8307 Molecularly Targeted Therapy in Oncogene-Driven Human Thyroid Cancer Cell Models: Assay Selection and Response Assessment

Abstract

Abstract Disclosure: A.T. Yang: None. T.W. Laetsch: Consulting Fee; Self; Cellectis, Deciphera, GentiBio, Jazz Pharmaceuticals, Jumo Health, MassiveBio, Novartis Pharmaceuticals, Pyramid Biosciences, Y-mAbs Therapeutics, Advanced Microbubbles, AI Therapeutics, Bayer, Inc.. A.T. Franco: None. Background: The most common types of pediatric thyroid cancer (TC) demonstrate kinase-activating genomic alterations that are clinically inhibitor responsive – however, optimal integration of molecular therapy into practice remains uncertain. Human TC cell models can facilitate evaluation of combination therapies. However, prior studies vary from using traditional cell counts to newer reaction-based assays; we assessed whether a linear relationship between cell count and alternative assays exist for human TC cell models. Methods: Standardized curves for Cell TiterGlo (CTG; measures total ATP using luciferase-based luminescence) and NucBlue (DAPI; binds to DNA with nuclear fluorescence) were generated using TC cells seeded at escalating concentrations (5k to 300k cells/well). A linear equation was fitted to the CTG data and used to estimate cell count. DAPI counts were obtained with EVOS automation. Human TC cell lines TPC1 (RET fusion) / CUTC5 (BRAF V600E) were seeded at low (1.5k/2k) and high (3k/4k) cells per well, exposed for 3 days to 0-256 nM selpercatinib (RET inhibitor) or 0-192 nM dabrafenib (BRAF inhibitor), respectively, and response was measured using CTG and DAPI. All were measured in triplicate. Results: Standardized curves of CTG and DAPI demonstrated a 9-23% difference between known and measured cell counts. CTG was accurate within 20k-40k cells/well; DAPI showed linearity within 10k-80k cells/well. TC cells exhibited different IC50 depending on the assay and seeding concentration. Prior studies for selpercatinib and dabrafenib reported in vitro IC50 of 0.4-67 nM and 0.5-6 nM, respectively. In this study, IC50 for CTG/DAPI were closer at higher cell counts: TPC1 was 6.1 / 8.2 nM respectively; CUTC5 was 10.6 / 5.1 nM. Wells seeded at lower counts demonstrated larger inter-assay difference: IC50 for TPC1 was 10.1 / 5.9 nM respectively; CUTC5 was 38.4 / 5.9 nM. Conclusion: IC50 values varied by cell seeding concentrations and assay used, with a two to six fold difference at lower seeding and loss of linearity between luminescence measurements and cell count. This variability complicates evaluating the concordance among data from different laboratories, and in vitro and clinical response. Alternative assays intended to assess viability of inhibitor-exposed human TC cell models may require additional evaluation to ensure measured values demonstrate the expected relationship to the intended measure (ie cell number) and ultimately clinical outcome. Presentation: 6/3/2024