Generation of 3D Tumor Spheroids with Encapsulating Basement Membranes for Invasion Studies

In the past, in vitro studies of invasion and tumor progression were performed primarily using cancer cells cultured on a flat, two‐dimensional (2D) surface in a monolayer. In recent years, however, many studies have demonstrated differences in cell signaling and cell migration between 2D and 3D cell cultures. Traditional 2D monolayer cancer cell invasion models do not fully recapitulate 3D cell‐to‐cell and cell−to−extracellular matrix interactions that in vivo models can provide. Moreover, although in vivo animal models are irreplaceable for studying tumor biology and metastasis, they are costly, time‐consuming, and impractical for answering preliminary questions. Thus, emergent and evolving 3D spheroid cell culture models have changed the way we study tumors and their interactions with their surrounding extracellular matrix. In the case of breast cancer, metastasis of breast cancer tumors results in high mortality rates, and thus development of robust cell culture models that are reproducible and practical for studying breast cancer progression is important for ultimately developing preventatives for cancer metastasis. This article provides a set of protocols for generating uniform spheroids with a thin sheet of basement membrane for studying the initial invasion of mammary epithelial cells into a surrounding collagen‐rich extracellular matrix. Details are provided for generating 3D spheroids with a basement membrane, polymerizing collagen I, embedding the spheroids in the 3D collagen gel, and immunostaining the spheroids for invasion studies. Published 2020. U.S. Government. Basic Protocol 1: Growth of uniformly sized tumor spheroids with an encapsulating basement membrane Basic Protocol 2: Polymerization and embedding of tumor spheroids in a 3D type I collagen gel Alternate Protocol: Embedding of tumor spheroids in collagen gels using a sandwich method Basic Protocol 3: Fixing and immunostaining of tumor spheroids embedded in 3D collagen gels