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

  • Nat Protoc. 2020 Aug;15(8):2413-2442. doi: 10.1038/s41596-020-0335-3.
Veena Padmanaban  1 ,  Eloise M Grasset  1 ,  Neil M Neumann  1 ,  Andrew K Fraser  1 ,  Elodie Henriet  1 ,  William Matsui  2  3 ,  Phuoc T Tran  2  4 ,  Kevin J Cheung  1  2  5 ,  Dan Georgess  1  6 ,  Andrew J Ewald  7  8
Affiliations
  • 1. Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • 2. Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • 3. Department of Oncology and Livestrong Cancer Institutes, Dell Medical School, University of Texas at Austin, Austin, TX, USA.
  • 4. Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • 5. Translational Research Program, Public Health Sciences and Human Biology Divisions, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
  • 6. Department of Natural Sciences, School of Arts & Sciences, Lebanese American University, Beirut, Lebanon.
  • 7. Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. [email protected].
  • 8. Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. [email protected].
PMID: 32690957 DOI: 10.1038/s41596-020-0335-3
Abstract

Cancer invasion and metastasis are challenging to study in vivo since they occur deep inside the body over extended time periods. Organotypic 3D culture of fresh tumor tissue enables convenient real-time imaging, genetic and microenvironmental manipulation and molecular analysis. Here, we provide detailed protocols to isolate and culture heterogenous organoids from murine and human primary and metastatic site tumors. The time required to isolate organoids can vary based on the tissue and organ type but typically takes <7 h. We describe a suite of assays that model specific aspects of metastasis, including proliferation, survival, invasion, dissemination and colony formation. We also specify comprehensive protocols for downstream applications of organotypic cultures that will allow users to (i) test the role of specific genes in regulating various cellular processes, (ii) distinguish the contributions of several microenvironmental factors and (iii) test the effects of novel therapeutics.