Results

Initially, 80 NOD/LtSz-scid IL2Rγnullmice were used for this study. Three weeks after xenotransplantation intraperitoneal sarcomatosis could be induced in 68 mice. No tumor occurred in five mice, while extraperitoneal tumor bulks were seen in seven mice after failed i.p. injection. No mouse was prematurely sacrificed prior to the treatment due to tumor progression. In one mouse HYP-PVP25 was injected subcutaneously. Five mice died prematurely during treatment. The remaining 62 mice were included into the statistical analysis.
Anesthesia, HIPEC and application of HYP-PVP25 were feasible without acute side effects in 93% of cases. Five mice died during treatment due to respiratory failure. Low body weight was noted in these five mice. Due to strict monitoring of body temperature, usage of a physical heat source and fast heat loss in anesthetized mice hypothermia (body temperature 34 – 36 °C) or hyperthermia (body temperature > 39 °C) during HIPEC did not occur. After i.p. injection of HYP-PVP25 all mice were observed regarding acute or delayed adverse reactions, which did not occur. No mouse died after the application of HYP-PVP25 or during HYP-PVP25-based PDD/PDT.
Conventional laparotomy after treatment revealed an extensive peritoneal dissemination of RMS in 81% of the cases. Tumor bulks could be identified by their specific morphology. Without PDD a median PCI of 7 (+/- 2.8) was measured. Due to its strong red fluorescence under blue excitation light the selective uptake of HYP in the tumor bulks, which already have been documented without PDD, could be proved. Healthy tissue did not show any HYP-PVP25 affinity. HYP-PVP25 showed stable and selective accumulation in the tumor nodules showing the full extent of the intraperitoneal tumor spread. Even smallest tumor nodules of less than one millimeter could easily be detected only by the red fluorescence. Using the KARL STORZ D-light C system surgical and photodynamic procedures were feasible without any difficulties. Clear PDD guided tumor identification and resection followed by PDT were carried out by easily switching between the illumination modes if needed. Under HYP-PVP25-based fluorescence guidance more tumors could be identified and distinguished from surrounding healthy tissue than without PDD. Consequently, we documented a statistically significant higher median PDD-PCI of 8.5 (+/- 2.9) (***P < 0.001).
The histological examination confirmed RMS cells in the observed tumors. The most and largest tumor masses (> 3 mm size) were typically found in the epigastric region on the greater curvature of the stomach followed by smaller tumors (1 – 3 mm size) in the right and left upper region, perihepatic and perisplenic. Under PDD a massive tumor affection by numerous small tumor nodules (< 1 mm size) occurred at the mesentery of the small and large intestine as well as at the peritoneum of the abdominal wall (Figure 1B).
Besides the macroscopic visible selective uptake and accumulation of HYP in tumor bulks we could demonstrate the penetration of HYP across the tumor surface using immunofluorescence microscopy. Thereby we observed a pronounced accumulation of HYP at superficial tumor cell layers followed by decreased intensity towards inner layers (Figure 2A).
Proliferation and apoptotic effects on the tumor were measured before and ten minutes after PDT. Early apoptotic effects at the outer tumor surface could be detected via TUNEL-assay (Figure 2B). Immunofluorescence microscopy revealed an increase in apoptotic cell layers after PDT. Superficial cell layers of 30 µm depth have been affected by photoactivated-HYP. Evaluation of tumor proliferation by Ki-67 immunohistochemistry and digital image analysis showed early PDT-dependent effects (Figure 2C and D). The statistical analysis revealed a significant reduction of tumor proliferation after ten minutes of PDT compared to the proliferation index of tumor harvested before PDT (***P < 0.001) (Figure 2D).
The combination of HIPEC and i.p. applicated HYP-PVP25 was feasible. The up taken HYP persisted after HIPEC in the tumor nodules independent from the cisplatin dosage in all groups (Figure 3). Additionally, hyperthermia of 42° C leaded to deeper penetration and better distribution of HYP across the tumor surface with still decreased intensity towards inner cell layers. Whereas the outer tumor surface has been affected by HIPEC without PDT, increased early apoptotic effects to deeper cell layers were seen after ten minutes of PDT (Figure 4). Especially PDT in combination with 60 mg/m² cisplatin and hyperthermia of 42 °C led to scattered sectors of apoptosis with lost tumor structure. Sections showed outstanding untypical alterations in the Ki-67 staining, which was estimated as reduction of tumor proliferation and seen in the first 9 to 11 cell layers of the surface. The affected cell layers appeared as sharply bounded stripe. This observation was made exclusively in the PDT exposed tumor area in all groups combining HIPEC and PDT (Figure 5). Statistical analysis did not show any significant difference between the HIPEC subgroups with or without PDT.
Penetration depth of PDT after HIPEC was measured to be 35 to 50 µm depending on cisplatin concentration combined with hyperthermia (Supplemental Figure S1A). Statistical analysis revealed a significant difference in penetration depth of PDT following HIPEC of 60 mg/m² cisplatin at 42 °C compared to PDT after HIPEC with lower cisplatin dosage given with lower temperature (30 mg/m² cisplatin at 37 °Cversus (vs .). 60 mg/m² cisplatin at 42 °C, *P = 0.013; 30 mg/m² vs . 60 mg/m² cisplatin at 42 °C, ***P < 0.001; 60 mg/m² cisplatin at 37 °C vs . 42 °C, **P < 0.007). Consequently, the deepest penetration depth of PDT was seen after HIPEC with cisplatin concentration of 60 mg/m² heated up to 42 °C. (Supplemental Figure S1C)
By HE-staining, no cell morphological changes were evident in the tissues of the tumors (Supplemental Figure S2 and S3) and the representatively examined control organs (liver, spleen, and peritoneum) after treatment (Supplemental Figure S4).