Summary Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications.
Summary Patient-derived tumor organoid cultures are an essential and innovative methodology for translational research. However, current techniques to establish these cultures are cumbersome, expensive, and often require irreplaceable clinical tissue from surgery or core biopsies. Fine-needle aspiration (FNA) provides a minimally invasive biopsy technique commonly performed in clinical settings. Here, we provide a protocol for FNA. We have found that FNA provides a cost-effective, rapid, and streamlined method for tissue acquisition for cancer organoid culture. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020) and Vilgelm et al. (2020) .
Thyroid cancer has the fastest growing incidence of any cancer in the United States, as measured by the number of new cases per year. Despite advances in tissue culture techniques, a robust model for thyroid cancer spheroid culture is yet to be developed. Using eight established thyroid cancer cell lines, we created an efficient and cost-effective 3D culture system that can enhance our understanding of in vivo treatment response. We found that all eight cell lines readily form spheroids in culture with unique morphology, size, and cytoskeletal organization. In addition, we developed a high-throughput workflow that allows for drug screening of spheroids. Using this approach, we found that spheroids from K1 and TPC1 cells demonstrate significant differences in their sensitivities to dabrafenib treatment that closely model expected patient drug response. In addition, K1 spheroids have increased sensitivity to dabrafenib when compared to monolayer K1 cultures. Utilizing traditional 2D cultures of these cell lines, we evaluated the mechanisms of this drug response, showing dramatic and acute changes in their actin cytoskeleton as well as inhibition of migratory behavior in response to dabrafenib treatment. Our study is the first to describe the development of a robust spheroid system from established cultured thyroid cancer cell lines and adaptation to a high-throughput format. We show that combining 3D culture with traditional 2D methods provides a complementary and powerful approach to uncover drug sensitivity and mechanisms of inhibition in thyroid cancer.
Summary Generation of fine-needle aspiration (FNA)-derived cancer organoids has allowed us to develop a number of downstream applications. In this protocol, we start with organoids cultured in a semi-solid format. We dissociate organoids into single cells and then plate in a 384-well format for high-throughput drug screening. While this method must be fine-tuned for each individual organoid culture, it offers a format well suited for rapidly screening medium-sized drug/compound libraries (500–5,000 molecules) and generating dose-response curves to measure relative efficacy. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020) and Vilgelm et al. (2020) .
Summary Our organoid generation technique has allowed for the development of downstream organoid applications. Here, we detail an accessible, straightforward protocol for immunofluorescent staining and imaging of thyroid cancer organoids, particularly those with tumor de-differentiation. Immunofluorescence is a powerful tool to help understand the localization of cell types within organoids and determine the interactions between those cells. As organoids have been shown to recapitulate patient tumor morphology, immunofluorescent staining and imaging of organoids allows for enhanced understanding of near in vivo structures. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020) and Vilgelm et al. (2020) .
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