A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Trisomy 16 mice have cardiovascular abnormalities thought to arise from altered development and maturation of the cardiac cushions. Cell-cell and cell-extracellular matrix (ECM) interactions play critical roles in heart morphogenesis. To begin to examine the potential involvement of cell-ECM interactions in abnormal trisomy 16 heart development, fibroblasts were isolated from normal and trisomy 16 embryonic mouse hearts. Behavior of these cells was compared in bioassays involving cell-ECM interactions including cell attachment and collagen gel contraction. Significant differences in cell-ECM interactions were found between fibroblasts isolated from normal and trisomy 16 embryonic hearts. Trisomy 16 cells attached poorly to collagen and laminin compared to normal fibroblasts. Trisomy 16 heart fibroblasts also contracted collagen gels less effectively than normal heart fibroblasts. Cell-ECM interactions are largely mediated by ECM receptors of the integrin family. Expression of beta 1 integrins was examined at the mRNA and protein levels in normal and trisomy 16 fibroblasts. Analyses of integrin expression indicated the pattern of integrins produced by normal and trisomy 16 fibroblasts to be similar. These results indicate that fibroblasts isolated from embryonic trisomy 16 mouse hearts interact with several ECM components including collagen and laminin less efficiently than fibroblasts from normal mouse embryos. As cell-ECM interactions play significant roles in cardiac cushion development, abnormal interactions may contribute to defective atrioventricular septal morphogenesis in the trisomy 16 mouse.

The differentiation and morphogenesis of neural tissues involves a diversity of interactions between neural cells and their environment. Many potentially important interactions occur with the extracellular matrix (ECM), a complex association of extracellular glycoproteins organized into aggregates and polymers. In this article, we discuss recent findings on neuronal interactions with the ECM and their roles in neural cell migration and neurite growth. First, we examine the expression and putative functions of the molecules of the neural ECM. Second, we discuss cell surface molecules that mediate neural interactions with ECM components. Last, we address proteoglycans (PGs), a diverse class of glycoproteins, present both as ECM components and as cell surface molecules, which may mediate neural interactions with their environment. The best-understood cellular interactions with the ECM are adhesive, mediated by binding between specific cell surface molecules and cell binding domains of ECM components (Strittmater and Fishman, 199 1; Damsky and Werb, 1992). Cellsubstratum adhesion is necessary for major cell movements of neuron morphogenesis, that is, the migrations of neural cells and their precursors and the migratory behavior ofgrowth cones at the extending tips of axons and dendrites. As cells move, adhesive molecules at the surface of the leading edge of a migrating cell or growth cone bind to ligands on other cell surfaces or ECM components. These bonds stabilize filopodia and lamellipodia, and, in some cases, provide anchorage against which cytoskeletal filaments, associated with the plasma membrane, exert forces to pull the cell or growth cone forward. Thus, ECM has been primarily viewed as an adhesive substratum to provide traction for migrating cells and to stabilize the position and, perhaps, the state of differentiation of nonmotile cells. However, the interactions between neural cells and the ECM are not longer regarded as only adhesive or mechanical. Two points are now clear. First, some of these interactions are definitely not adhesive, but, rather, they may even be antiadhesive (Chiquet-Ehrismann, 199 1). Second, evidence has accumulated to indicate that the cell surface molecules that mediate cell-cell and cell-ECM interactions (immunoglobulin superfamily, cad

Characterization of the Binding of Serum Amyloid P to Laminin

LIM-kinase is critical for the mesenchymal-to-amoeboid cell morphological transition in 3D matrices

Human Caco-2 cells transfected with c-Ha-Ras as a model for endocrine differentiation in the large intestine

Hepatic stellate cells (HSC) changed their morphology and function including production of matrix metalloproteinases (MMPs) in response to extracellular matrix (ECM) component used as a substratum in culture. We examined in this study the regulatory role of ECM component on expression of MMPs and tissue inhibitor of metalloproteinase (TIMP) in rat HSCs cultured on polystyrene, type I collagen-coated surface, type I collagen gel, or Matrigel, respectively. When cultured on type I collagen gel, HSCs showed the asteroid cell shape and MMP-1 activity, as detected by in situ zymography. Expression of MMP-1 protein and mRNA were examined by using immunofluorescence staining and RT-PCR analysis in HSCs cultured on type I collagen gel. Active form of MMP-2 was detected by gelatin zymography in the conditioned medium of HSCs cultured on type I collagen gel, whereas it was not detected when HSCs were cultured on polystyrene, type I collagen-coated surface, or Matrigel. Increased MMP-2 mRNA was detected by RT-PCR in HSCs cultured on type I collagen gel. Increased MT1-MMP proteins were shown to localize on the cell membrane by using immunofluorescence staining in HSCs cultured on type I collagen gel. Elevated expression of membrane-type matrix metallproteinase-1 (MT1-MMP) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR in HSCs cultured on type I collagen-coated surface or type I collagen gel. These results indicate that expression of MMPs and TIMP-2 is regulated by ECM components in cultured HSCs, suggesting an important role of HSCs in the remodeling of liver tissue.

In vitro breast cancer tissue models play key roles in studying cancer biology and drug discovery. In efforts to recreate native breast cancer spatial organization, 3D in vitro models have received increased attention. These models can be categorized into two major classes: scaffold-based and scaffold-free systems. Scaffold based systems, such as pre-fabricated scaffolds and assembled hydrogels composed of natural and/or synthetic materials, promote cell-extracellular matrix (ECM) interactions. In the case of scaffolds, cells are seeded on the surface of a matrix, whereas in hydrogels, cells are surrounded by a matrix in all dimensions. In both cases, the cells can receive important physical and biochemical cues from the scaffolds that impact their function. In contrast, scaffold-free systems, such as cancer cell spheroids, promote extensive cell-cell interactions, as cells are densely packed in aggregate forms via cell-cell adhesion ligands. These cell-cell interactions via direct contact, in addition to secreted paracrine factors, are also important signals that regulate cell behavior. Despite substantial progress in developing 3D breast cancer models, significant challenges still remain. The breast cancer microenvironment typically possesses both strong cell-cell and cell-ECM interactions, and recapitulating them in in vitro 3D models is essential. However, in most in vitro systems, enhancing one of these interactions often results in decreasing the other. Collagen fibers are one of the major ECM molecules in breast tumors, and analyses of patient biopsies indicate that breast cancer cells often reside in collagen fiber-rich ECMs. Collagen fibers are highly ordered and hierarchical. In nature, collagen molecules assemble into fibrils with diameters on the order of a hundred nanometers. These fibrils bundle to form fibers with diameters of ~1-20 microns. Collagen fibers provide structural, mechanical and biochemical signaling to resident cells, which influences their behavior. However, few biomaterial systems have been developed based on natural collagen fibers for 3D cell growth and tissue formation. Here, we developed a strategy for 3D breast tissue model construction in vitro using extracted collagen fibers from decellularized natural tissues. In this platform, breast cancer cells and supporting cells are cultured within the gaps between individual collagen fibers, which resembles natural conditions. This system maintains strong cell-ECM and cell-cell interactions for resident breast cancer cells and the surrounding stromal fibroblasts or mesenchymal cells. Using this platform, a number of in vitro breast cancer models have been established, including inflammatory breast carcinoma, ductal carcinoma, and pleomorphic breast carcinoma. Importantly, implanting the model tissue onto the chicken chorioallantoic membrane for 9 days resulted in tissue histologically resembling ECM-rich patient breast cancer biopsy tissues. In summary, the extracted native collagen fibers enable the construction of breast cancer models in vitro through maintained cell-cell and cell-ECM interactions. These models histologically resemble in vivo tumor models and patient biopsies. With simple preparation, this platform can be easily scaled up for rapid deployment for downstream applications, such as drug discovery and mechanistic studies of tumor cell interactions as well as cancer progression. With the flexibility to change the cancer cell and surrounding cell types, this system is expected to have great utility for the study of other cancers as well. Citation Format: Rui Tang, Aixiang Ding, Marvin Rivera, Eben Alsberg. Modeling breast cancer tissue in vitro using extracted native collagen fibers [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-53.

Cell-cell and cell-ECM interactions are important regulators of cohesion, a property previously demonstrated to mediate malignant invasion. Intermixing of the epithelial and stromal compartments is often associated with increased invasive capacity and poor prognosis. The studies reported here propose a novel role for α5β1 integrin, the principle mediator of fibronectin matrix assembly (FNMA), as an invasion suppressor in prostate cancer cells. Employing a combination of biophysical and cell biological techniques we explore the relationship between cohesion, invasion, FNMA and tumor-stromal interactions, using a well-characterized prostate cancer model. We show that cohesion is inversely proportional to invasive capacity. We also show that the most invasive cells are deficient in FNMA and express reduced levels of α5β1 integrin. We generated cells over-expressing either wild-type α5 integrin or a non-functional α5 in which the cytoplasmic domain was replaced with that of α2. We show that only wild-type α5 was able to increase cohesion and reduce the invasiveness of aggressive cells. In order to further explore the influence of FNMA on tumor-stromal cell interaction we employed a liquid miscibility model in which interactions between tumor and stroma are rooted in the same thermodynamic principles that govern the mixing of simple fluids. In this model, whether tumor and stromal cells segregate or intermix depends on the ratio of the strength of self-cohesion to cross-adhesion; a high ratio corresponds with segregation, whereas a lower ratio denotes intermixing. We hypothesized that segregation between tumor and stroma can be promoted by increasing the strength of tumor cell cohesion. To test this hypothesis, we first showed that aggressive prostate cancer cells intermix with prostate fibroblasts. We then treated the cancer cells with MEK inhibitors (MEKi) and showed that treatment restored FNMA, leading to increased cell-cell cohesion. When mixed with prostate stromal cells, MEKi-treated cells segregated, suggesting that increasing cohesion between tumor cells is sufficient to reduce their affinity for stromal cells. These data suggest that by increasing tumor cell cohesion through a fibronectin matrix mediated adhesion mechanism, it may be possible to effectively reduce affinity of tumor cells for stroma, thus inhibiting invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5171. doi:1538-7445.AM2012-5171

A Novel Organotypic Model Mimics the Tumor Microenvironment

Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells

A model system for tumor angiogenesis: Involvement of transforming growth factor-α in tube formation of human microvascular endothelial cells induced by esophageal cancer cells

Collagen I gels with protein concentrations of 1, 2, and 3.5 mg/ml were prepared and embedded in a porous polylactide scaffold to reduce their contraction. Concentration of the gel did not affect its degradation. Collagen gels promoted the formation of cell networks. The cells in the collagen gel with a concentration of 1 mg/ml embedded in polylactide scaffold had elongated spindle-like shape, in contrast to flattened cells in collagen gel of the same concentration not embedded in the scaffold. Stabilization of the collagen gel in the polylactide scaffold promoted active synthesis of laminin and fibronectin by cells as soon as on day 5 of culturing in comparison with that in free collagen substrate.

Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: Involvement of transforming growth factor-α

Hepatic stellate cells (HSCs), a mesenchymal cell type in hepatic parenchyma, have unique features with respect to their cellular origin, morphology, and function. Normal, quiescent HSCs function as major vitamin A-storing cells containing over 80% of total vitamin A in the body to maintain vitamin A homeostasis. HSCs are located between parenchymal cell plates and sinusoidal endothelial cells, and extend well-developed, long processes surrounding sinusoids in vivo as pericytes. However, HSCs are known to be 'activated' or 'transdifferentiated' to myofibroblast-like phenotype lacking cytoplasmic lipid droplets and long processes in pathological conditions such as liver fibrosis and cirrhosis, as well as merely during cell culture after isolation. HSCs are the predominant cell type producing extracellular matrix (ECM) components as well as ECM degrading metalloproteases in hepatic parenchyma, indicating that they play a pivotal role in ECM remodeling in both normal and pathological conditions. Recent findings have suggested that HSCs have a neural crest origin from their gene expression pattern similar to neural cell type and/or smooth muscle cells and myofibroblasts. The morphology and function of HSCs are regulated by ECM components as well as by cytokines and growth factors in vivo and in vitro. Liver regeneration after partial hepatectomy might be an invaluable model to clarify the HSC function in elaborate organization of liver tissue by cell-cell and cell-ECM interaction and by growth factor and cytokine regulation.

Multicellular tumor spheroids are the most well-characterized organotypic models for cancer research. Generally, scaffold-based and scaffold-free techniques are widely used for culturing spheroids. In scaffold-free techniques, the hanging drop (HD) method is a more versatile technique, but the retrieval of three-dimensional (3D) cell spheroids in the hanging drop method is usually labor-intensive. We developed oil-coated polystyrene nanofiber-based reusable slippery surfaces for the generation and easy retrieval of 3D spheroids. The developed slippery surfaces facilitated the rolling and gliding of the cell medium drops as well as holding the hydrophilic drops for more than 72 h by the virtue of surface tension as in the hanging drop method. In this study, polystyrene nanofibers were developed by the facile technique of electrospinning and the morphological evaluation was performed by scanning electron microscopy (SEM) and cryo-FESEM. We modeled the retrieval process of 3D spheroids with the ingredients of 3D spheroid generation, such as water, cell culture media, collagen, and hyaluronic acid solution, demonstrating the faster and easy retrieval of 3D spheroids within a few seconds. We created MCF-7 spheroids as a proof of concept with a developed slippery surface. 3D spheroids were characterized for their size, homogeneity, reactive oxygen species, proliferative marker (Ki-67), and hypoxic inducing factor 1ά (HIF-1ά). These 3D tumor spheroids were further tested for evaluating the cellular toxicity of the doxorubicin drug. Hence, the proposed slippery surfaces demonstrated the potential alternative of culturing 3D tumor spheroids with an easy retrieval process with intact 3D spheroids.