Legends
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).