LAUSR

Over the past 10 years, we have developed and characterized over 280 orthotopic patient derived xenografts (O-PDXs) representing 23 different types of pediatric solid tumors. The O-PDXs have undergone comprehensive characterization including genomics, transcriptomics, epigenomics, histologic review and analysis of cellular heterogeneity. All data, tissue samples and cryopreserved O-PDXs are freely available with no obligation to collaborate through the Childhood Solid Tumor Network (CSTN) at www.stjude.org/CSTN/. While these in vivo models are useful for preclinical testing of drugs and immunotherapy, researchers are limited by throughput, expense and expertise. High throughput ex vivo culture systems that more faithfully recapitulate the cellular heterogeneity and arrested differentiation in the patient tumors are urgently needed to test new drug combinations. Solid tumor cell lines have undergone extensive selection and adaptation in culture, and most have lost the cellular heterogeneity found in patient tumors. In order to overcome this barrier in the field, we have developed a 3D-to-2D workflow. Previous attempts to produce 2D ex vivo cultures from patient tumors or O-PDXs have had limited success because most cells die upon initial plating, they are difficult to cryopreserve for future studies and they lose key cell populations found in the patient tumors. Our new workflow was designed to overcome two sources of stress when adapting patient tumors or O-PDXs to culture. The first source of stress is transitioning from the in vivo environment to ex vivo in culture medium. The second source of stress is the transition from extensive 3D cell-cell contacts in vivo to a more limited cell-cell contact ex vivo. To overcome these two sources of stress, we adapted cultures to 2D in a stepwise manner. First, we dissociate O-PDX tumors into single cell suspension and then rapidly re-aggregate them in 3D spheres for ex vivo culture. After the spheres have grown for 7 days, they are transitioned to 2D by seeding cells in culture dishes coated with extracellular matrix components. Importantly, the 3D and 2D cultures preserve the cellular heterogeneity of the O-PDX and patient tumor and they can be cryopreserved. When injected back into immunocompromised mice, they recapitulate the molecular and cellular features of the original tumors. We have shown that the 3D and 2D cultures can be genetically manipulated using lentiviral vectors and can be used for high-throughput drug screening. Matched 3D and 2D cryovials are now available to the international biomedical research community for rhabdomyosarcoma through the CSTN. Neuroblastoma, osteosarcoma, Ewing sarcoma and retinoblastoma are expected to be available in late 2023. We anticipate this will accelerate the testing of oncology drugs for pediatric solid tumors in response to the RACE act through the PIVOT program. Citation Format: Justina D. McEvoy, Melody Allensworth, Mariajose Franco, Natalie McDonald, Brittney Gordon, Anand Patel, Elizabeth Stewart, Michael Dyer. Preservation of cellular heterogeneity in 3D and 2D cultures of pediatric solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3570.