Peritoneal Tumorigenesis and Inflammation are Ameliorated by Humidified-Warm Carbon Dioxide Insufflation in the Mouse

Peritoneal Tumorigenesis and Inflammation are Ameliorated by Humidified-Warm Carbon Dioxide Insufflation in the Mouse Sandra Carpinteri 2 Shienny Sampurno (Hons) 2 Maria-Pia Bernardi MBBS 1 2 Markus Germann 2 Jordane Malaterre 2 Alexander Heriot MA MB BChir FRCS (Gen.) FRCSEd FRACS 1 Brenton A. Chambers 0 Steven E. Mutsaers 4 Andrew C. Lynch MMedSci FRACS FCSSANZ FASCRS(INT) 1 Robert G. Ramsay 3 R. G. Ramsay e-mail: 0 Faculty of Veterinary Science, University of Melbourne , Parkville, VIC , Australia 1 Department of Surgical Oncology, Peter MacCallum Cancer Centre , Melbourne, VIC , Australia 2 Cancer Research Division, Peter MacCallum Cancer Centre , Melbourne, VIC , Australia 3 Peter MacCallum Cancer Centre, Sir Peter MacCallum Department of Oncology, The University of Melbourne , Melbourne, VIC , Australia 4 Lung Institute of Western Australia and Centre for Cell Therapy and Regenerative Medicine , Nedlands, WA , Australia Background. Conventional laparoscopic surgery uses CO2 that is dry and cold, which can damage peritoneal surfaces. It is speculated that disseminated cancer cells may adhere to such damaged peritoneum and metastasize. We hypothesized that insufflation using humidified-warm CO2, which has been shown to reduce mesothelial damage, will also ameliorate peritoneal inflammation and tumor cell implantation compared to conventional dry-cold CO2. Methods. Laparoscopic insufflation was modeled in mice along with anesthesia and ventilation. Entry and exit ports were introduced to maintain insufflation using dry-cold or humidified-warm CO2 with a constant flow and pressure for 1 h; then 1000 or 1 million fluorescent-tagged murine colorectal cancer cells (CT26) were delivered into the peritoneal cavity. The peritoneum was collected at intervals up to 10 days after the procedure to measure inflammation, mesothelial damage, and tumor burden using fluorescent detection, immunohistochemistry, and scanning electron microscopy. - Results. Rapid temperature control was achieved only in the humidified-warm group. Port-site tumors were present in all mice. At 10 days, significantly fewer tumors on the peritoneum were counted in mice insufflated with humidified-warm compared to dry-cold CO2 (p \ 0.03). The inflammatory marker COX-2 was significantly increased in the dry-cold compared to the humidified-warm cohort (p \ 0.01), while VEGFA expression was suppressed only in the humidified-warm cohort. Significantly less mesothelial damage and tumor cell implantation was evident from 2 h after the procedure in the humidified-warm cohort. Conclusions. Mesothelial cell damage and inflammation are reduced by using humidified-warm CO2 for laparoscopic oncologic surgery and may translate to reduce patients risk of developing peritoneal metastasis. Laparoscopy is the preferred minimally invasive approach used in a range of abdominal procedures reported to decrease the volume of intraoperative bleeding, reduce the risk of infections and shorten postoperative hospital stays.1 Laparoscopy uses CO2 gas that is dry and cold to insufflate the abdomen. This may have negative impact on the patient in the short and longer term, as it has been found to increase hypothermia and to exacerbate intra-abdominal adhesions and damage of the mesothelial lining of the peritoneum.2,3 Up to 35 % of patients die of peritoneal tumor recurrence after curative colorectal cancer resection, often resulting in locoregional morbidity without systemic metastasis.4,5 Laparoscopic surgery has generated controversy, as it has been argued that disseminated tumor cells may arise during the procedure and may have an increased propensity to metastasize to the peritoneum.6,7 The underlying basis of this is thought to be due to tissue trauma caused by desiccation, predisposing to tumor cell adhesion potentiated by exposure to dry-cold CO2 during laparoscopy. This leads to the proposal that these events might be reduced by using humidified-warm CO2 gas for abdominal insufflation. Testing these concepts in mice is effective because large cohort sizes can be used using low cell numbers to generate a minimal cancer burden to reflect the tumor recurrence rates seen in patients. A traceable colorectal cancer model, combined with a shorter time to generate metastasis data after laparoscopy, provide tools to directly test the effects of different insufflation modalities on peritoneal carcinomatosis. The parietal peritoneum is composed of a single mesothelial layer that covers connective tissue and regulates angiogenesis, fibrinolysis, inflammation, and tissue repair.8,9 Microvilli projections from a monolayer of mesothelial cells provide a frictionless surface between the peritoneum and visceral lining of the abdominal organs, allowing transport of nutrients and growth factors across the peritoneum.10 For sustained hydration, tissue remodeling and regulating inflammation, a thin fluid film, the glycocalyx, overlays the mesothelium.8 The first sign of peritoneal damage after injury is a change in mesothelial microvilli, with shortening and progressive disappearance evident during peritoneal dialysis and peritonitis.10,11 Conversely, during peritoneal recovery, microvilli are more abundant and may ameliorate further damage of the mesothelium, promoting repair by maintaining protection by the glycocalyx.12,13 The second sign of damage is a change in mesothelial cell morphology, characterized by rounding up of the cells and detachment from the basal lamina.12 Events are potentiated by desiccation due to dry-cold CO2 exposure.14 This combined injury may facilitate mesothelial breach, cancer cell adherence, and implantation on the basement membrane.15 Laparoscopy minimizes peritoneal desiccation compared to laparotomy.16 However, the use of dry-cold CO2 for insufflation may itself modify the mesothelium and promote metastasis.6 Peritoneal trauma during surgery elicits the release of proinflammatory and proangiogenic mediators such as cyclooxygenase-2 (COX-2) and vascular endothelial growth factor A (VEGFA) to facilitate wound repair. COX-2 is an enzyme that synthesizes prostaglandins as part of the normal inflammatory response. It also induces angiogenesis after tissue injury.17 An increase in VEGFA similarly stimulates angiogenesis after tissue damage to promote reoxygenation for repair.18 Increases in these factors may also create a protumorigenic environment, enhancing adhesion of disseminated cancer cells, resulting in peritoneal metastasis and are poor prognosis markers in cancer.19,20 The aim of the study was to explore the impact of humidification and warming of CO2 used for laparoscopic insufflation on peritoneal damage, inflammation and the potential to develop peritoneal metastases in a colorectal mouse model. Animals and Husbandry The study was performed in accordance with the animal ethics guidelines of the National Health and Medical Research Council (NHMRC) (Australia) and approved by the Peter MacCallu (...truncated)