Human-derived osteoblast-like cells and pericyte-like cells induce distinct metastatic phenotypes in primary breast cancer cells.

Approximately 70% of advanced breast cancer patients will develop bone metastases, which accounts for ∼90% of cancer-related mortality. Breast cancer circulating tumor cells (CTCs) establish metastatic tumors in the bone after a close interaction with local bone marrow cells including pericytes and osteoblasts, both related to resident mesenchymal stem/stromal cells (BM-MSCs) progenitors. In vitro recapitulation of the critical cellular players of the bone microenvironment and infiltrating CTCs could provide new insights into their cross-talk during the metastatic cascade, helping in the development of novel therapeutic strategies. Human BM-MSCs were isolated and fractionated according to CD146 presence. CD146+ cells were utilized as pericyte-like cells (PLCs) given the high expression of the marker in perivascular cells, while CD146- cells were induced into an osteogenic phenotype generating osteoblast-like cells (OLCs). Transwell migration assays were performed to establish whether primary breast cancer cells (3384T) were attracted to OLC. Furthermore, proliferation of 3384T breast cancer cells was assessed in the presence of PLC- and OLC-derived conditioned media. Additionally, conditioned media cultures as well as transwell co-cultures of each OLCs and PLCs were performed with 3384T breast cancer cells for gene expression interrogation assessing their induced transcriptional changes with an emphasis on metastatic potential. PLC as well as their conditioned media increased motility and invasion potential of 3384T breast cancer cells, while OLC induced a dormant phenotype, downregulating invasiveness markers related with migration and proliferation. Altogether, these results indicate that PLC distinctively drive 3384T cancer cells to an invasive and migratory phenotype, while OLC induce a quiescence state, thus recapitulating the different phases of the in vivo bone metastatic process. These data show that phenotypic responses from metastasizing cancer cells are influenced by neighboring cells at the bone metastatic niche during the establishment of secondary metastatic tumors.

Modular Microphysiological System for Modeling of Biologic Barrier Function.

Microphysiological systems, also known as organs-on-chips, are microfluidic devices designed to model human physiology in vitro. Polydimethylsiloxane (PDMS) is the most widely used material for organs-on-chips due to established microfabrication methods, and properties that make it suitable for biological applications such as low cytotoxicity, optical transparency, gas permeability. However, absorption of small molecules and leaching of uncrosslinked oligomers might hinder the adoption of PDMS-based organs-on-chips for drug discovery assays. Here, we have engineered a modular, PDMS-free microphysiological system that is capable of recapitulating biologic barrier functions commonly demonstrated in PDMS-based devices. Our microphysiological system is comprised of a microfluidic chip to house cell cultures and pneumatic microfluidic pumps to drive flow with programmable pressure and shear stress. The modular architecture and programmable pumps enabled us to model multiple in vivo microenvironments. First, we demonstrate the ability to generate cyclic strain on the culture membrane and establish a model of the alveolar air-liquid interface. Next, we utilized three-dimensional finite element analysis modeling to characterize the fluid dynamics within the device and develop a model of the pressure-driven filtration that occurs at the glomerular filtration barrier. Finally, we demonstrate that our model can be used to recapitulate sphingolipid induced kidney injury. Together, our results demonstrate that a multifunctional and modular microphysiological system can be deployed without the use of PDMS. Further, the bio-inert plastic used in our microfluidic device is amenable to various established, high-throughput manufacturing techniques, such as injection molding. As a result, the development plastic organs-on-chips provides an avenue to meet the increasing demand for organ-on-chip technology.

Enzymatically crosslinked gelatin-laminin hydrogels for applications in neuromuscular tissue engineering.

We report a water-soluble and non-toxic method to incorporate additional extracellular matrix proteins into gelatin hydrogels, while obviating the use of chemical crosslinkers such as glutaraldehyde. Gelatin hydrogels were fabricated using a range of gelatin concentrations (4%-10%) that corresponded to elastic moduli of approximately 1 kPa-25 kPa, respectively, a substrate stiffness relevant for multiple cell types. Microbial transglutaminase was then used to enzymatically crosslink a layer of laminin on top of gelatin hydrogels, resulting in 2-component gelatin-laminin hydrogels. Human induced pluripotent stem cell derived spinal spheroids readily adhered and rapidly extended axons on GEL-LN hydrogels. Axons displayed a more mature morphology and superior electrophysiological properties on GEL-LN hydrogels compared to the controls. Schwann cells on GEL-LN hydrogels adhered and proliferated normally, displayed a healthy morphology, and maintained the expression of Schwann cell specific markers. Lastly, skeletal muscle cells on GEL-LN hydrogels achieved long-term culture for up to 28 days without delamination, while expressing higher levels of terminal genes including myosin heavy chain, MyoD, MuSK, and M-cadherin suggesting enhanced maturation potential and myotube formation compared to the controls. Future studies will employ the superior culture outcomes of this hybrid substrate for engineering functional neuromuscular junctions and related organ on a chip applications.

Engineering anisotropic cardiac monolayers on microelectrode arrays for non-invasive analyses of electrophysiological properties.

A standard culture of cardiac cells as unorganized monolayers on tissue culture plastic or glass does not recapitulate the architectural or the mechanical properties of native myocardium. We investigated the physical and protein cues from the extracellular matrix to engineer anisotropic cardiac tissues as highly aligned monolayers on top of the microelectrode array (MEA). The MEA platform allows non-invasive measurement of beating rate and conduction velocity. The effect of different extracellular proteins was tested by using the most common extracellular matrix proteins in the heart, fibronectin and gelatin, after aligning myocytes using a microcontact (µC) printing technique. Both proteins showed similar electrophysiological results before the monolayer began to delaminate after the sixth day of culture. Additionally, there were no significant differences on day 4 between the two microcontact printed proteins in terms of sarcomere alignment and gap junction expression. To test the effect of substrate stiffness, a micromolded (µM) gelatin hydrogel was fabricated in different concentrations (20% and 2%), corresponding to the elastic moduli of approximately 33 kPa and 0.7 kPa, respectively, to cover both spectra of the in vivo range of myocardium. Cardiac monolayers under micromolded conditions beat in a much more synchronized fashion, and exhibited conduction velocity that was close to the physiological value. Both concentrations of gelatin hydrogel conditions yielded similar sarcomere alignment and gap junction expression on day 4 of culture. Ultimately, the 3D micromolded gelatin hydrogel that recapitulated myocardial stiffness improved the synchronicity and conduction velocity of neonatal rat ventricular myocytes (NRVM) without any stimulation. Identifying such microenvironmental factors will lead to future efforts to design heart on a chip platforms that mimic in vivo environment and predict potential cardiotoxicity when testing new drugs.