(F-207) Expansion of patient-derived tumoroids in a novel serum-free, Wnt-free, suspension culture system

Tumoroid (or cancer organoid) technology enables culture of patient tissue-derived cancer cells in 3D, with retention of key characteristics from the original patient tumor. Tumoroid models are often more representative of patient tumors than long-established 2D cancer cell lines. However, current tumoroid culture relies on labor-intensive media formulations and culture methods. To address these issues, we have developed a novel serum-free, Wnt agoinst-free, and conditioned medium-free tumoroid culture medium, named Gibco™ OncoPro™ Tumoroid Culture Medium. The system has been optimized to retain key patient-specific genotypic and phenotypic characteristics during in vitro culture. We hypothesized that, as opposed to the traditional embedded culture, we could add diluted basement membrane extract (BME) to propagate cells in suspension. Suspension culture enables a single user to generate hundreds of millions of cells, a feat that would require hundreds of BME domes using standard tumoroid culture methods. Our data confirm that, when paired with our novel tumoroid culture medium, suspension culture maintains patient-specific characteristics comparably to embedded culture when passaged side-by-side for multiple months. Additionally, we demonstrate that our novel medium and culture method is compatible with colorectal, lung, pancreatic, and head and neck tumoroid lines that were derived in embedded culture with various complete media formulations.

OncoPro Tumoroid Culture Medium was developed using an iterative design of experiments (DOE) approach to optimize basal medium, supplements, and growth factors. By supplementing the base medium with indication-specific growth factors, tumoroid lines were derived from colorectal, lung, breast, and endometrial cancers from both fresh surgical resections and cryopreserved primary cancer cells. For select lines, tumoroids were cultured for up to 50 passages, with the majority of growth occurring in the suspension culture format. Additionally, to test the applicability of our system with previously established tumoroid/cancer organoid models, we procured publicly available cancer models from the National Cancer Institute Patient-Derived Models Repository (NCI PDMR). Multiple colorectal, lung, pancreas, and head and neck tumoroid models were tested for growth in both embedded and suspension formats, including tumoroids originally derived in media systems with conditioned medium containing Wnt-3A, R-spondin 3, and noggin. Brightfield and fluorescent microscopy, cell counting, and next-generation sequencing (NGS) were used to assess maintenance of tumoroid morphology, growth rate, and gene expression patterns and genomic mutational profiles.

Tumoroid cultures derived in OncoPro Tumoroid Culture Medium adopted donor-specific morphologies that were maintained during long-term culture. Cell doubling time for derived tumoroid lines was donor-dependent and averaged around 65 hours for colorectal tumoroids – on par with that of traditional 2D cancer cell lines – and 90 hours for lung tumoroids, with stable growth rates for 100+ days and through cryopreservation cycles. The allelic frequency of single nucleotide variations in 161 highly relevant cancer genes was also highly correlated (R²>0.9) between resection tissue and late-passage tumoroids, with retention of single base substitution patterns and key oncogenic driver mutations (Figure 1A). Importantly, tumoroid lines maintained their global gene expression levels for over 20,000 human RefSeq genes during long-term culture, with correlation between initial tumor material and late-passage samples of >0.8 and conservation of donor- and indication-specific gene expression profiles. To eliminate bias due to stable housekeeping or lowly-expressed genes, a cancer-specific gene panel was also compared with similar results. Interestingly, the largest changes in gene expression or genomic mutations tended to be observed during culture initiation and the first few passages, where depletion of EpCAM-negative cells was also observed. Tumoroid lines purchased from the NCI PDMR or alternative commercial sources showed comparable growth rates and morphologies in the OncoPro suspension and embedded culture systems and in the original NCI PDMR-recommended or homebrew culture system. Both single nucleotide variant allelic frequency and ploidy values were conserved from initial starting material following expansion to cryopreservation-competent banks ( >10e6 cells) (Figure 1B). Gene expression levels across over >20,000 human RefSeq genes were compared between culture methods and showed high ( >0.9) correlation (Figure 1C), and Wnt-related signaling pathways were not differentially regulated between culture systems.

We thank Discovery Life Sciences for providing the dissociated tumor cells used to generate data in this study. Established tumoroid models provided by the NCI PDMR. We thank the PDMR and their Contributing Institutions for their contributions to this work.