Previously no mouse gastric cancer cell lines have been available for transplantation into C57BL/6 mice. However, a gastric cancer model in immunocompetent mice would be useful for analyzing putative therapies. N‐Methyl‐N‐nitrosourea (MNU) was given in drinking water to C57BL/6 mice and p53 heterozygous knockout mice. Only 1 tumor from a p53 knockout mouse could be cultured and the cells s.c. transplanted into a C57BL/6 mouse. We cultured this s.c. tumor, and subcloned it. mRNA expression in the most aggressive YTN16 subline was compared to the less aggressive YTN2 subline by microarray analysis, and fibroblast growth factor receptor 4 ( FGFR4) in YTN16 cells was knocked out with a CRISPR/Cas9 system and inhibited by an FGFR4 selective inhibitor, BLU9931. These transplanted cell lines formed s.c. tumors in C57BL/6 mice. Four cell lines (YTN2, YTN3, YTN5, YTN16) were subcloned and established. Their in vitro growth rates were similar. However, s.c. tumor establishment rates, metastatic rates, and peritoneal dissemination rates of YTN2 and YTN3 were lower than for YTN5 and YTN16. YTN16 established 8/8 s.c. tumors, 7/8 with lung metastases, 3/8 with lymph node metastases and 5/5 with peritoneal dissemination. FGFR4 expression by YTN16 was 121‐fold higher than YTN2. FGFR4‐deleted YTN16 cells failed to form s.c. tumors and showed lower rates of peritoneal dissemination. BLU9931 significantly inhibited the growth of peritoneal dissemination of YTN16. These studies present the first transplantable mouse gastric cancer lines. Our results further indicate that FGFR4 is an important growth signal receptor in gastric cancer cells with high FGFR4 expression.
Background: IL-12 is a pro-inflammatory cytokine with antitumor potential that activates both innate and adaptive immunity. However, IL-12 shows poor pharmacokinetics and intolerable toxicities due to unspecific distribution. Herein, we present pH-sensitive polymeric micelles loading IL-12 (IL-12/m) that release fully active IL-12 after sensing intratumoral pH to safely potentiate immunotherapy. Methods: IL-12/m with 40 nm in diameter were prepared by self-assembly in phosphate buffer. The IL-12 release was tested at different pH. The stimulation of IFN-γ release from IL-12 and IL-12/m was studied in mouse splenocytes. Pharmacokinetic, pharmacodynamic, and antitumor activity were studied in immunosuppressed (B16F10 and 4T1) mouse tumor models (staged ≥ 200 mm3) and in a lung metastasis model (4T1). The safety of IL-12 was evaluated by tracing levels of cytokines and blood markers of toxicity. Results: IL-12/m selectively activated at intratumoral pH 6.5, releasing more than 80% of IL-12 in 24 h. While IL-12/m did not induce IFN-γ production in splenocytes, the released IL-12 showed similar activity to native IL-12. In mice, IL-12/m prolonged blood circulation with minimal leakage, yielding a 3-fold longer half-life than IL-12. Moreover, the accumulation in tumor for IL-12/m (7.5%ID/g of tumor) was 3-fold higher compared that of free IL-12, with more than 90% of IL-12/m being activated. The tumors treated with IL-12/m showed 2-3-fold higher levels of IFN-γ, IL-6 and TNF-α than those treated with IL-12, whereas anti-inflammatory IL-10 was 2-fold lower for IL-12/m. In blood and organs, IL-12/m lowered the cytokine levels compared to IL-12. Also, IL-12/m avoided the tachyphylaxis of IL-12 without peak effects of IFN-γ, and the blood markers of toxicity, i.e., BUN, ALT and AST, remained close to control levels, supporting the tumor selective activation of IL-12/m. IL-12/m was efficacious in the B16F10 tumor model (10 µg iv 3 injections every 4 days) as monotherapy, and in combination with anti-PD-1 (10 mg/kg IP 3 injections every 4 days), which led to 6 of 10 complete regressions (CR). IL-12/m was also effective at 1 µg (5 injections every 2 days) against 4T1 tumors. IL-12/m increase the number and activation of CD8+ T cells and NK cells in tumors. The combination of IL-12/m (10 µg iv 3 injections every 4 days) with anti-PD1 and anti-CTLA-4 (both 10 mg/kg IP 3 injections every 4 days) achieve complete CR in a spontaneous model of lung metastasis. Responders showed immunological memory after being rechallenged with fresh tumor cells. Conclusions: IL-12/m, a pH-activated polymeric micelle loading IL-12, masked the activity of IL-12 in healthy tissues, while it unleashed full potency of IL-12 in tumors, improving tolerability and efficacy. IL-12/m presented greater antitumor activity than IL-12 as monotherapy and in combination with immune checkpoint inhibitors. Citation Format: Horacio Cabral, Pengwen Chen, Kazuhiko Kakimi, Kazunori Kataoka, Takuya Miyazaki, Koji Nagaoka. pH-activatable IL-12-loaded polymeric micelles safely enhance antitumor efficacy as monotherapy and in combination with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2072.
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