Modeling open surgery in mice to explore peritoneal damage, carbon dioxide humidification and desmoidogenesis.

BACKGROUND: The exposure of the peritoneum to desiccation during surgery generates lasting damage to the mesothelial lining which impacts inflammation and tissue repair. We have previously explored open abdominal surgery in mice subjected to passive airflow however, operating theatres employ active airflow. Therefore, we sought an engineering solution to recapitulate the active airflow in mice. Similarly, to the passive airflow studies we investigated the influence of humidified-warm carbon dioxide (CO2) on this damage in the context of active airflow. Additionally, we addressed the controversial role of surgery in exacerbating desmoidogenesis in a mouse model of familial adenomatous polyposis. METHODS: An active airflow mouse-operating module manufactured to produce the equivalent downdraft airflow to that of a modern operating theatre was employed. We quantified mesothelial cell integrity by scanning electron microscopy (SEM) sampled from the peritoneal wall that was subjected to mechanical damage or not, with and without the delivery of humidified-warm CO2. To explore the role of open and laparoscopic surgery in the process of desmoidogenesis we crossed Apcmin/ + C57Bl/6 mice with p53 +/- mice to generate animals that developed desmoid tumors with 100% penetrance. RESULTS: One hour of active airflow generates substantial damage to peritoneal mesothelial cells and their microvilli as measured at 24 h post intervention, which is significantly greater than that generated by passive airflow. Use of humidified-warm CO2 mostly protects the mesothelium that had not experienced additional mechanical (surgical) damage at 24 h. Maximal damage was evident in all treatment groups regardless of flow or use of gas. At day 10 mechanically-damaged peritoneum remains in mice but is essentially repaired in the gas-treated groups. Regarding desmoidogenesis, operating procedures did not increase the frequency of desmoid tumors but their frequency correlated with time following surgery but not age of mice. CONCLUSIONS: Active airflow generates more peritoneal damage than passive airflow and is reduced significantly by the use of humidified-warm CO2. Introduced peritoneal damage is largely repaired in mice by day 10 with gas. Desmoid tumor incidence is not increased substantially by surgery itself but rises over time following surgery compared to non-surgery mice.

Tumor-Infiltrating Lymphocyte Function Predicts Response to Neoadjuvant Chemoradiotherapy in Locally Advanced Rectal Cancer.

PURPOSE: The presence of tumor-infiltrating lymphocytes (TILs) in tumors is superior to conventional pathologic staging in predicting patient outcome. However, their presence does not define TIL functionality. Here we developed an assay that tests TIL cytotoxicity in patients with locally advanced rectal cancer before definitive treatment, identifying those who will obtain a pathologic complete response (pCR). We also used the assay to demonstrate the rescue of TIL function after checkpoint inhibition blockade (CIB). PATIENTS AND METHODS: Thirty-four consecutive patients were identified initially, with successful completion of the assay before surgery in those 17 patients who underwent full treatment. An in vitro cytotoxic assay of rectal cancer tumoroids cocultured with patient-matched TILs was established and validated. Newly diagnosed patients were recruited with pretreatment biopsy specimens processed within 1 month. Evaluation of TIL-mediated tumoroid lysis was performed by measuring the mean fluorescence intensity of cell death marker, propidium iodide. CIB (anti-programmed cell death protein 1 [anti-PD-1] antibody) response was also assessed in a subset of patient specimens. RESULTS: Six of the 17 patients achieved an objective pCR on final evaluation of the resected specimen after neoadjuvant chemoradiotherapy. Cytotoxic killing identified the pCR group with a higher mean fluorescence intensity (27,982 [95% CI, 25,340 to 30,625]) compared with the non-pCR cohort (12,428 [95% CI, 9,434 to 15,423]; p < .001). Assessment of the effectiveness of CIB revealed partial restoration of cytotoxicity in TILs with increased PD-1 expression with anti-PD-1 antibody exposure. CONCLUSION: Evaluating TIL function can be undertaken within weeks of the diagnostic biopsy, affording the potential to alter patient management decisions and refine selection for a watch-and-wait protocol. This cytotoxic assay also has the potential to serve as a platform to assist in the additional development of CIB.