关键词: 胰腺癌, Kirsten大鼠肉瘤病毒癌基因同源物(KRAS) G12C, 细胞系建立, sotorasib, RMC-6236, 耐药

Abstract: Objective To establish a human pancreatic cancer cell line harboring a Kirsten rat sarcoma viral proto-oncogene (KRAS) G12C mutation and to construct isogenic single-cell clonal strains with stable Cas9 expression, thereby to provide a standardized cellular model for the study of targeted therapy resistance of pancreatic cancer. Methods Cells were isolated from surgically resected tumor tissue of a pancreatic cancer patient and subjected to primary culture and serial passaging to establish a stable cell line. Systematic characterization was performed through morphological observation, short tandem repeat (STR) profiling, mycoplasma testing, subcutaneous tumor formation in NOD/SCID mice, histopathological analysis, and whole-exome sequencing (WES). A Cas9-stably expressing cell line was subsequently constructed, and single-cell clones were screened. The half-maximal inhibitory concentration (IC₅₀) of each clone against sotorasib and RMC-6236 was determined by CCK-8 assay. RNA sequencing(RNA-seq) combined with gene set enrichment analysis (GSEA) was employed to evaluate signaling pathways associated with differential drug sensitivity. Results A human pancreatic cancer cell line, designated PUMC-PAAD7, and was successfully established. The cell line was continuously passaged for more than 40 times in vitro, formed subcutaneous xenograft tumors in NOD/SCID mice, which exhibited histopathological features consistent with pancreatic cancer and harbored a KRAS G12C mutation. Seven isogenic Cas9-stably expressing single-cell clones were obtained, among which PUMC-PAAD7-C6 and PUMC-PAAD7-C7 displayed marked drug sensitivity heterogeneity. C6 was highly sensitive to both inhibitors, whereas C7 exhibited prominent resistance, with IC₅₀ values 16-fold and 23-fold higher than those of C6, respectively. RNA-seq identified 362 differentially expressed genes. GSEA revealed significant enrichment differences between the two clones in the NF-κB signaling, sphingolipid signaling and podosome assembly pathways. Conclusions A KRAS G12C-mutant pancreatic cancer cell line is successfully established. And seven isogenic Cas9-stably expressing single-cell clonal strains are constructed. These strains exhibit intrinsic drug sensitivity heterogeneity, provide an ideal isogenic paired model for investigating the mechanisms underlying primary resistance to KRAS inhibitors.

Key words: pancreatic cancer, Kirsten rat sarcoma viral oncogene homolog (KRAS) G12C, cell line establishment, sotorasib, RMC-6236, drug resistance