Tumoroid cultures, also known as cancer organoids, have been shown to maintain patient-specific mutational and gene expression profiles over the course of long-term culture better than traditional cancer lines. Despite the physiological relevance of tumoroid models, they have yet to supplant cancer lines, particularly for high-throughput screening (HTS) applications, in large part due to the relative difficulty of the culture workflow. The current gold standard tumoroid culture method involves embedding the cells into a scaffold (most commonly basement membrane extract or BME), which is highly manual, costly in time and resources, and difficult to implement in HTS workflows. We have developed a novel tumoroid culture medium, named GibcoTM OncoProTM Tumoroid Culture Medium, and method in which the addition of diluted BME to tumoroid suspension cultures preserves apical-in polarity (equivalent to that of embedded cultures) while leveraging the benefits of a suspension culture workflow. Additionally, histology for a subset of suspension cultures have been compared with xenograft tumors formed in mice and demonstrate that the suspension culture maintains similar cellular organization. Our system 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, and has been shown compatible with colorectal, lung, pancreatic, and head and neck tumoroid lines that were derived in embedded culture with various complete media formulations. Critically, 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. Tumoroid lines representing the four unique cancer indications evaluated maintained patient-specific mutational and gene expression profiles (both ≥ 90% correlated with the original material) over the course of culture. An evaluation of differentially expressed genes from a panel of over 20,000 human RefSeq genes showed that there was no change (greater than a two-fold increase/decrease; p-value < 0.05) in gene expression for ≥ 98% of genes, on average, between embedded and suspension culture. Following scale up in suspension culture, we also show that tumoroids can be plated using automated liquid handling techniques for downstream HTS assays. Taken together, our data indicate that this suspension culture workflow paired with OncoPro Tumoroid Culture Medium maintains all critical characteristics of tumoroid lines grown in the traditional embedded culture workflow, while providing the scalability and compatibility with liquid handling that will enable greater adoption of these more physiologically relevant cancer models. Citation Format: Brittany N. Balhouse, Colin Paul, Chris Yankaskas, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Anthony Chatman, Amber Bullock, Matthew Dallas, David Kuninger. How low can you go: Maintenance of tumoroid phenotype with a highly scalable and automation-compatible reduced-ECM suspension culture method [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 158.

In vitro cancer research often fails to translate to the clinic, in part due to the use of traditional 2D cancer cell lines as models, which fail to resemble primary cancer cells by a variety of measures, including high mutational burden. An emerging solution to this problem is to replace traditional cell lines with patient tissue-derived cells expanded in 3D, also known as tumoroids or cancer organoids. We developed a defined, serum- and conditioned medium-free system, GibcoTM OncoProTM Tumoroid Culture Medium, that can be used to derive stable tumoroid lines from a variety of tissue sources and maintains the phenotype and genotype of patient-derived tumor cells. By supplementing the base medium with indication-specific growth factors, tumoroid lines were derived from colorectal, lung, and endometrial cancers from both fresh surgical resections and cryopreserved primary cancer cells. To demonstrate the utility of these patient-derived cells as long-term in vitro models, colorectal and lung tumoroid lines were derived from multiple donors and cultured for up to 50 passages. Brightfield microscopy, cell counts, and next-generation sequencing were used to assess maintenance of tumoroid morphology, growth rate, gene expression patterns, and genomic mutations. Patient-derived tumoroid cultures adopted donor-specific morphologies that were maintained during long-term culture. Cell doubling time tended to stabilize within the first few passages as cultures established, was donor-dependent, and averaged around 65 hours for colorectal tumoroids - on par with that of traditional 2D cancer cell lines - and 90-100 hours for lung and endometrial tumoroids, respectively. Importantly, tumoroid lines maintained their gene expression pattern for over 20,000 human RefSeq genes during long-term culture, with correlation between initial tumor material and late-passage samples of R>0.8. Distinct molecular subtypes of colorectal cancer were preserved in cultured tumoroids. The allelic frequency of single nucleotide variations (SNVs) in 161 highly relevant cancer genes was also highly correlated (R>0.9) between uncultured tissue and late-passage tumoroids. Within SNVs, transition/transversion mutation ratios were conserved. Tumoroids were cryopreserved and recovered during this study, demonstrating that biobanking of colorectal and lung tumoroids should not impact their long-term stability. Finally, a subset of the derived colorectal and lung tumoroids were tested and shown to be tumorigenic in mice, where subsequent histology of the tumor was similar to that of in vitro cultures. Altogether, tumoroid derivation and culture in this novel medium enables the long-term preservation of patient-specific cellular genotype and phenotype, which should allow for expansion, biobanking, and performance of experimental repeats within the same patient tissue-derived cultures across labs and over time. Citation Format: Chris Yankaskas, Brittany Balhouse, Colin Paul, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Mark Kennedy, Matt Dallas, David Kuninger. Derivation and long-term maintenance of patient-derived tumoroid lines in a defined, serum-free medium [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 160.

Cancer drug development is an extremely challenging and resource-consuming process with a dismal success rate of 5%. The high failure rate is partly due to the inadequacy of traditional 2D cell culture model to predict drug efficacy and toxicity. A highly accurate and representative system is therefore urgently needed to improve cancer drug discovery and development process. The emerging 3D cancer models can better recapitulate the in vivo tumor microenvironment and have shown closer gene expression profiles to clinical samples compared to 2D models. Another crucial element of in vitro cancer models is the source of cancer cells: patient-derived models have shown advantages to better capture tumor heterogeneity and clinically relevant genetic alterations of the tissue origin compared to traditional cancer cell lines. Several studies showed that response of patient-derived models to treatment reflects the response of patients from which the organoids are derived, suggesting they are great platform for studies of personalized medicine. Hereby, we performed a proof-of-concept high throughput multiplexed plate-reader based drug screening assay on 3D patient-derived tumoroids. Methods: Patient-derived tumoroids were sequenced for cancer relevant mutations, copy number variants, and altered gene expression using multi-biomarker targeted next generation sequencing (NGS) and RNA-seq. 3D tumoroid lines derived from three different colorectal cancer patient samples, together with a colorectal cancer cell line were treated with chemotherapy, and targeted therapy agents. The same tumoroid and cell lines were cultured and treated in 2D as experimental control. Drug response readout was multiplexed using three different plate reader-based assays, measuring the reducing power, ATP, and the release of lactate dehydrogenase from treated cells. Results: Comparable drug responses were observed from three different viability cytotoxicity assays. Different patient-derived tumoroids demonstrated differential drug sensitivity that correlates with their clinical cancer stages. Both patient-derived and cancer cell lines cultured in 3D models showed markedly increased drug resistance compared 2D adherent culture. Differential response was also observed between cancer cell lines vs patient-derived lines. Conclusion: Taken together, these findings suggest the potential advantages of 3D patient-derived tumoroid models over 2D culture and cancer cell lines for accurate prediction of drug response. Through targeted NGS and RNA-seq, patient-specific drug targets could be identified for personalized drug screening. Selected drug candidates could be further subjected to in vivo drug validation using patient-derived xenograft model generated from the same donor. In combination with other derived models or large donor banks, this workflow provides a platform for large-scale drug discovery workflows and precision medicine. Citation Format: Xiaoyu Yang, Andrew Tsao, Garrett Wong, Chris Yankaskas, Colin Paul, Brittany Balhouse, Amber Bullock, Anthony Chatman, Shyanne Salen, Sybelle Djikeng, Matt Dallas, David Kuninger. Multiplexed plate-reader based drug screening of 3D-tumoroid models [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 2750.

Tumoroid technology enables culture of patient tissue-derived cancer cells in 3D, with retention of key characteristics from the original patient tumor. However, current tumoroid culture relies on labor-intensive media formulations and culture workflows. To address these issues, we have developed the serum- and conditioned medium-free Gibco™ OncoPro™ Tumoroid Culture Medium to enable derivation and expansion of patient-derived lines from multiple cancer indications. The system has been optimized to retain key patient genotypic and phenotypic characteristics during in vitro culture in a Wnt agonist-free system that can be easily adopted and transferred between labs. 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, including tumoroids originally derived in conditioned medium containing Wnt-3A, R-spondin 3, and Noggin. Cells were assessed for survival, growth, and fidelity to starting material in both OncoPro Tumoroid Culture Medium and in PDMR-recommended homebrew media. Growth and morphology of tumoroid cells was monitored and was comparable between culture conditions. To test for genetic stability of cultures over time, the mutational status of organoids expanded in each condition was characterized using targeted NGS. Both SNV allelic frequency and ploidy values were conserved from initial starting material following expansion to cryopreservation-competent banks (>10e6 cells). Gene expression levels across over >20,000 human RefSeq genes were compared between culture methods and showed high (>0.9) correlation, and Wnt-related signaling pathways were not differentially regulated between media types. In select cases, tumoroid cells expanded in each condition were also analyzed by flow cytometry using optimized antibody panels and protocols. Total cell viability and expression of EpCAM, CD45, CD31, smooth muscle actin, vimentin, CDX2, CEACAM, and cytokeratin 7 was nearly identical across culture conditions. Taken together, our approach represents a simple and effective method to expand and maintain patient-derived human cancer organoids in vitro, with no clear adverse effects from moving to a Wnt-free system. Citation Format: Colin Paul, Brittany Balhouse, Chris Yankaskas, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Expansion of established patient-derived tumoroids in a novel serum-free, Wnt-free media system [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 177.