Multimodal Techniques to Study Tumor Growth, Basement Membrane Breaching, and Invasion in 3D Matrices

Daan Smits, Antoine Khalil

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Cancer-derived organoids and three-dimensional (3D) extracellular matrix (ECM) are taking center stage as in vitro models to study neoplastic cell behavior, since they recapitulate the heterogeneous cellular composition of tumors and their extracellular environment. In combination with imaging and molecular/biochemical techniques, 3D organoid models have contributed substantially to our knowledge about the cellular and molecular mechanisms that regulate the growth of tumors and invasion into the surrounding tissue. We here outline a set of protocols that describe culturing of cancer-derived organoids in 3D matrices and various strategies that allow modeling of tumor growth, tumor cell penetration into basement membranes, and invasion into Collagen I-rich ECM. Furthermore, we specify protocols for subsequent handling of organoids cultured in 3D ECM for confocal microscopy and analysis of gene expression at the protein and mRNA level. Although we here use breast cancer-derived organoids, these protocols can be directly applied or adapted for organoids derived from other cancer types or healthy tissues. Thus, in addition to investigating cell behavior of multiple cancer types, the combination of protocols described here may be used to study processes such as cell differentiation and migration during homeostasis and normal development.
Original languageEnglish
Title of host publicationMethods in Molecular Biology
EditorsC. Margadant
PublisherHumana New York
Number of pages23
ISBN (Electronic)978-1-0716-2887-4
ISBN (Print)978-1-0716-2886-7, 978-1-0716-2889-8
Publication statusPublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • 3D cultures
  • Cancer cell invasion
  • Confocal microscopy
  • ECM remodeling
  • Extracellular matrix
  • Immunofluorescence
  • Organoids
  • Protein extraction
  • RNA extraction


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