Abstract 5499: A comparison of spheroid formation using AcroCyte‘s 3-dimensional R3CE spheroid culture platform <i>versus</i> traditional 3D culture methods

Abstract Three-dimensional (3D) spheroid culture systems have gained attention due to their evident advantages in mimicking the in vivo tumor microenvironment, making them highly valuable for drug testing and cancer research, as the behavior of 3D-cultured cells is more reflective of in vivo cellular responses. However, traditional spheroid methods, including ultra-low attachment plates, Matrigel, and soft agar, have limitations, especially for certain cell lines that fail to form stable spheroids. Here, we introduce a novel 3D spheroid culture method using the AcroCyte R3CE plate, and compare it to the generation of spheroids using traditional 3D culture methods. R3CE is a revolutionary scaffold-free single-cell proliferation 3D cell culture platform that can quickly and reliably culture spheroids from a variety of samples including cell lines, circulating tumor cells and biopsies. This technology is highly efficient and can help researchers gain insight into cancer research, clinical treatments, and drug discovery. 20 cell lines with historically poor spheroid formation in standard methods were subjected to the novel AcroCyte R3CE plate culture system which enabled robust and consistent spheroid formation. RNA sequencing (RNA-seq) was performed to compare gene expression profiles between 3D spheroids cultured using the R3CE plate and traditional 2D monolayer cultures. Our analysis identified distinct biomarker expression profiles associated with spheroid cultures, including cancer stem cell-related proteins and key signaling pathways relevant to cell differentiation and tumor biology. The unique biomarker expression profiles of the cancer spheroid cells were validated at the protein level by way of immunofluorescence or western blot. This innovative approach utilizing the R3CE 3D cell culture platform, not only improves spheroid formation across difficult-to-culture cell lines, but also preserves critical biomarker expression profiles and pathways linked to tumorigenesis. These findings highlight the potential of the R3CE 3D culture system to establish a reliable and versatile spheroid panel for high-throughput drug screening, providing a more physiologically relevant model to evaluate drug efficacy and mechanistic insights. Citation Format: Zichun Wang, Di Zhan, Qikuan Chen, Chao-Jie Yang, Jia-Yang Chen, Ying C Chang, Yinfei Yin. A comparison of spheroid formation using AcroCyte‘s 3-dimensional R3CE spheroid culture platform versus traditional 3D culture methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5499.