Fig. 6

circCAR-MS10-NK cells maintain superior antitumor activity under CAF-induced shedding conditions in vitro and in vivo. a, b Soluble MSLN (solMSLN) levels were elevated in Capan-2 cells cocultured with human pancreatic primary fibroblasts (HPPFs) or CAFs (CAF5, CAF6, CAF16, and CAF22), as shown by western blotting and quantified relative to those in cancer-only controls (n = 3). c In vitro real-time cytotoxicity of circCAR-NK cells cocultured with mCherry-expressing Capan-2 cancer cells, in the absence or presence of CAFs. CAFs were coincubated to mimic the tumor microenvironment characterized by shed MSLN secretion. Cancer cell survival was monitored by quantifying mCherry/RFP fluorescence, presented as relative cancer viability, which was defined as the mCherry signal normalized to the 0 h value (set to 1.0) (n = 3). Statistical comparisons were performed between the circCAR-SS1 and circCAR-MS10 groups at each indicated time point. d IHC of MSLN expression in human pancreatic tumor tissues using a CLMS10-derived antibody across stages 1A, 2A, and 4. e, f In vivo therapeutic effects of CAR-NK cells were evaluated in a metastatic mouse model with intraperitoneal AsPC-1-MSLN-Luc tumors containing CAF6 cells. f Representative IVIS images of mice treated with mock, linCAR-NK (SS1, 15B6, MS10), circCAR-MS10, or _LVCAR-MS10 cells (n = 4 per group); all mRNA-CAR groups were coelectroporated with IL-21 mRNA. g Body weight monitoring during treatment. Tumor burden quantified via IVIS imaging over time (h) and on day 21 (i) (n = 4). The data are presented as the means ± s.d. Statistical analyses: one-way ANOVA for (b, i) and two-way ANOVA for (c, g, h); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001