Astrocytes from the brain microenvironment alter migration and morphology of metastatic breast cancer cells.

Tumor cell metastasis to the brain involves cell migration through biochemically and physically complex microenvironments at the blood-brain barrier (BBB). The current understanding of tumor cell migration across the BBB is limited. We hypothesize that an interplay between biochemical cues and physical cues at the BBB affects the mechanisms of brain metastasis. We found that astrocyte conditioned medium(ACM) applied directly to tumor cells increased tumor cell velocity, induced elongation, and promoted actin stress fiber organization. Notably, treatment of the extracellular matrix with ACM led to even more significant increases in tumor cell velocity in comparison with ACM treatment of cells directly. Furthermore, inhibiting matrix metalloproteinases in ACM reversed ACM's effect on tumor cells. The effects of ACM on tumor cell morphology and migration also depended on astrocytes' activation state. Finally, using a microfluidic device, we found that the effects of ACM were abrogated in confinement. Overall, our work demonstrates that astrocyte-secreted factors alter migration and morphology of metastatic breast tumor cells, and this effect depends on the cells' mechanical microenvironment.-Shumakovich, M. A., Mencio, C. P., Siglin, J. S., Moriarty, R. A., Geller, H. M., Stroka, K. M. Astrocytes from the brain microenvironment alter migration and morphology of metastatic breast cancer cells. FASEB J. 31, 5049-5067 (2017). www.fasebj.org.

Fabrication and evaluation of 3D printed BCP scaffolds reinforced with ZrO2 for bone tissue applications.

Fused deposition modeling (FDM) is a promising 3D printing and manufacturing step to create well interconnected porous scaffold designs from the computer-aided design (CAD) models for the next generation of bone scaffolds. The purpose of this study was to fabricate and evaluate a new biphasic calcium phosphate (BCP) scaffold reinforced with zirconia (ZrO2 ) by a FDM system for bone tissue engineering. The 3D slurry foams with blending agents were successfully fabricated by a FDM system. Blending materials were then removed after the sintering process at high temperature to obtain a targeted BCP/ZrO2 scaffold with the desired pore characteristics, porosity, and dimension. Morphology of the sintered scaffold was investigated with SEM/EDS mapping. A cell proliferation test was carried out and evaluated with osteosarcoma MG-63 cells. Mechanical testing and cell proliferation evaluation demonstrated that 90% BCP and 10% ZrO2 scaffold had a significant effect on the mechanical properties maintaining a structure compared that of only 100% BCP with no ZrO2 . Additionally, differentiation studies of human mesenchymal stem cells (hMSCs) on BCP/ZrO2 scaffolds in static and dynamic culture conditions showed increased expression of bone morphogenic protein-2 (BMP-2) when cultured on BCP/ZrO2 scaffolds under dynamic conditions compared to on BCP control scaffolds. The manufacturing of BCP/ZrO2 scaffolds through this innovative technique of a FDM may provide applications for various types of tissue regeneration, including bone and cartilage.

Matrix stiffness regulates the tight junction phenotypes and local barrier properties in tricellular regions in an iPSC-derived BBB model.

The blood-brain barrier (BBB) can respond to various mechanical cues such as shear stress and substrate stiffness. In the human brain, the compromised barrier function of the BBB is closely associated with a series of neurological disorders that are often also accompanied by the alteration of brain stiffness. In many types of peripheral vasculature, higher matrix stiffness decreases barrier function of endothelial cells through mechanotransduction pathways that alter cell-cell junction integrity. However, human brain endothelial cells are specialized endothelial cells that largely resist changes in cell morphology and key BBB markers. Therefore, it has remained an open question how matrix stiffness affects barrier integrity in the human BBB. To gain insight into the effects of matrix stiffness on BBB permeability, we differentiated brain microvascular endothelial-like cells from human induced pluripotent stem cells (iBMEC-like cells) and cultured the cells on extracellular matrix-coated hydrogels of varying stiffness. We first detected and quantified the junction presentation of key tight junction (TJ) proteins. Our results show matrix-dependent junction phenotypes in iBMEC-like cells, where cells on softer gels (1 kPa) have significantly lower continuous and total TJ coverages. We also determined that these softer gels also lead to decreased barrier function in a local permeability assay. Furthermore, we found that matrix stiffness regulates the local permeability of iBMEC-like cells through the balance of continuous ZO-1 TJs and no junction regions ZO-1 in tricellular regions. Together, these findings provide valuable insights into the effects of matrix stiffness on TJ phenotypes and local permeability of iBMEC-like cells. STATEMENT OF SIGNIFICANCE: Brain mechanical properties, including stiffness, are particularly sensitive indicators for pathophysiological changes in neural tissue. The compromised function of the blood-brain barrier is closely associated with a series of neurological disorders often accompanied by altered brain stiffness. In this study, we use polymeric biomaterials and provide new evidence that biomaterial stiffness regulates the local permeability in iPSC-derived brain endothelial cells in tricellular regions through the tight junction protein ZO-1. Our findings provide valuable insights into the changes in junction architecture and barrier permeability in response to different substrate stiffnesses. Since BBB dysfunction has been linked to many diseases, understanding the influence of substrate stiffness on junction presentations and barrier permeability could lead to the development of new treatments for diseases associated with BBB dysfunction or drug delivery across BBB systems.

RNA localization in confined cells depends on cellular mechanical activity and contributes to confined migration.

Cancer cells experience mechanical confining forces during metastasis and, consequently, can alter their migratory mechanisms. Localization of numerous mRNAs to cell protrusions contributes to cell polarization and migration and is controlled by proteins that can bind RNA and/or cytoskeletal elements, such as the adenomatous polyposis coli (APC). Here, we demonstrate that peripheral localization of APC-dependent RNAs in cells within confined microchannels is cell type dependent. This varying phenotype is determined by the levels of a detyrosinated tubulin network. We show that this network is regulated by mechanoactivity and that cells with mechanosensitive ion channels and increased myosin II activity direct peripheral localization of the RAB13 APC-dependent RNA. Through specific mislocalization of the RAB13 RNA, we show that peripheral RNA localization contributes to confined cell migration. Our results indicate that a cell's mechanical activity determines its ability to peripherally target RNAs and utilize them for movement in confinement.

Recent progress and new challenges in modeling of human pluripotent stem cell-derived blood-brain barrier.

The blood-brain barrier (BBB) is a semipermeable unit that serves to vascularize the central nervous system (CNS) while tightly regulating the movement of molecules, ions, and cells between the blood and the brain. The BBB precisely controls brain homeostasis and protects the neural tissue from toxins and pathogens. The BBB is coordinated by a tight monolayer of brain microvascular endothelial cells, which is subsequently supported by mural cells, astrocytes, and surrounding neuronal cells that regulate the barrier function with a series of specialized properties. Dysfunction of barrier properties is an important pathological feature in the progression of various neurological diseases. In vitro BBB models recapitulating the physiological and diseased states are important tools to understand the pathological mechanism and to serve as a platform to screen potential drugs. Recent advances in this field have stemmed from the use of pluripotent stem cells (PSCs). Various cell types of the BBB such as brain microvascular endothelial cells (BMECs), pericytes, and astrocytes have been derived from PSCs and synergistically incorporated to model the complex BBB structure in vitro. In this review, we summarize the most recent protocols and techniques for the differentiation of major cell types of the BBB. We also discuss the progress of BBB modeling by using PSC-derived cells and perspectives on how to reproduce more natural BBBs in vitro.

Mechanosensing of Mechanical Confinement by Mesenchymal-Like Cells.

Mesenchymal stem cells (MSCs) and tumor cells have the unique capability to migrate out of their native environment and either home or metastasize, respectively, through extremely heterogeneous environments to a distant location. Once there, they can either aid in tissue regrowth or impart an immunomodulatory effect in the case of MSCs, or form secondary tumors in the case of tumor cells. During these journeys, cells experience physically confining forces that impinge on the cell body and the nucleus, ultimately causing a multitude of cellular changes. Most drastically, confining individual MSCs within hydrogels or confining monolayers of MSCs within agarose wells can sway MSC lineage commitment, while applying a confining compressive stress to metastatic tumor cells can increase their invasiveness. In this review, we seek to understand the signaling cascades that occur as cells sense confining forces and how that translates to behavioral changes, including elongated and multinucleated cell morphologies, novel migrational mechanisms, and altered gene expression, leading to a unique MSC secretome that could hold great promise for anti-inflammatory treatments. Through comparison of these altered behaviors, we aim to discern how MSCs alter their lineage selection, while tumor cells may become more aggressive and invasive. Synthesizing this information can be useful for employing MSCs for therapeutic approaches through systemic injections or tissue engineered grafts, and developing improved strategies for metastatic cancer therapies.

Physical confinement alters sarcoma cell cycle progression and division.

Tumor cells experience physical confinement on one or multiple axes, both in the primary tumor and at multiple stages during metastasis. Recent work has shown that confinement in a 3D spheroid alters nucleus geometry and delays cell division, and that vertical confinement impairs mitotic spindle rounding, resulting in abnormal division events. Meanwhile, the effects of bi-axial confinement on cell cycle progression has received little attention. Given the critical role of nuclear shape and mechanics in cell division, we hypothesized that bi-axial physical confinement of the cell body and nucleus would alter cell cycle progression. We used sarcoma cells stably expressing the fluorescence ubiquitination cell cycle indicator (FUCCI), along with fibronectin-coated microchannel devices, and explored the impact of bi-axial physical confinement on cell cycle progression. Our results demonstrate that bi-axial physical confinement reduces the frequency of cell division, which we found to be attributed to an arrest in the S/G2/M phase of the cell cycle, and increases the frequency of abnormal division events. Cell and nuclear morphology were both altered in confinement, with the most confining channels preventing cells from undergoing the normal increase in size from G1 to S/G2/M during cell cycle progression. Finally, our results suggest that confinement induces a mechanical memory to the cells, given our observation of lasting effects on cell division and morphology, even after cells exited confinement. Together, our results provide new insights into the possible impact of mechanical forces on primary and secondary tumor formation and growth.

Collagen hydrogel scaffold promotes mesenchymal stem cell and endothelial cell coculture for bone tissue engineering.

The generation of functional, vascularized tissues is a key challenge for the field of tissue engineering. Before clinical implantations of such tissue engineered bone constructs can succeed, tactics to promote neovascularization need to be strengthened. We have previously demonstrated that the tubular perfusion system (TPS) bioreactor is an effective culturing method to augment osteogenic differentiation and maintain viability of human mesenchymal stem cells (hMSC). Here, we devised a strategy to address the need for a functional microvasculature by designing an in vitro coculture system that simultaneously cultures osteogenic differentiating hMSCs with endothelial cells (ECs). We utilized the TPS bioreactor as a dynamic coculture environment, which we hypothesize will encourage prevascularization of endothelial cells and early formation of bone tissue and could aid in anastomosis of the graft with the host vasculature after patient implantation. To evaluate the effect of different natural scaffolds for this coculture system, the cells were encapsulated in alginate and/or collagen hydrogel scaffolds. We discovered the necessity of cell-to-cell proximity between the two cell types as well as preference for the natural cell binding capabilities of hydrogels like collagen. We discovered increased osteogenic and angiogenic potential as seen by amplified gene and protein expression of ALP, BMP-2, VEGF, and PECAM. The TPS bioreactor further augmented these expressions, indicating a synergistic effect between coculture and applied shear stress. The development of this dynamic coculture platform for the prevascularization of engineered bone, emphasizing the importance of the construct microenvironments and will advance the clinical use of tissue engineered constructs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1123-1131, 2017.

Dynamic Bioreactor Culture of High Volume Engineered Bone Tissue.

Within the field of tissue engineering and regenerative medicine, the fabrication of tissue grafts of any significant size--much less a whole organ or tissue--remains a major challenge. Currently, tissue-engineered constructs cultured in vitro have been restrained in size primarily due to the diffusion limit of oxygen and nutrients to the center of these grafts. Previously, we developed a novel tubular perfusion system (TPS) bioreactor, which allows the dynamic culture of bead-encapsulated cells and increases the supply of nutrients to the entire cell population. More interestingly, the versatility of TPS bioreactor allows a large range of engineered tissue volumes to be cultured, including large bone grafts. In this study, we utilized alginate-encapsulated human mesenchymal stem cells for the culture of a tissue-engineered bone construct in the size and shape of the superior half of an adult human femur (∼ 200 cm(3)), a 20-fold increase over previously reported volumes of in vitro engineered bone grafts. Dynamic culture in TPS bioreactor not only resulted in high cell viability throughout the femur graft, but also showed early signs of stem cell differentiation through increased expression of osteogenic genes and proteins, consistent with our previous models of smaller bone constructs. This first foray into full-scale bone engineering provides the foundation for future clinical applications of bioengineered bone grafts.