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/).