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Fig. 1 (A) Schematic illustration of the HIPEC and HYP-PVP25-based PDD and PDT treatment procedure. Treatment experiments were performed 21 days after RH-30 tumor cell xenotransplantation. Four hours prior PDD and PDT, HYP-PVP25 (100 µg/200 µl) was injected i.p.. The first group received no HIPEC, the remaining four groups underwent i.p. lavage with a perfusion rate of 180 ml per hour over 60 minutes with cisplatin (30 or 60 mg/m2) heated up to 37 or 42 °C three hours after the HYP-PVP25 injection. Immediately afterwards, a median laparotomy was performed and tumor dissemination was documented visually using the peritoneal carcinomatosis index (PCI) according to the principle of Jacquet and Sugarbaker et al. (1996) and repeated under HYP-PVP25-based fluorescence guidance (PDD) with blue light and recorded as PDD-PCI. Subsequently HYP-PVP25-based photodynamic therapy (PDT) was performed for 10 minutes. (B) Representative images of native and Hypericin-fluorescence guided (PDD) tumor and lymph node detection. As a characteristic of the RMS tumor model used, which was described previously in detail, the tumor foci are always found at intra-abdominal sites, which are preferentially located on the large curvature of the stomach, peri-hepatic, peri-splenic, mesenteric and peritoneally on the abdominal wall . With aid of PDD, the tumor sites and metastases can be much better delineated and detected. (C)Overview about the used scheme for peritoneal carcinomatosis index (PCI) calculation. Total and PCI of the respective organs was recorded according to the principle of Jacquet and Sugarbaker et al.(1996), which has been previously adapted for the HIPEC animal model . PCI lesion size score: 0 = no tumor; 1 = tumor ≤ 1 mm; 2 = tumor > 1 mm ≤ 3 mm; 3 = tumor > 3 mm.
Fig. 2 Evaluation of the Hypericin-Polyvinylpyrrolidone 25-monotherapy. (A) Analysis of HYP-PVP25-uptake and distribution within the RMS tumor via red autofluorescence signal of HYP. Both HYP-PVP25-treated tumors with and without subsequent PDT treatment for 10 minutes showed a good HYP-uptake capacity into the deeper tumor layer. A somewhat more pronounced HYP-fluorescence signal in the outer layers of the tumor after PDT treatment is striking.(B) Terminal desoxynucleotidyl transferase (TUNEL)-labeled DNA fragmentation as a sign of apoptosis induction (green fluorescence signal) is visible in the marginal areas of the HYP-PVP25-based PDT-treated tumors (10 min), whereas HYP-PVP25-treated tumors without subsequent PDT (Non-PDT) showed no evidence of apoptosis induction. The nuclei were counterstained in blue with DAPI. (C)Immunohistochemical analysis of the proliferation marker Ki-67 (brown) showed no obvious changes in protein expression through the HYP-PVP25-monotherapy without (Non-PDT) and with 10-minute PDT.(D) Determination of the Ki-67 proliferation index using a Ki-67 quantifier software module (Cognition Master Professional Suite: Ki67 Quantifier, VMscope GmbH, Berlin, Germany) showed a significant (*** P value = 0.0003; unpaired t-test) slight reduction in the proliferation capacity during HYP-PVP25-based PDT.
Fig. 3 Analysis of HYP uptake and distribution within the tumor. The red fluorescence of HYP was determined by wide field microscopy. Red fluorescence signal is strong within the outer tumor margins and is seen down to the deeper layers of the tumor regardless of the combined HIPEC- HYP-PVP25 treatment with and without PDT. The cell nucleus was counterstained with Hoechst (blue).
Fig. 4 Investigation of apoptosis induction by terminal desoxynucleotidyl transferase (TUNEL)-assay after combined HIPEC treatment with HYP-PVP25-based PDT. TUNEL-labelled fragmented DNA as a sign of apoptosis induction is evident by a green fluorescence signal. Green fluorescence signals are evident in the outer margins of tumors treated with combined HIPEC- HYP-PVP25 therapy across multiple cell layers. The expression of which is dose- and temperature-dependent and moreover apoptosis induction is again significantly enhanced by addition of a 10-minute PDT. The cell nuclei were counterstained with DAPI (blue).
Fig. 5 Evaluation of changes in tumor cell proliferation in case of combinatorial HIPEC- HYP-PVP25 treatment by analysis of Ki-67 protein marker expression (brown signal). Immunohistochemical analysis of the proliferation marker Ki-67 revealed no obvious changes in protein expression through the individual treatments without addition of the 10-minute HYP-PVP25-based PDT (Non-PDT). Subsequent PDT reduced the expression capacity of the proliferation marker Ki-67 in the tumor margins by an average of 8-10 layers of tumor cells, depending on the temperature and dose of the previous HIPEC treatment.
TABLE 1.  Overview of the treatment groups (each n = 16).