Correlation between Spheroid Formation Ability and Reported Invasiveness of HEK-293, HT-29, MDA-MB-231, and HeLa Cancer Cell Lines in Commercial Well Plates

Three-dimensional (3D) cell cultures, such as spheroids, are essential for replicating in vivo tumor environments, offering a more accurate model for cancer research and drug testing. Spheroids form through the self-aggregation of cells under specific conditions, enabling the study of cellular behavior, including invasiveness. In this study, we investigated the correlation between spheroid formation ability and the reported invasiveness of four widely used human cancer cell lines — Human Embryonic Kidney 293 (HEK-293), Human Colorectal Adenocarcinoma (HT-29), Human Breast Cancer (MDA-MB-231), and Human Cervical Cancer (HeLa) — using a commercial round bottom 96-well microplate and AggreWell™ microwells with varying cell seeding concentrations. Cells were cultured in highly viscous media and seeded at varying densities (1,000, 2,000, and 3,000 cells per well) to assess the effect of cell number on spheroid size over time. Microscopic analysis revealed distinct differences among the cell lines; HEK-293 and HT-29 cells formed compact, well-defined spheroids, with larger spheroids observed at higher seeding densities. In contrast, the more aggressive and invasive MDA-MB-231 and HeLa cells failed to form spheroids under these conditions. These findings demonstrate the intricate relationship between cancer cell aggressiveness, seeding density, and spheroid formation ability, which are critical factors in optimizing 3D culture-based drug development and cancer research