Abstract 1220: Cryopreserved 3D human tumor spheroid platform in 96-well format for robust &amp; scalable discovery and screening
Authors: Madhu Nag, Simon Ströbel, Simon Hutter, Tala Issa, Laure-Anne Ligeon, Nils Goedecke, Olivier Frey
Journal: Cancer Research (2025)

Development of new therapies relies on rigorous testing in physiologically relevant, predictive in vitro models. This approach reduces the attrition of preclinical candidates going into animal studies and onto Phase 1 trials. While 2D tumor cell cultures have traditionally been the norm in early drug screening due to their automation compatibility and ease of use, they are no longer the optimal choice for drug development. This is due to their lack in translatability to human physiology, which is essential for assessing aspects of the cellular microenvironment, intercellular interactions, and the role of immune cells. The generation of 3D in vitro tumor spheroids for high-throughput screening campaigns can be labor-intensive, particularly at the lead discovery stage. The availability of a constant, reproducible supply of a panel of tumor spheroids for target ID and target validation studies represents a significant time and resource saving. To provide more flexibility in large-scale screening for therapeutic candidates in oncology, we developed a new platform offering a wide range of cryopreserved 3D tumor models in microplate format, enabling off-the-shelf high-content screening. The platform allows users to begin testing compounds within 3-5 days by just thawing an ARCTis™ plate (Always Ready Cryo Tissues) and following a few instructions. Another advantage of these cryopreserved 3D models is that they are manufactured in larger batches under highly standardized processes, ensuring a consistent starting point for compound testing. This results in enhanced data quality, an extended test window length, and data that is comparable across workgroups. Our current portfolio comprises 20 tumor cell lines from ATCC, representing the most common solid tumors. Staurosporine has been used as a positive control to detect a dose-dependent effect on cell viability. Following this treatment the dose-response indicated growth inhibition, shrinkage or tumor cell killing effects based on size and metabolic analysis (ATP). Furthermore, we demonstrated the robust performance of these assays for plates stored up to 6 months at -80 °C. This study demonstrates the efficacy of selected small molecules to show the functionality of the spheroids and to compare their functionality across different tumor models. Therefore, we will present applications for ARCTis™ plates that proof their relevance for pre-clinical testing. In conclusion, this innovative approach addresses the limitations of traditional 2D cell cultures by providing a high-throughput, in-plate frozen, 3D cell culture platform and access to a large tumor panel for parallel testing, offering a more physiologically relevant representation of tumor diversity. Our robust frozen flexible assay platform offers a promising new approach to advancing cancer drug discovery, streamlining the exploration of complex drug testing screens in vitro. Citation Format: Madhu Nag, Simon Ströbel, Simon Hutter, Tala Issa, Laure-Anne Ligeon, Nils Goedecke, Olivier Frey. Cryopreserved 3D human tumor spheroid platform in 96-well format for robust &amp; scalable discovery and screening [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 1220.