Deciphering the Divergent Transcriptomic Landscapes of Cervical Cancer Cells Grown in 3D and 2D Cell Culture Systems

Cervical cancer remains a significant health challenge for women worldwide, with a disproportionate impact on developing regions like sub-Saharan Africa. Taking advantage of recent advancements in developing suitable preclinical models to study cell proliferation, differentiation, and gene expression, we used RNA sequencing to compare the transcriptomic profiles of SiHa cervical cancer cells grown in 3D versus 2D culture systems. For SiHa cells, the culture conditions involved using DMEM supplemented with 10% FBS. For 2D culture experiments, 5 ml of SiHa cells at a concentration of 2 x 10^5 cells/ml were seeded into 10 cm dishes and overlaid with an additional 5 ml of their respective growth media. For 3D cultures, 5000 SiHa cells were seeded in each well of a 96-well Nunclon Sphera, U-bottom plate (ThermoFisher), then centrifuged at 70 × g for 5 min before placing in the incubator. Media replacement was performed every 2-3 days, and spheroid formation was monitored for 7-9 days. The 2D monolayer cultures for SiHa included 3 distinct cell passages, with 2 replicates per passage. The 2D biological replicates were generated in 2 separate batches: passage 1 replicates (2D_P1_R1, 2D_P1_R2) in Batch 1 and passage 2 and 3 replicates (2D_P2_R1, 2D_P2_R2, 2D_P3_R1, 2D_P3_R2) in Batch 2. For 3D spheroid SiHa cultures, 3 biological replicate samples were generated (3D_R1, 3D_R2, 3D_R3) separately.