It has been more than 20 years since it was first demonstrated that endothelial cells will rapidly form capillary-like structures in vitro when plated on top of a reconstituted basement membrane extracellular matrix (BME, Matrigel, EHS matrix, etc.). Subsequently, this morphological differentiation has been demonstrated with a variety of endothelial cells; with endothelial progenitor cells; and with transformed/immortalized endothelial cells. The differentiation process involves several steps in blood vessel formation, including cell adhesion, migration, alignment, protease secretion, and tubule formation. Because the formation of vessel structures is rapid and quantifiable, endothelial cell differentiation on basement membrane has found numerous applications in assays. Such differentiation has been used (1) to study angiogenic and antiangiogenic factors, (2) to define mechanisms and pathways involved in angiogenesis, and (3) to define endothelial cell populations. Further, the endothelial cell differentiation assay has been successfully used to study processes ranging from wound repair and reproduction to development and tumor growth. The assay is easy to perform and is the most widely used in vitro angiogenesis assay.

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Significant advances in our understanding of cancer cell behavior, growth, and metastasis have been facilitated by studies using a basement membrane-like extracellular matrix extract, also known as Matrigel. The basement membrane is a thin extracellular matrix that is found in normal tissues and contacts epithelial and endothelial cells, smooth muscle, fat, Schwann cells, etc. It is composed of mainly laminin-111, collagen IV, heparan sulfate proteoglycan, entactin/nidogen, and various growth factors (fibroblast growth factor, transforming growth factor beta, epidermal growth factor, etc.). Most tumors of epithelial origin produce significant amounts of basement membrane matrix and interact with it particularly during metastasis. Cancer cells metastasize via degradation of the vessel basement membrane matrix to extravasate into the blood stream and colonize distant sites. This review will focus on the interaction of cancer cells and cancer stem cells with the basement membrane-like matrix and the various uses of this interaction to accelerate tumor growth in vivo and to develop in vitro assays for invasion, morphology, and dormancy. Such assays and methods have advanced our understanding of the process of cancer progression, the genes and pathways that are involved, the potential of various therapeutic agents, the effects of neighboring cells, and the role of stem cells.

Many cells in tissues are in contact with a highly specialized extracellular matrix, termed the basement membrane. Basement membranes have certain common components, including collagen IV, laminins, heparan sulfate proteoglycans, and growth factors which have a wide variety of biological activities. Extracts of basement membrane-rich tissue have yielded material suitable for studying cell-basement membrane interactions. Cells cultured in a 3D basement membrane matrix allow the in vitro modeling of cell behavior, including differentiation, apoptosis, steps in capillary formation, cancer growth, invasion, etc. It has also led to the development of widely used assays for invasion and angiogenesis and more recently for tumor cell dormancy. Importantly, stem cell culture in 3D basement membrane matrices has provided important advances that allow for expansion of these cells in feeder layer-free cultures and for studying their differentiation. 3D basement membrane culture has allowed the molecular dissection of pathways and genes important in differentiation, aided in the identification of progenitor cells, and led to the development of tissue constructs which may be models for regenerative medicine. This review will outline how this technology has led to important research assays and findings that have advanced our understanding of tissue development and disease and aided in the preclinical development of various therapeutics.

This protocol requires 2-4 h and presents a method for injecting tumor cells, cancer stem cells or dispersed biopsy material into subcutaneous or orthotopic locations within recipient mice. The tumor cells or biopsy are mixed with basement membrane matrix proteins (CultrexBME or Matrigel) at 4 °C and then injected into recipient animals at preferred anatomical sites. Tumor cells can also be co-injected with additional cell types, such as fibroblasts, stromal cells, endothelial cells and so on. Details are given on appropriate cell numbers, handling and concentration of the basement membrane proteins, recipient animals, injection location and techniques. This procedure enables the growth of tumors from cells or biopsy material (tumor graft) with greater efficiency of take and growth, and with retention of the primary tumor phenotype based on histology. Co-injection with additional cell types provides more physiological models of human cancers for use in drug screening and studying cancer biology.

Many anti-cancer drugs fail in human trials despite showing efficacy in preclinical models. It is clear that the in vitro assays involving 2D monoculture do not reflect the complex extracellular matrix, chemical, and cellular microenvironment of the tumor tissue, and this may explain the failure of 2D models to predict clinical efficacy. We first optimized an in vitro microtumor model using a tumor-aligned ECM, a tumor-aligned medium, MCF-7 and MDA-MB-231 breast cancer spheroids, human umbilical vein endothelial cells, and human stromal cells to recapitulate the tissue architecture, chemical environment, and cellular organization of a growing and invading tumor. We assayed the microtumor for cell proliferation and invasion in a tumor-aligned extracellular matrix, exhibiting collagen deposition, acidity, glucose deprivation, and hypoxia. We found maximal proliferation and invasion when the multicellular spheroids were cultured in a tumor-aligned medium, having low pH and low glucose, with 10% fetal bovine serum under hypoxic conditions. In a 7-day assay, varying doses of fluorouracil or paclitaxel had differential effects on proliferation for MCF-7 and MDA-MB-231 tumor spheroids in microtumor compared to 2D and 3D monoculture. The microtumors exhibited a tumor morphology and drug response similar to published xenograft data, thus demonstrating a more physiologically predictive in vitro model.

This study determines the role of laminin-1 in promoting metastatic colonization during breast cancer. For this purpose, human mammary epithelial cell lines representing normal (MCF-10A), adenocarcinoma (MCF-7), and malignant carcinoma (MDA-MB-231) were propagated in 3-dimensional cultures composed of laminin-1, collagen I, or mixtures of the two, and analyzed by Western blot, immunocytochemistry, semiquantitative reverse transcription polymerase chain reaction, and methylation-specific PCR. Here we demonstrate that laminin-1 decreases methylation of the E-cadherin promoter, resulting in increased mRNA and protein expression for malignant mammary epithelial cells. This decreased methylation is associated with dramatic changes in the cellular and structural morphology as well as a 70-fold decrease in DNA methyltransferase 1 (DNMT1) and a 6-fold decrease in cadherin 11 protein expression. To control for specificity of laminin-1 interactions, cells were also cultured on 2-dimensional plastic substrata and collagen I hydrogels for analysis, and the MCF-10A and MCF-7 were used as nonmalignant controls. Using a 3-dimensional model, we present evidence that laminin-1 is capable of inducing epigenetic change by inhibiting expression of DNMT1 and preventing methylation of the E-cadherin promoter, resulting in E-cadherin expression and the formation of cell-cell bonds in malignant carcinoma.

The utilization of basement membrane matrix has helped to overcome many of the obstacles associated with stem cell research. Initially, there were several problems with investigating stem cells, including difficult extraction from tissues, the need for feeder layers, poor survival, minimal proliferation, limited differentiation in vitro, and inadequate survival when injected or transplanted in vivo. Given that the basement membrane is the first extracellular matrix that is produced by the developing embryo, it was quickly identified as an important factor for modulating stem cell behavior, and since then, basement membrane extract (BME) has been successfully employed in numerous methods as a substratum in vitro and as a bioactive support in vivo to overcome many of these problems. A thin BME coating is sufficient to maintain an undifferentiated phenotype during embryonic stem cell expansion, while a thick BME hydrogel may be employed to induce stem cell differentiation. BME also promotes stem cell survival for in vivo applications and provides a physiological environment for evaluating stem cell co-culture with other cell types. The present article provides a concise review of current methodologies utilizing BME for stem cell research.