Organotypic culture assays for murine and human primary and metastatic-site tumors

Published in Protocols & Methods

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We need simplified model systems to study complex biological processes like metastasis. In the last decade, organoids have provided unique advantages that complement the strengths of 2D culture and in vivo mouse models. Organoids are composed of a few hundred cells and are able to self-organize into structures that resemble the corresponding tissue architecture in vivo. Organoids have successfully been derived from many organs, including normal and cancerous mammary gland, prostate, brain, stomach, liver, kidney, lung, and intestine. They are generated using two broad categories – (1) clonal organoids are derived from rare stem cell populations and (2) freshly isolated and therefore heterogenous tissue organoids. We have utilized tissue organoids to study various aspects of cancer progression and metastasis including proliferation, survival, invasion, dissemination, and colony formation.


Isolation of heterogenous tissue organoids applies three key principles – (1) physical and enzymatic breakdown of the tissue, (2) depletion of stromal cells or single epithelial cells, and (3) enrichment of epithelial organoids consisting of ~100-500 cells each. These principles can be readily applied to other epithelial tissues. We have successfully used these methods to isolate organoids from primary and metastatic tumors of the mammary gland, pancreas, and liver across murine, human, and PDX models. This workflow also allows for a convenient and controlled assessment of stromal influences on key cancer cell phenotypes. We have evaluated the effects of immune cells, fibroblasts, and ECM composition on tumor organoids. For example, a single change from a basement membrane-like ECM (Matrigel) to a stromal ECM (collagen I) is sufficient to induce invasion in a tumor organoid. Furthermore, 3D organotypic culture systems are amenable to downstream genetic, cellular, and molecular applications including flow cytometry, immunofluorescence, viral transduction, and Western blotting.    


A significant limitation in studying metastasis has been the lack of in vitro assays that model complex processes such as invasion and local dissemination, tumor cell seeding and metastatic outgrowth. To overcome this difficulty, we designed and developed 3D culture-based assays that model specific aspects of tumor progression in vivo. Organoids embedded in 3D Matrigel grow several-fold while retaining epithelial organization, making it ideal for assessing tumor growth. Using cold-polymerized 3D collagen I, we provide a microenvironment in which most tumor organoids invade and disseminate locally. We also model cellular phenotypes of survival, seeding, and outgrowth in a modified colony formation assay; making it a great surrogate for in vivo tail vein injections.


We anticipate that these methods can readily be extended to various other tissue types and be effective tools in identifying genetic, molecular, or microenvironmental factors that regulate tumor progression. You can learn more about our protocols for variations of 3D organotypic culture systems and their downstream analyses in our recent publication (

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