Abstract 369: Elucidation of intratumoral heterogeneity using patient-derived lung cancer cell line.

Abstract Intratumoral heterogeneity has been reported to be associated with treatment resistance in various cancers, including lung cancer. Elucidating intratumoral heterogeneity is a critical issue that must be addressed to improve patient prognosis. Previous studies on intratumoral heterogeneity have primarily focused on collecting and analyzing patient-derived cancer cells, classifying them into subclones with distinct characteristics, and conducting comparative analyses. In contrast, few studies have focused on the early phase of intratumoral heterogeneity. We established a patient-derived cancer cell line (KTOR83) using pleural effusion collected from a lung cancer patient harboring a BRAF gene mutation. From KTOR83, we generated single-cell-derived clones and established five subclones (clones A, B, C, D, and E). In drug sensitivity assays using the BRAF inhibitor dabrafenib, clones A and B exhibited resistance, whereas clones C, D, and E were sensitive. In cell migration assays, clones D and E demonstrated high migratory capacity. In xenograft models, clones A, B, and C showed strong tumorigenic potential, while clones D and E exhibited low tumor-forming ability. Furthermore, flow cytometric analysis revealed that in clone C, the expression pattern of the cell surface marker EpCAM shifted from a unimodal to a bimodal distribution before and after dabrafenib exposure. To investigate whether this bimodal expression of EpCAM corresponds to two phenotypically distinct subclones, we plan to isolate EpCAM-high and EpCAM-low populations using flow cytometry and compare their phenotypic characteristics. Through cloning of patient-derived cancer cells, we confirmed the presence of subclones with distinct phenotypes. Moreover, in one of these clones, exposure to an anticancer drug induced a shift to bimodal expression of a cell surface marker, capturing an early event of intratumoral heterogeneity in which a single cell differentiates into phenotypically distinct populations. This study presents a novel approach to capturing the early phase of intratumoral heterogeneity and provides new insights into the mechanisms underlying tumor progression. Citation Format: Tomoko Funazo, Hiroaki Ozasa, Takahiro Tsuji, Kazutaka Hosoya, Yusuke Shima, Masahiro Ooi, Keiichiro Suminaga, Kentaro Hashimoto, Hiroshi Yoshida, Hitomi Ajimizu, Takashi Nomizo, Hironori Yoshida, Toyohiro Hirai. Elucidation of intratumoral heterogeneity using patient-derived lung cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 369.