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Abstract The DNA mismatch repair (MMR) system is a highly conserved pathway crucial for maintaining the genomic integrity. MMR impairment in sporadic tumors is due to MLH1 epigenetic silencing or mutations in other MMR genes (such as MSH2, MSH6, PMS2) and leads to microsatellite instability (MSI). Two independent molecular classifications reported the existence of a MSI subgroup in gastric cancer (GC), including 22-23% of all cases. GC MSI inter and intra-tumoral heterogeneity represents a significant challenge for precise patient stratification and treatment. Even if MSI status in GC is usually associated with a good prognosis, some reports described a subpopulation of MSI GC patients displaying a worse outcome. Moreover, while clinical trials testing immune checkpoint inhibitors in MSI tumors showed effectiveness across all treatment lines, the outcomes in GC patients were not as remarkable as in other cancer types. From the analysis of MSI GC patients enrolled in phase III clinical trials testing anti-PD1 monotherapy, it emerged that nearly half of the tumors are intrinsically resistant and complete and long-lasting responses are achieved in very few cases. Tumor mouse immunocompetent models allow to mimic the complex interactions between a fully functional immune system and a developing tumor. MSI GC syngeneic mouse models could thus represent a valuable tool to better understand the molecular complexity and predict the clinical behavior of this subgroup. To obtain MSI GC mouse models, we generated non-transformed organoids from normal gastric mucosa of BALB/c mice as a starting point for the induction of a neoplastic transformation ex vivo. To induce MMR deficiency, we inactivated either Mlh1 or Msh2 genes using CRISPR/Cas9 genome editing. Importantly, we did not introduce any other alteration in known oncogenes and tumor suppressor genes, thus allowing the natural emergence of driver mutations in the context of the genetic instability. Over time, MMR deficient models, but not the WT controls, exhibited MSI and increased their mutational burden. Furthermore, when cultured in stringent culture conditions (two-dimensional culture, growth factor deprivation), only MMR deficient cells generated tumor masses upon subcutaneous injection in immunodeficient NOD SCID mice. Importantly, cells retrieved from the tumor masses (n = 8) exhibited mutations in a relevant fraction of genes associated with the MSI subgroup by TCGA (21 out of 35) – including Kras, Arid1a, Erbb3 and Tpr53 – thus offering a good representation of the human MSI GC mutational landscape. Given the immunogenicity of the Cas9 protein, we finalized our models optimizing the genome editing protocol to avoid a persistent Cas9 expression. Cas9-free MMR deficient models were able to generate tumors in immunocompetent BALB/c mice, thus representing a valuable resource to investigate tumor development, progression and responses to immunotherapy in vivo. Citation Format: Daniela Conticelli, Gabriele Picco, Cristina Migliore, Claudia Orrù, Fabrizio Maina, Emanuela Boccuni, Simona Corso, Silvia Giordano. Gastric cancer with microsatellite instability: Ex vivo generation of syngeneic mouse models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1446.
Conticelli et al. (Fri,) studied this question.