Metastasis is a major cause of mortality and remains a hurdle in the search for a cure for cancer. Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for transfer of microRNAs. The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of ‘metastatic hijack’: cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in vivo. Targeting these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer.

Tissue loss due to oral diseases requires the healing and regeneration of tissues of multiple lineages. While stem cells are native to oral tissues, a current major limitation to regeneration is the ability to direct their lineage-specific differentiation. This work utilizes polymeric scaffold systems with spatiotemporally controlled morphogen cues to develop precise morphogen fields to direct mesenchymal stem cell differentiation. First, a simple three-layer scaffold design was developed that presented two spatially segregated, lineage-specific cues (Dentinogenic TGF-β1 and Osteogenic BMP4). However, this system resulted in diffuse morphogen fields, as assessed by the in vitro imaging of cell-signaling pathways triggered by the morphogens. Mathematical modeling was then exploited, in combination with incorporation of specific inhibitors (neutralizing antibodies or a small molecule kinase inhibitor) into each morphogen in an opposing spatial pattern as the respective morphogen, to design a five-layer scaffold that was predicted to yield distinct, spatially segregated zones of morphogen signaling. To validate this system, undifferentiated MSCs were uniformly seeded in these scaffold systems, and distinct mineralized tissue differentiation were noted within these morphogen zones. Finally, to demonstrate temporal control over morphogen signaling, latent TGF-β1 was incorporated into one region of a concentric scaffold design, and laser treatment was used to activate the morphogen on-demand and to induce dentin differentiation solely within that specific spatial zone. This study demonstrates a significant advance in scaffold design to generate precise morphogen fields that can be used to develop in situ models to explore tissue differentiation and may ultimately be useful in engineering multi-lineage tissues in clinical dentistry.

Culture of endothelial cells (ECs) embedded in 3D scaffolds of denatured collagen has shown tremendous therapeutic potential in clinical trials of tissue repair. It is postulated that these matrix-embedded ECs (MEECs) attain a differential phenotype similar to early progenitor forms, which cannot be attained in 2D culture. MEECs are compared to 2D-ECs and endothelial progenitor cells (EPCs) by secretome, phenotype, and genetic fingerprint, and are found to be altered from 2D-ECs on all levels, adopting an EPC-like phenotype. This manifests in elevation of CD34 expression—a progenitor cell marker—and protein secretion and gene expression pro-files that are similar to EPCs. Even more striking is that EPCs in 2D lose their phenotype, evident by the loss of CD34 expression, but are able to regain expression over time when embedded in the same 3D matrices, suggesting that future in vitro EPC work should use ME-EPCs to recapitulate in vivo phenotype. These findings elucidate the relationship between EPCs and the substratum-dependent regulation imparted by ECs which is critical to understand in order to optimize MEEC therapy and propel it into the clinic.

Endothelial cells (EC) are state-dependent regulators of the tumor ecosystem: quiescent ECs promote homeostasis; proliferative ECs stimulate tumor growth. Tumors, in turn, promote pro-tumorigenic EC phenotype. We studied functional and phosphorylative transformations on EC state in cancer. Quiescent HUVECs cultured in breast cancer cell-conditioned media displayed marked elongation and impaired wound healing. Quantitative mass spectrometry identified phosphorylative regulators of this dysfunctional transformation. Growth factor receptor kinases showed decreased, rather than increased activity, suggesting that EC regulation in tumors can arise other than from classic growth-factor-mediated angiogenesis alone. Of the 152 kinases and phosphatases across 62 families, six were chosen for functional validation using pharmacologic inhibitors. Inhibiting Akt and Ptp1b restored EC regulatory state, warranting further investigation as therapeutic targets; Src inhibition, however, promoted the dysfunctional phenotype, suggesting caution for Src inhibitors as EC-regulating therapies. Mapping phosphorylative drivers reveals complex relationships between EC phenotype, transformation, and regulation, and may shed light on how existing cancer-targeting inhibitors affect tumor endothelium. Data are available via ProteomeXchange with identifier PXD020333.

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