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)