A novel 3D in vitro metastasis model elucidates differential invasive strategies during and after breaching basement membrane

Guzman, Asja; Alemany, Víctor Sánchez; Yen, Nguyen Thi Hai; Zhang, Catherine Ruiqi; Kaufman, Laura J.

doi:10.1016/j.biomaterials.2016.11.014

Cited by 31 publications

(32 citation statements)

References 70 publications

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“…Other current 3D spheroid models do not possess an intact, well‐formed basement membrane surrounding the spheroid. Most 3D models model the 3D microenvironment by mixing Matrigel and collagen I (Berens, Holy, Riegel, & Wellstein, 2015) or by using a mixture of agarose and Matrigel to from spheroids (Li et al., 2011)—or they form a thicker and more diffuse layer around the spheroids that is not organized into a discrete, thin sheet of basement membrane (Guzman, Sanchez Alemany, Nguyen, Zhang, & Kaufman, 2017). With the protocols in this article, collagen IV and laminin are observed by immunostaining to be co‐localized, with fibronectin decorating collagen I everywhere in the ECM hydrogel, surrounding the spheroid.…”

Section: Commentarymentioning

confidence: 99%

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

Nazari

2020

CP Cell Biology

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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

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Section: Commentarymentioning

confidence: 99%

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

Nazari

2020

CP Cell Biology

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“…With these caveats being raised, these results support the idea that multiple aspects of cancer cell migration are highly conserved across cancer types, and microenvironmental, casespecific factors might set the conditions for a more individual or collective migration by regulating cancer cell heterogeneity in terms of EMT and migration, as effectively represented by the variation of the parameters in the model. Supporting this picture, it has been recently shown that multiple breast cancer cell lines with varying origin and morphological features can switch from solitary to collective migration by manipulating the physical properties of the extracellular medium in vitro (40,41).…”

Section: Emt-induction In Nearest Neighborsmentioning

confidence: 94%

A biophysical model of Epithelial-Mesenchymal Transition uncovers the frequency and size distribution of Circulating Tumor Cell clusters across cancer types

Bocci

2019

Preprint

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The gain of cellular motility via the epithelial-mesenchymal transition (EMT) is considered crucial in the metastatic cascade. Cells undergoing EMT to varying extents are launched into the bloodstream as single circulating tumor cells (CTCs) or multi-cellular clusters. The frequency and size distributions of these multicellular clusters has been recently measured, but the underlying mechanisms enabling these different modes of migration remain poorly understood. We present a biophysical model that couples the epithelialmesenchymal phenotypic transition and cell migration to explain these different modes of cancer cell migration. With this reduced physical model, we identify a transition from individual migration to clustered cell migration that is regulated by the rate of EMT and the degree of cooperativity between cells during migration. This single cell to clustered migration transition can robustly recapitulate cluster size distributions observed experimentally across several cancer types, thus suggesting the existence of common features in the mechanisms of cell migration during metastasis. Furthermore, we identify three main mechanisms that can facilitate the formation and dissemination of large clusters: first, mechanisms that prevent a complete EMT and instead increase the population of hybrid Epithelial/Mesenchymal (E/M) cells; second, multiple intermediate E/M states that give rise to heterogeneous clusters formed by cells with different epithelial-mesenchymal traits; and third, non-cell-autonomous induction of EMT via cell-tocell signaling that gives rise to spatial correlations among cells in a tissue. Overall, this biophysical model represents a first step toward bridging the gap between the molecular and biophysical understanding of EMT and various modes of cancer cell migration, and highlights that a complete EMT might not be required for metastasis. Statement of significanceThe Epithelial-Mesenchymal Transition (EMT) confers motility and invasive traits to cancer cells. These cells can then enter the circulatory system both as single cells or as multi-cellular clusters to initiate metastases.We develop a biophysical model to investigate how EMT at the single cell level can give rise to a solitary or clustered cell migration. This model quantitatively reproduces cluster size distributions reported in human circulation and mouse models, therefore suggesting similar mechanisms in cancer cell migration across different cancer types. Moreover, we show that a partial EMT to a hybrid epithelial/mesenchymal cell state is sufficient to explain both single cell and clustered migration, therefore questioning the necessity of a complete EMT for cancer metastasis.

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“…117 First, dispersed mammary epithelial cells and diluted rBM were seeded in a concave, low-adhesion well and centrifuged, causing them to self-assemble into a tumor spheroid with a rBM coating after 24 h (Figure 6C). Tumor spheroids were then embedded into collagen I.…”

Section: Enter the Matrix: Epithelial Morphogenesis And Disseminationmentioning

confidence: 99%

“…One advantage of engineered biomaterial platforms is their experimental accessibility relative to animal models, which is particularly useful for optical microscopy. In the publications highlighted here, fluorescence microscopy was utilized to reveal cell migration at the periphery, 87,89,109,115,117,118,150 distinct biomarker expression, 88 disorganization of epithelial architecture, 112,113,119,120,122–124 as well as coordination between tumor and stromal cells. 128–131,133 However, many of these publications utilized manual analysis of selected cells or tissues, which samples a limited subset of the population with low throughput.…”

Section: Future Directionsmentioning

confidence: 99%

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Multicellular tumor invasion and plasticity in biomimetic materials

2017

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Cancer cell invasion through the extracellular matrix is associated with metastatic spread and therapeutic resistance. In carcinomas, the detachment and dissemination of individual cells has been associated with an epithelial-mesenchymal transition, but tumors can also invade using collective, multicellular phenotypes. This malignant tumor progression is also associated with alignment and stiffening of the surrounding extracellular matrix. Historically, tumor invasion has been investigated using 2D monolayer culture, small animal models or patient histology. These assays have been complemented by the use of natural biomaterials such as reconstituted basement membrane and collagen I. More recently, engineered materials with well-defined physical, chemical and biomolecular properties have enabled more controlled microenvironments. In this review, we highlight recent developments in multicellular tumor invasion based on microfabricated structures or hydrogels. We emphasize the role of interfacial geometries, biomaterial stiffness, matrix remodeling, and co-culture models. Finally, we discuss future directions for the field, particularly integration with precision measurements of biomaterial properties and single cell heterogeneity, standardization and scale-up of these platforms, as well as integration with patient-derived samples.

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A novel 3D in vitro metastasis model elucidates differential invasive strategies during and after breaching basement membrane

Cited by 31 publications

References 70 publications

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

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

A biophysical model of Epithelial-Mesenchymal Transition uncovers the frequency and size distribution of Circulating Tumor Cell clusters across cancer types

Multicellular tumor invasion and plasticity in biomimetic materials

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