Animal selection

Female New Zealand rabbits, aged 4 months (weighing approximately 2 kg) were purchased from an approved supplier (Gontran Achard Breeding Center for Sale with Controlled Health Status [CEGAV SSC], Argenvilliers, France). This study was carried out with the ethical approval from the French National Ministry of Research (Authorization of Projects for Scientific Purposes #28502-2020120314287244) and complied with ethical regulations (decree no. 2001-131, European directive 86-609-CEE 1986) and the ARRIVE guidelines 2.0.12

Preliminary Study: Safety of Physiological Serum Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC)Experimental Protocol for PIPAC in Rabbits

After a 1-week acclimatization period, three rabbits were treated by physiological serum PIPAC, while a fourth rabbit served as a control to obtain organ biopsies without PIPAC treatment. The rabbits were fed a standard diet and housed under a 12-h light/dark cycle. The animals were euthanized at the end of the experiment and immediately autopsied. The PIPAC experimental procedure in rabbits was based on clinical treatment protocols.13 The animals were anesthetized with an intramuscular injection of ketamine (Renaudin Laboratory, Itxassou, France) at a dose of 15–20 mg/kg combined with xylazine (Rompun 2%; Elanco, Cuxhaven, Germany) at 4 mg/kg and acepromazine (Calmivet; Vetoquinol, Tarare, France) at 0.85 mg/kg. The abdomen was shaved and disinfected. An open laparoscopy was performed under visual control, and a 12 mm balloon trocar was inserted. Abdominal exploration was carried out using an optical camera (zero-degree angle) after obtaining a constant capnoperitoneal pressure of 8 mmHg. A second 10 mm trocar was placed in the midline and secured. The nebulizer nozzle (770–12, Reger Medizintechnik, Rottweil, Germany) was then connected to the high-pressure injector via a high-pressure line and inserted into the abdominal cavity through the trocar. The abdominal cavity was checked for tightness (absence of CO₂ leakage). The high-pressure injector was then activated with the injection parameters set at a flow rate of 0.8 mL/s and a maximum upstream injection pressure of 20 bars (electronic supplementary material [ESM] Figs. 1a–c).

Fig. 1

A HES staining and multiphoton imaging of rabbit visceral and parietal peritoneum with and without PIPAC. Scale bar in (a) = 50 µm. B Photographs of blue staining after PIPAC in intra-abdominal cavities illustrate the extent of nebulization diffusion (black arrowheads). M muscularis, PP parietal peritoneum, SHG second harmonic generation, StM striated muscle, SM submucosa, VP visceral peritoneum, SmM smooth muscle, Ve blood vessel, HES hematoxylin-eosin-saffron, PIPAC pressurized intraperitoneal aerosol chemotherapy

The PIPAC procedure (50 mL of physiological serum – 0.9% NaCl) was performed under clinical practice conditions over a period of 30 min on days 8, 15, and 21.13 On day 26, indigo carmine (10 mg, Carmyne®; SERB, France) and 0.9% NaCl (50 mL) were mixed and injected via the PIPAC procedure into the peritoneal cavity for 30 min. An abdominal cone beam computed tomography (CT; Artis Icono, Siemens Healthineers, France) was performed after the third PIPAC. Euthanasia was then carried out using an intravenous injection of 1–2 mL of T-61 (embutramide 200 mg/mL + mebezonium 26.92 mg/mL + tetracaine 4.39 mg/mL). A xiphopubic laparotomy was performed and two researchers visually assessed the distribution and intensity of blue staining on the visceral and parietal peritoneum of the rabbits (ESM Fig. 1d).

Criteria Analysis

The well-being score was analyzed, including assessments of physical activity, facial expressions, resting posture, appearance, nutrition, hydration, and responsiveness to stimuli (scored from 0 to 10). Morbidity and mortality were also evaluated. Histological analysis (hematoxylin-eosin-saffron [HES] staining, Tissue-Tek Prisma™, Sakura Finetek, The Netherlands) and microscopic multiphoton analysis of the liver and visceral and parietal peritoneum were conducted by two expert surgeon researchers in the field.14

Oxaliplatin and Cisplatin-Doxorubicin PIPAC Study

This experimental feasibility study in a rabbit model with peritoneal metastases compared the effects of PIPAC oxaliplatin (n = 5) with PIPAC cisplatin-doxorubicin (n = 5) and a control group treated with physiological saline (n = 5).

Rabbit Model of Peritoneal Metastasis

The VX2 tumor was serially transmitted into the hind leg of New Zealand white rabbits (n= 2) following the procedure described by Mei et al.10 and Pascale et al.15 After 15 days, the tumors were placed into cell suspension and implanted via mini laparotomy in the stomach, omentum, visceral peritoneum (small intestine), and parietal peritoneum using an 18G spinal needle (Becton Dickinson, Franklin Lakes, NJ, USA). The tumors were allowed to grow for 8 days before treatment.

Experimental Design

The body surface area of a 3.5 kg rabbit is 0.233 m2. Three groups of five rabbits were randomized on day 8 after tumor implantation into the following groups: control (PIPAC with 50 mL physiological saline); oxaliplatin (92 mg/m2); and cisplatin (10.5 mg/m2) – doxorubicin (2.1 mg/m2).

The PIPAC procedures were performed on days 8, 15, and 21 after tumor implantation, and euthanasia was performed on day 26 (ESM Fig. 1e). An abdominal CT scan was performed prior to each PIPAC procedure and euthanasia.

Criteria Analysis

The well-being score was calculated before each PIPAC procedure. At each PIPAC, the volume of ascites was measured and the Peritoneal Cancer Index (PCI) was visually calculated. Biopsies of at least three nodules were taken and fixed in 4% paraformaldehyde and embedded in paraffin. Sections (4-μm-thick) were cut and stained with HES using an automated Benchmark XT instrument (Roche), for mouse monoclonal Ki67/MKI67 antibody (8D5, NBP2-22112, Bio-Techne, France), and TUNEL assay kit (HRP DAB, ab206386, Abcam, France). The histological response (HR) score is visually assessed according to the Peritoneal Regression Grading Score (PRGS). A surface index based on Ki-67 and TUNEL status was established to compare proliferation and dead cell rates (%) with Fiji/ImageJ (National Institutes of Health). Circulating DNA isolation and quantification were performed as previously described.16 For DNA isolation, we followed the manufacturer’s instructions regarding the use of RNA carrier but omitted the concentration step using Agencourt AMPure beads (Beckman Coulter). Quantification of VX2 ctDNA was performed as previously described, with the following modifications: quantitative polymerase chain reaction (qPCR) was carried out in triplicate in 20 µL reactions, each consisting of MeltDoctor HRM Master Mix (Applied Biosystems), 200 nM oligos, and 1.5 µL of purified DNA.

Statistical Analysis

Data were analyzed using a one- or two-way repeated measure of analysis of variance (ANOVA), with post hoc tests to locate the source of significant differences using Bonferroni or Sidak corrections for multiple comparisons (GraphPad Prism). The results are presented as means with standard deviation (SD). Asterisks in the figures indicate statistically significant differences compared with controls where the probability of falsely rejecting the null hypothesis was <5% (p < 0.05).