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During angiogenesis, endothelial cells (ECs) from intact blood vessels quickly infiltrate avascular regions via vascular sprouting. This process is fundamental to many normal and pathological processes such as wound healing and tumor growth, but its initiation and control are poorly understood. Vascular endothelial cell growth factor (VEGF) can promote vessel dilation and angiogenic sprouting, but given the complex nature of vascular morphogenesis, additional signals are likely necessary to determine, for example, which vessel segments sprout, which dilate, and which remain quiescent. Fluid forces exerted by blood and plasma are prime candidates that might codirect these processes, but it is not known whether VEGF cooperates with mechanical fluid forces to mediate angiogenesis. Using a microfluidic tissue analog of angiogenic sprouting, we found that fluid shear stress, such as exerted by flowing blood, attenuates EC sprouting in a nitric oxide-dependent manner and that interstitial flow, such as produced by extravasating plasma, directs endothelial morphogenesis and sprout formation. Furthermore, positive VEGF gradients initiated sprouting but negative gradients inhibited sprouting, promoting instead sheet-like migration analogous to vessel dilation. These results suggest that ECs integrate signals from fluid forces and local VEGF gradients to achieve such varied goals as vessel dilation and sprouting.

Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBMxenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.

The ability to properly model intravascular steps in metastasis is essential in identifying key physical, cellular, and molecular determinants that can be targeted therapeutically to prevent metastatic disease. Research on the vascular microenvironment has been hindered by challenges in studying this compartment in metastasis under conditions that reproduce in vivo physiology while allowing facile experimental manipulation. We present a microfluidic vasculature system to model interactions between circulating breast cancer cells with microvascular endothelium at potential sites of metastasis. The microfluidic vasculature produces spatially-restricted stimulation from the basal side of the endothelium that models both organ-specific localization and polarization of chemokines and many other signaling molecules under variable flow conditions. We used this microfluidic system to produce site-specific stimulation of microvascular endothelium with CXCL12, a chemokine strongly implicated in metastasis. When added from the basal side, CXCL12 acts through receptor CXCR4 on endothelium to promote adhesion of circulating breast cancer cells, independent of CXCL12 receptors CXCR4 or CXCR7 on tumor cells. These studies suggest that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers and highlight the unique capabilities of our microfluidic device to advance studies of the intravascular microenvironment in metastasis.

Current approaches for single molecule force spectroscopy are typically constrained by low throughput and high instrumentation cost. Herein, a low-cost, high throughput technique is demonstrated using microfluidics for multiplexed mechanical manipulation of up to ~4000 individual molecules via molecular fluid loading on-a-chip (FLO-Chip). The FLO-Chip consists of serially connected microchannels with varying width, allowing for simultaneous testing at multiple loading rates. Molecular force measurements are demonstrated by dissociating Biotin-Streptavidin and Digoxigenin-AntiDigoxigenin interactions along with unzipping of double stranded DNA of varying sequence under different dynamic loading rates and solution conditions. Rupture force results under varying loading rates and solution conditions are in good agreement with prior studies, verifying a versatile approach for single molecule biophysics and molecular mechanobiology. FLO-Chip enables straightforward, rapid, low-cost, and portable mechanical testing of single molecules that can be implemented on a wide range of microscopes to broaden access and may enable new applications of molecular force spectroscopy. Single molecule force spectroscopy methods are often low throughput and have high instrument cost. Here the authors report FLO-Chip, a low-cost, high throughput technique using microfluidics for multiplexed mechanical manipulation of many individual molecules via molecular fluid loading on-a-chip.

The intricate interplay between malignant cells and host cellular and non-cellular components play crucial role in different stages of tumor development, progression, and metastases. Tumor and stromal cells communicate to each other through receptors such as integrins and secretion of signaling molecules like growth factors, cytokines, chemokines and inflammatory mediators. Chemokines mediated signaling pathways have emerged as major mechanisms underlying multifaceted roles played by host cells during tumor progression. In response to tumor stimuli, host cells-derived chemokines further activates signaling cascades that support the ability of tumor cells to invade surrounding basement membrane and extra-cellular matrix. The host-derived chemokines act on endothelial cells to increase their permeability and facilitate tumor cells intravasation and extravasation. The tumor cells-host neutrophils interaction within the vasculature initiates chemokines driven recruitment of inflammatory cells that protects circulatory tumor cells from immune attack. Chemokines secreted by tumor cells and stromal immune and non-immune cells within the tumor microenvironment enter the circulation and are responsible for formation of a “pre-metastatic niche” like a “soil” in distant organs whereby circulating tumor cells “seed’ and colonize, leading to formation of metastatic foci. Given the importance of host derived chemokines in cancer progression and metastases several drugs like Mogamulizumab, Plerixafor, Repertaxin among others are part of ongoing clinical trial which target chemokines and their receptors against cancer pathogenesis. In this review, we focus on recent advances in understanding the complexity of chemokines network in tumor microenvironment, with an emphasis on chemokines secreted from host cells. We especially summarize the role of host-derived chemokines in different stages of metastases, including invasion, dissemination, migration into the vasculature, and seeding into the pre-metastatic niche. We finally provide a brief description of prospective drugs that target chemokines in different clinical trials against cancer.

Background Molecular mechanisms underlying inflammation-associated breast tumor growth are poorly studied. S100A7, a pro-inflammatory molecule has been shown to enhance breast cancer growth and metastasis. However, the S100A7-mediated molecular mechanisms in enhancing tumor growth and metastasis are unclear. Methods Human breast cancer tissue and plasma samples were used to analyze the expression of S100A7, cPLA2, and PGE2. S100A7-overexpressing or downregulated human metastatic breast cancer cells were used to evaluate the S100A7-mediated downstream signaling mechanisms. Bi-transgenic mS100a7a15 overexpression, TNBC C3 (1)/Tag transgenic, and humanized patient-derived xenograft mouse models and cPLA2 inhibitor (AACOCF3) were used to investigate the role of S100A7/cPLA2/PGE2 signaling in tumor growth and metastasis. Additionally, CODEX, a highly advanced multiplexed imaging was employed to delineate the effects of S100A7/cPLA2 inhibition on the recruitment of various immune cells. Results In this study, we found that S100A7 and cPLA2 are highly expressed and correlate with decreased overall survival in breast cancer patients. Further mechanistic studies revealed that S100A7/RAGE signaling promotes the expression of cPLA2 to mediate its oncogenic effects. Pharmacological inhibition of cPLA2 suppressed S100A7-mediated tumor growth and metastasis in multiple pre-clinical models including transgenic and humanized patient-derived xenograft (PDX) mouse models. The attenuation of cPLA2 signaling reduced S100A7-mediated recruitment of immune-suppressive myeloid cells in the tumor microenvironment (TME). Interestingly, we discovered that the S100A7/cPLA2 axis enhances the immunosuppressive microenvironment by increasing prostaglandin E2 (PGE2). Furthermore, CO-Detection by indEXing (CODEX) imaging-based analyses revealed that cPLA2 inhibition increased the infiltration of activated and proliferating CD4 + and CD8 + T cells in the TME. In addition, CD163 + tumor associated-macrophages were positively associated with S100A7 and cPLA2 expression in malignant breast cancer patients. Conclusions Our study provides new mechanistic insights on the cross-talk between S100A7/cPLA2 in enhancing breast tumor growth and metastasis by generating an immunosuppressive TME that inhibits the infiltration of cytotoxic T cells. Furthermore, our studies indicate that S100A7/cPLA2 could be used as novel prognostic marker and cPLA2 inhibitors as promising drugs against S100A7-overexpressing aggressive breast cancer.

Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), with each EC residing in the vessel wall integrating local signals to determine whether to remain quiescent or undergo morphogenesis. These signals include vascular endothelial growth factor (VEGF) and flow-induced mechanical stimuli such as interstitial flow, which are both elevated in the tumor microenvironment. However, it is not clear how VEGF signaling and mechanobiological activation due to interstitial flow cooperate during angiogenesis. Here, we show that endothelial morphogenesis is histone deacetylase-1- (HDAC1) dependent and that interstitial flow increases the phosphorylation of HDAC1, its activity and its export from the nucleus. Furthermore, we show that HDAC1 inhibition decreases endothelial morphogenesis and matrix metalloproteinase-14 (MMP14) expression. Our results suggest that HDAC1 modulates angiogenesis in response to flow, providing a new target for modulating vascularization in the clinic.

Background In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility. Methods We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands. Results We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not. Conclusions As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood.

Sprouting angiogenesis—the infiltration and extension of endothelial cells from pre-existing blood vessels—helps orchestrate vascular growth and remodeling. It is now agreed that fluid forces, such as laminar shear stress due to unidirectional flow in straight vessel segments, are important regulators of angiogenesis. However, regulation of angiogenesis by the different flow dynamics that arise due to vessel branching, such as impinging flow stagnation at the base of a bifurcating vessel, are not well understood. Here we used a recently developed 3-D microfluidic model to investigate the role of the flow conditions that occur due to vessel bifurcations on endothelial sprouting. We observed that bifurcating fluid flow located at the vessel bifurcation point suppresses the formation of angiogenic sprouts. Similarly, laminar shear stress at a magnitude of ~3 dyn/cm2 applied in the branched vessels downstream of the bifurcation point, inhibited the formation of angiogenic sprouts. In contrast, co-application of ~1 µm/s average transvascular flow across the endothelial monolayer with laminar shear stress induced the formation of angiogenic sprouts. These results suggest that transvascular flow imparts a competing effect against bifurcating fluid flow and laminar shear stress in regulating endothelial sprouting. To our knowledge, these findings are the first report on the stabilizing role of bifurcating fluid flow on endothelial sprouting. These results also demonstrate the importance of local flow dynamics due to branched vessel geometry in determining the location of sprouting angiogenesis.

Continuous perfusion is necessary to sustain microphysiological systems and other microfluidic cell cultures. However, most of the established microfluidic perfusion systems, such as syringe pumps, peristaltic pumps, and rocker plates, have several operational challenges and may be cost-prohibitive, especially for laboratories with no microsystems engineering expertise. Here, we address the need for a cost-efficient, easy-to-implement, and reliable microfluidic perfusion system. Our solution is a modular pumpless perfusion assembly (PPA), which is constructed from commercially available, interchangeable, and aseptically packaged syringes and syringe filters. The total cost for the components of each assembled PPA is USD 1–2. The PPA retains the simplicity of gravity-based pumpless flow systems but incorporates high resistance filters that enable slow and sustained flow for extended periods of time (hours to days). The perfusion characteristics of the PPA were determined by theoretical calculations of the total hydraulic resistance of the assembly and experimental characterization of specific filter resistances. We demonstrated that the PPA enabled reliable long-term culture of engineered endothelialized 3-D microvessels for several weeks. Taken together, our novel PPA solution is simply constructed from extremely low-cost and commercially available laboratory supplies and facilitates robust cell culture and compatibility with current microfluidic setups.