49 Dimensionality provides translational insights into hormone-responsive behaviors in breast cancer

Objectives/Goals: We aim to understand how estradiol (E2) modulates proliferation and migration in distinct breast cancer subtypes within a collagen-based 3D platform that serves to better recapitulate human tissue architecture. This work seeks to bridge in vitro hormone biology with translational modeling of hormone-driven tumor behaviors. Methods/Study Population: MDA-MB-231 (triple-negative) and MCF-7 (hormone receptor-positive) breast cancer cells, tagged with red fluorescent protein, were exposed to varying E2 concentrations (0 to 10-4 M) in both 2D and 3D culture. In 2D, 2,000 cells/well were seeded in 96-well plates. Wells were imaged on Day 1 using confocal fluorescent microscopy, treated with E2, and reimaged on Day 5 to assess post-treatment counts. Normalized proliferation was calculated as Day 5/Day 1 cell count. In 3D, per well, 60,000 cells were embedded in a 12 µL base layer of 0.3% type I collagen, overlaid with 40 µL plain collagen, and topped with 150 µL E2-containing media. On Day 5, microscopy and a machine learning algorithm assessed cell counts and migration, defining migratory cells as those >1 standard deviation from the mean z-position of the tumor body. Results/Anticipated Results: In 2D, MCF-7 cells demonstrated significant dose-dependent proliferation to E2. Normalized cell counts increased progressively from 0 M to 10-10 M, at which point proliferation peaked, followed by a significant decline at higher concentrations. In contrast, MDA-MB-231 cells showed stable proliferation across the 0 to 10-6 M range, with a marked reduction in count at 10-4 M. In 3D, MDA-MB-231 cell proliferation remained stable across low and intermediate E2 doses, with a marked reduction at 10-4 M. MCF-7 cells, however, showed no significant differences in normalized proliferation across any E2 concentration in 3D. Quantitative migration analysis revealed that the percentage of migratory cells remained statistically unchanged across all E2 concentrations for both MCF-7 and MDA-MB-231 cells in the 3D matrix. Discussion/Significance of Impact: Dimensionality influences hormone responsiveness in breast cancer, highlighting limitations of 2D assays in modeling clinical behaviors and predicting outcomes. Advancing biomimetic 3D platforms like these may ultimately enable patient-specific screening of endocrine response and guide personalized therapy for hormone-sensitive cancers.