Methods

Cell Culture Procedure

The human alveolar RMS cell line RH-30 (No. ACC-489, DSMZ, Braunschweig, Germany) was obtained from the German biological resource bank ´DSMZ´(https://www.dsmz.de). The RH-30 cells were cultured in Dulbecco’s modified Eagle’s medium plus Ultraglutamine 1 (Lonza, Verviers, Belgium) with 10% fetal calf serum (FCS) (PAN Biotech GmbH, Aidenbach, Germany) and 1% antibiotic-antimycotic solution (Gibco, Paisley, UK) under humidified conditions at 37 °C and 5% CO2 atmosphere. All cells were mycoplasma negative. Every second day the culture medium was changed, and confluent cancer cells were treated with 0.05% trypsin – 0.02% EDTA (Lonza, Verviers, Belgium).

Animal model

Eight- to eleven-week-old NOD/LtSz-scid IL2Rγnullmice, weighing 25 to 30g, were used for all experiments. This immunodeficient mouse strain was initially described and established by Shultz et al. . The animals were initially sourced from Jackson Laboratories (Bar Harbor, ME, USA) for the establishment of an own breeding and subsequently obtained from our own animal facility. A total number of 100 animals were used for all the experiments. Animals were kept under specific pathogen-free conditions, fed an autoclaved standard diet and given free access to sterilized water. All animal experiments were approved by the local government ethics committee for animal studies (Regierungspräsidium Gießen, V54 – 19 c 20 15 h 01 MR 20/14, No. G38/2017). To ensure a comparable tumor growth, only RH-30 tumor cell passages 2-5 were used for the xenotransplantation. The RH-30 tumor cells were grown and trypsinized as described above and then resuspended in 0.9% sterile sodium chloride solution (Ecolav® B.Braun, Melsungen, Germany). A total number of 2 x 106 RH-30 tumor cells were injected into the lower left flank of the animals.

Intraperitoneal lavage/HIPEC

Intraperitoneal lavage treatment was performed 21 days after xenotransplantation (RH-30). In the control group, a continuously intraperitoneal lavage with isotonic saline solution (37 or 42 °C, each group n=16) was used for 60 minutes. In the treatment group, animals were treated with intraperitoneal cisplatin (30 or 60mg/m2) for 60 minutes at 37 or 42 °C (each group n=16). The groups (each group n = 16) with the corresponding dosages and temperatures are given in Table 1. An inflow catheter was introduced into the right and an outflow catheter into the left lower abdomen. The lavage was administered continuously into the abdominal cavity via the inflow catheter (perfusion rate: 180 ml per hour). The fluids of the outflow were trapped by drainage bags. The lavage fluids were heated up to 37 or 42 °C using a water bath. Figure 1 gives an overview about the experimental setup.
All experiments were performed under general anesthesia and prophylactic pain treatment according to the guidelines of the local government ethics committee for animal studies (Regierungspräsidium Gießen, V54 – 19 c 20 15 h 01 MR 20/14, No. G38/2017). Initial sedation was performed in a whole-body chamber with 4-5% isoflurane (AbbVie Inc., Ludwigshafen, Germany). Further anesthesia and prophylactic pain management were performed with 0.05 mg/kg body weight fentanyl (Hameln Pharma plus GmbH, Hameln, Germany) + 5 mg/kg body weight midazolam (F. Hoffmann-La Roche AG, Basel, Switzerland) + 0.5 mg/kg body weight dexmedetomidine (EVER Valinject GmbH, Gröbenzell, Germany) subcutaneously and maintained after 10-15 min by a continuous supply of 2% isoflurane mixed with oxygen (Linde AG, Hanover, Germany). Body temperature of mice was monitored using an anal temperature probe. After 60 minutes of intraperitoneal lavage, a median laparotomy was performed. Tumor dissemination was documented by the peritoneal carcinomatosis index (PCI) according to the principle of Jacquet and Sugarbaker et al. (2006) and adapted for the animal model (Fig. 2) . Tumors and its corresponding regions were photographed using the camera of a 3 mm 0° laparoscope (KARL STORZ GmbH & Co. KG, Tuttlingen, Germany).
Finally, the animals were sacrificed by administration of a lethal dose of isoflurane.

Magnetic resonance imaging (MRI)

Exemplary magnetic resonance images of four animals were taken before, during and after HIPEC with and without the administration of a Gadolinium-containing contrast agent (dosage: 0.1 ml/kg body weight; Gadovist, Bayer Vital GmbH, Leverkusen, Germany) into the tail vein. MR imaging was performed with the experimental 7 Tesla magnetic resonance imaging machine (ClinScan 70/30 USR, Bruker BioSpin MRI GmbH, Ettlingen, Germany). The system consists of a magnetic tube with an inner diameter of 30.2 cm and a preparation plate with a rail for attaching an animal bed. The examined animals were positioned and fixed in the center of the MRI for the exclusion of movement artefacts. They were continuously sedated with an isoflurane-oxygen mixture (AbbVie Inc., Ludwigshafen, Germany) via an anesthetic mask and the respiratory rate was recorded. The MRI examination protocol includes several horizontal and frontal measurements for morphological imaging.

Histological analysis and Immunohistochemistry

After laparotomy, healthy control (liver, spleen, peritoneum) and tumor tissues were harvested for the histological work-up. The tissues were fixed in 3.7% formalin, paraffin-embedded (Tissue-Tek® TECTM blocking station, Sakura Finetek Co., Ltd., Japan), cut into 3-5 μm thin tissue sections (Leica SM 2000 R Sliding Benchtop Microtome, Leica Camera AG, Wetzlar, Germany), stretched (paraffin stretching bath type 1052, Gesellschaft für Labortechnik mbH, Germany) and mounted on SuperFrost® Plus slides (Gerhard Menzel B.V. & Co. KG, Germany). Subsequently, the sections were dried and fixed over night at 60 °C (heating cabinet type BK3064, Dipl. Ing. W. Ehret GmbH, Germany), deparaffinized with xylol (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and rehydrated in a descending ethanol alcohol series (Otto Fischar GmbH + Co. KG, Saarbrücken, Germany). A standard hematoxylin (MEDITE GmbH, Burgdorf, Germany) and eosin (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) staining was carried out for the evaluation of necrosis and histological changes. For immunohistochemical analysis of proliferation and apoptosis protein marker expression, the tumor tissues were labeled with primary antibodies detecting Ki-67 (#M7240, Dako Cytoformation, Glostrup, Denmark; dilution 1:100) as proliferation marker or Cleaved Caspase-3 (#9661, Cell Signaling, Beverly, USA; dilution 1:400) as marker for apoptosis induction. Primary antibodies were diluted in a 2% BSA solution and incubated for one hour at room temperature, followed by another one-hour incubation with a secondary antibody−labeled, polymer−horseradish peroxidase (HRP) (Dako Envision+ Kit; Dako, Glostrup, Denmark). AEC (3-amino-9-ethylcarbazol) served as chromogenic agent. All sections were counterstained with Mayer’s hemalum solution (Merck KGaA, Darmstadt, Germany). Each section was digitalized as a whole slide image (WSI) by using a PreciPoint M8 microscope with an integrated scanner and analyzed with the microscopy software ViewPoint Light (PreciPoint, Freising, Germany). The Ki-67 proliferation index was recorded via automated analysis of WSI with a Ki-67 quantifier module of a pathology software (Cognition Master Professional Suite: Ki67 Quantifier, VMscope GmbH, Berlin, Germany).

TUNEL assay

For investigations on induced apoptosis in tumors and surrounding tissues, terminal desoxyribosyl-transferasemediated dUTP nick-end labeling test (In Situ Cell Death Detection Kit, Fluorescein, Roche Diagnostics GmbH, USA) was performed. For the TUNEL method, the 3 µm tissue sections were also deparaffinized and rehydrated in a descending alcohol series, followed by a permeabilization step in permeabilization buffer (0.1% Triton X-100, 0.1% tri-sodium citrate dihydrate) for 8 min at room temperature and twice washing step with PBS. The enzyme and labelling solutions provided in the TUNEL-assay kit were diluted with each other in a ratio of 1:10 and 30-50 µl of this TUNEL reaction mixture was added and the samples were incubated for 60 min at 37 °C. Afterwards the samples were washed thrice with PBS. For positive control of apoptosis, a preparation was treated with 0.3 mg/ml DNase I (Roche Diagnostics GmbH) in PBS with magnesium and calcium for 15 min at room temperature and washed twice with PBS. For a negative control, only the labelling solution without enzyme was added to the preparation. The slides were mounted and nuclear counterstained with VECTASHIELD® HardSet™ Antifade Mounting Medium with DAPI (VECTOR Laboratories, Burlingame, USA). Fluorescence microscopic images were captured on a wide field microscope (Leica DM5500, Leica, Wetzlar, Germany).

Statistical analysis

All experiments were replicated at least three times, and data sets were expressed as means ± standard deviations (SD). Statistically significant differences were compared using the unpaired Student’s t-test. P values: ***P < 0.001; **P < 0.01; *P < 0.05 were considered statistically as significant. All analyses were performed with the software Microsoft Excel 2017 and Graphpad Prism Version 8 (http://www.graphpad.com/scientific-software/prism/).