Quantitative assessment of extravasation of IL-15-secreting MSLN-CAR-NK-92 cells using tumor transparency imaging

Background:The extravasation of anticancer immune cells is a very important issue that must be understood to improve the anticancer effect of chimeric antigen receptor (CAR)-expressing anticancer immune cell therapy.To date, no study has been reported to quantitatively evaluate the degree of extravasation of anticancer immune cells escaping from tumor blood vessels to the tumor microenvironment (TME) at the microscopic level.Methods: In this study, for the first time, using tumor transparency imaging, the extent of extravasation of CAR-NK and NK cells in pancreatic tumors was determined.we used tumor transparency imaging, which preserves intact vasculature, to accurately measure the extravasation and infiltration of established MSLN-CAR-NK-92 cells and unmodified NK-92 cells in an NSG mouse model of pancreatic cancer.Extravasation was quantified by calculating the volume ratio of cells located inside versus outside tumor vessels.Results: Following intravenous infusion, MSLN-CAR-NK-92 cells showed higher extravasation rates (85.3% vs. 57.4%),penetration depths (185 m vs. 128 m), and average extravasated cell counts (7,717 vs. 2,311) compared with NK-92 cells.Further measures of penetration and cytotoxicity also favored MSLN-CAR-NK-92 cells, with CPA50/CPD50 values of 6,887 cells at 88 m versus 3,509 cells at 45 m, and CDA50/CDD50 values of 6,350 cells at 102 m versus 2,023 cells at 48 m, respectively.These findings highlight the value of extravasation efficiency as a metric for assessing immune cell performance in solid tumors.Conclusion: Considering these results, the extravasation efficiency of anticancer immune cells can be regarded as a valuable indicator for evaluating the effectiveness of CAR constructs designed for NK cells target pancreatic cancer.In this study, we establish a quantitative extravasation imaging platform for evaluating CAR-NK cell trafficking in pancreatic and cholangiocarcinoma tumor models.This approach provides a structured framework for assessing immune cell delivery and therapeutic distribution within the tumor microenvironment.