HIPEC.…”

“…Adapted for the animal model, the abdominal cavity was divided into nine regions, in which the size of the largest tumor nodule was measured and scored from 0 to 3. The scores of all regions were added to calculate the PCI 16 (Figure 1C). Tumor detection was repeated under HYP‐PVP25‐based fluorescence guidance (PDD) with blue light and recorded as PDD–PCI.…”

“…Tissues (tumor, liver, spleen, peritoneum) were harvested after laparotomy for the histological workup as described previously 16 : tissues were fixed in 3.7% formalin, paraffin‐embedded, and cut into 3–5 μm thin sections. The sections were primary labeled with anti‐Ki‐67 (#M7240, Dako Cytoformation, Glostrup, Denmark, dilution 1:100, mouse‐monoclonal) antibody and secondary labeled with a polymer, horseradish peroxidase (HRP) antibody (Dako Envision+ Kit; Dako, Glostrup, Denmark), with AEC (3‐amino‐9‐ethylcarbazol).…”

“…For investigations on induced apoptosis, TUNEL assays were performed on 3‐μm thin tissue sections as reported previously using the terminal desoxyribosyl‐transferasemediated dUTP nick‐end labeling test (In Situ Cell Death Detection Kit, Fluorescein, Roche Diagnostics GmbH, USA) 16 . Control tissues were only treated with the labeling solution without enzyme as negative control and with 0.3 mg/ml DNase I (Roche Diagnostics GmbH) as positive control.…”