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

Abstract

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.

Fig. 1

Mouse model of human laparoscopic insufflation. a Images of preinsufflated and actively insufflated mice. b Tumor detection on peritoneum and at port sites. In pilot trials, we established that elevated gas flow rates, particularly using dry-cold CO₂, led to excessive and localized tumor development. c Work flow design of end point and time course experiments after insufflation designed to allow quantitation of tumor burden and peritoneal inflammatory responses, respectively

Fig. 2

Temperature regulation and tumor burden depending on CO₂ insufflation protocol. a Heat lamp intervention was used when maintaining normothermia within the range 36.5–37.5 °C (green zone) throughout procedure for no insufflation control (n = 3), dry-cold CO.2 (n = 14), and humidified-warm CO₂ (n = 14) cohorts. Significant hypothermia was evident in the dry-cold cohort. b Tumor masses were identified macroscopically (white circles) on the peritoneum of mice 10 days after insufflation with either dry-cold CO₂ (n = 14) or humidified-warm CO₂ (n = 14), or in mice that were not insufflated (n = 3). Only mice with port-site tumors (yellow broken circles) were used for analysis. Tumors derived from the injected CT26 cells were confirmed by Cherry-Red fluorescence (Supplementary Fig. 2). Number of tumors counted was significantly higher in the dry-cold cohort compared to humidified-warm cohort (p = 0.03)

Fig. 3

Mesothelial microvilli integrity is disrupted after CO₂ insufflation. a Regions where microvilli were essentially absent or were shorter than normal. Red arrow indicates typical example of shortened microvilli; black arrow, long microvilli. b Representative SEM images of the peritoneum at 24 h for each treatment group. c Quantitation of microvilli status in each cohort. Comparative statistical analyses between groups are tabulated (*p < 0.001; t test)

Fig. 4

Mesothelial layer integrity and cancer cell adhesion after CO₂ insufflation. a Representative SEM images of peritoneum captured for each treatment group at 8 h. b Quantitation of rounding up and retracting cells for different time points. c Tumor cell adhesion quantified for each treatment group showing elevated adhesion in the dry-cold CO₂ group (t test)

Fig. 5

Markers of inflammation and macrophage infiltration change after CO₂ insufflation. a Visualization of markers and semiquantitative analyses were performed by scoring extent by intensity of antigen expression (brown staining) averaged per mouse at five different areas of mesothelium in three mice per treatment per time point (original magnification, ×40). b COX-2. c VEGFA. d F4/80 macrophage marker (*p < 0.05; t test)