Objectives The purpose of this study was to examine the role of heparanase in controlling thrombosis following vascular injury or endovascular stenting. Background The use of endovascular stents are a common clinical intervention for the treatment of arteries occluded due to vascular disease. Both heparin and heparan sulfate are known to be potent inhibitors of thrombosis. Heparanase is the major enzyme that degrades heparan sulfate in mammalian cells. This study examined the role of heparanase in controlling thrombosis following vascular injury and stent-induced flow disturbance. Methods This study used mice overexpressing human heparanase and examined the time to thrombosis using a laser-induced arterial thrombosis model in combination with vascular injury. An ex vivo system was used to examine the formation of thrombus to stent-induced flow disturbance. Results In the absence of vascular injury, wild type and heparanase overexpressing (HPA Tg) mice had similar times to thrombosis in a laser-induced arterial thrombosis model. However, in the presence of vascular injury, the time to thrombosis was dramatically reduced in HPA Tg mice. An ex vivo system was used to flow blood from wild type and HPA Tg mice over stents and stented arterial segments from both animal types. These studies demonstrate markedly increased thromboses on stents with blood isolated from HPA Tg mice in comparison to blood from wild type animals. We found that blood from HPA Tg animals had markedly increased thrombosis when applied to stented arterial segments from either wild type or HPA Tg mice. Conclusions Taken together, this study’s results indicate that heparanase is a powerful mediator of thrombosis in the context of vascular injury and stent-induced flow disturbance.

β-Lap showed NQO1-dependent efficacy against two triple-negative breast cancer (TNBC) xenografts. NQO1 expression variations in human breast cancer patient samples were noted, where ~60% cancers over-expressed NQO1, with little or no expression in associated normal tissue. Differential DNA damage and lethality were noted in NQO1(+) versus NQO1-deficient (NQO1(-)) TNBC cells and xenografts after β-lap treatment. β-Lap-treated NQO1(+) cells died by programmed necrosis, whereas co-cultured NQO1(-) TNBC cells exhibited DNA damage and caspase-dependent apoptosis. NQO1 inhibition (dicoumarol) or H2O2 scavenging (catalase [CAT]) blocked all responses. Only NQO1(-) cells neighboring NQO1(+) TNBC cells responded to β-lap in vitro, and bystander effects correlated well with H2O2 diffusion. Bystander effects in NQO1(-) cells in vivo within mixed 50:50 co-cultured xenografts were dramatic and depended on NQO1(+) cells. However, normal human cells in vitro or in vivo did not show bystander effects, due to elevated endogenous CAT levels. Innovation and Conclusions: NQO1-dependent bystander effects elicited by NQO1 bioactivatable drugs (β-lap or deoxynyboquinone [DNQ]) likely contribute to their efficacies, killing NQO1(+) solid cancer cells and eliminating surrounding heterogeneous NQO1(low) cancer cells. Normal cells/tissue are protected by low NQO1:CAT ratios.

The shear stresses derived from blood flow regulate many aspects of vascular and immunobiology. In vitro studies on the shear stress-mediated mechanobiology of endothelial cells have been carried out using systems analogous to the cone-and-plate viscometer in which a rotating, low-angle cone applies fluid shear stress to cells grown on an underlying, flat culture surface. We recently developed a device that could perform high-throughput studies on shear-mediated mechanobiology through the rotation of cone-tipped shafts in a standard 96-well culture plate. Here, we present a model of the three-dimensional flow within the culture wells with a rotating, cone-tipped shaft. Using this model we examined the effects of modifying the design parameters of the system to allow the device to create a variety of flow profiles. We first examined the case of steady-state flow with the shaft rotating at constant angular velocity. By varying the angular velocity and distance of the cone from the underlying plate we were able to create flow profiles with controlled shear stress gradients in the radial direction within the plate. These findings indicate that both linear and non-linear spatial distributions in shear stress can be created across the bottom of the culture plate. In the transition and “parallel shaft” regions of the system, the angular velocities needed to provide high levels of physiological shear stress (5 Pa) created intermediate Reynolds number Taylor-Couette flow. In some cases, this led to the development of a flow regime in which stable helical vortices were created within the well. We also examined the system under oscillatory and pulsatile motion of the shaft and demonstrated minimal time lag between the rotation of the cone and the shear stress on the cell culture surface. Biotechnol. Bioeng. 2013;110: 1782-1793.

The metastatic spread of cancer is a major barrier to effective and curative therapies for cancer. During metastasis, tumor cells intravasate into the vascular system, survive in the shear forces and immunological environment of the circulation, and then extravasate into secondary tumor sites. Biophysical forces are potent regulators of cancer biology and are key in many of the steps of metastasis. In particular, the adhesion of circulating cells is highly dependent upon competing forces between cell adhesion receptors and the shear stresses due to fluid flow. Conventional in vitro assays for drug development and the mechanistic study of metastasis are often carried out in the absence of fluidic forces and, consequently, are poorly representative of the true biology of metastasis. Here, we present a novel high-throughput approach to studying cell adhesion under flow that uses a multi-well, mechanofluidic flow system to interrogate adhesion of cancer cell to endothelial cells, extracellular matrix and platelets under physiological shear stresses. We use this system to identify pathways and compounds that can potentially be used to inhibit cancer adhesion under flow by screening anti-inflammatory compounds, integrin inhibitors and a kinase inhibitor library. In particular, we identify several small molecule inhibitors of FLT-3 and AKT that are potent inhibitors of cancer cell adhesion to endothelial cells and platelets under flow. In addition, we found that many kinase inhibitors lead to increased adhesion of cancer cells in flow-based but not static assays. This finding suggests that even compounds that reduce cell proliferation might also enhance cancer cell adhesion during metastasis. Overall, our results validate a novel platform for investigating the mechanisms of cell adhesion under biophysical flow conditions and identify several potential inhibitors of cancer cell adhesion during metastasis.

Background: Heparanase cleaves heparan sulfate proteoglycans (HSPGs), key components of the extracellular matrix and critical endothelial mediators of hemostasis. Our previous work has shown that heparanase levels are elevated in atherosclerosis and following endovascular stenting. Methods: We used mice overexpressing human heparanase and examined the time to thrombosis using a laser induced arterial thrombosis model in combination with vascular injury. An ex-vivo system was used to examine the formation of thrombus to stent-induced flow disturbance. To evaluate thrombotic potential in wild-type (WT) or heparanase transgenic (HPA-Tg) mice, the left common carotid artery was isolated and a vascular flow probe was used to monitor blood flow. Rose Bengal dye was injected into the mice and the mid portion of the common carotid artery was then illuminated with a 1.5-mW green laser (540 nm) until an occlusive thrombus was formed. Arterial injury was also performed prior to measuring thrombosis to examine the effects of light and heavy injury on thrombosis in WT and HPA-Tg mice. A novel ex-vivo assay of stent thrombosis was performed in which mouse aortas were stented into a modified Chandler loop and the loops were perfused with blood from transgenic mice. Results were analyzed with electron microscopy and optical absorbance for clot formation. Results: In the absence of injury there was no difference in the time for formation of thrombus in the WT and HPA-Tg mice. However in the presence of arterial injury, thrombosis in HPA-Tg mice occurred significantly faster than in WT mice (light injury: 67.7 minutes vs. 39.8 minutes, p<0.05; heavy injury: 63.0 minutes vs. 41.8 minutes, p<0.05). The prothrombotic phenotype of heparanase transgenic mice is masked when heparin is administered prior to injury (WT: 68.5 minutes vs. HPA Tg: 69.2 minutes p=NS). Blood from heparanase transgenic mice also increased stent thrombosis threefold in flow loops as measured by optical density (2.8 vs. 1.0 optical density, p<0.05) and increased adherent platelets were found in electron microscopy. Conclusions: Increased arterial expression of heparanase leads to enhanced risk of thrombosis in the injured artery. In particular, endovascular stenting induces both injury and flow disturbance that exacerbate the thrombotic potential of blood with increased heparanase levels.

Introduction: Despite many advances in our understanding of breast cancer biology over the last decades, there are no therapies that can effectively prevent or treat metastatic cancer. During the growth of the primary tumor mass and its dissemination through the body during metastasis, cancer cells are exposed to a host of mechanical environments generated by the local matrix compliance, pressure and tension from tumor mass expansion, and fluidic shear stresses from interstitial and vascular fluid flow. These biophysical forces are emerging as powerful regulators of cancer growth, quiescence and metastasis; however, our understanding of the mechanisms of the biomechanical regulation of tumor biology remains very limited. The overall goal was to identify the role of mechanical forces in regulating key steps in tumor metastasis and progression. Results: We recently developed a mesofluidic system that allows the high throughput study of tumor cell interactions under flow. This device allows us to simulate the adhesion of cancer cells to endothelial cells under physiological flow conditions to model this step of the metastatic cascade and to apply shear forces to cells in a 96-well format. In addition, we have designed a system to apply mechanical stretch to cells in a high throughput format (576 wells simultaneously). Using these two systems, we examined the interplay between mechanical cues and the propensity of breast cancer cells to metastasize and undergo epithelial-to-mesenchymal transition (EMT). Mechanical strain increases circulating tumor cell adhesion to endothelial cells and extracellular matrix (ECM). We applied cyclic mechanical strain (5% maximal strain) to MDA-MB-231 and MCF-7 cancer cells for 24 hours and then performed adhesion assays of the cancer cells to activated and non-activated endothelial cells (ECS) and purified ECM proteins. We found that cyclic strain increased cancer cell adhesion to activated ECs in comparison to non-strained cells. In addition cancer cells exposed to mechanical strain adhered more to collagen I, laminin, and vitronectin, while they adhered less to collagen II and fibronectin in comparison to non-strained control cells. To determine which integrins were involved in the strain-induced change in adhesion, we treated the cells with a library of integrin inhibitors while applying strain for 24 hours. Cilengitide, P11, ATN-161, Bio 1211, and RGDS peptides reduced the adhesion of cancer cells back to the level of non-strained cancer cells, indicating the role of αvβ3, αvβ5, α5β1, and α4β1 integrins in the biophysical regulation of circulating tumor cell adhesion. Cyclic strain alters the ability of breast cancer cells to invade through an endothelial monolayer. We next used the high throughput system to apply multiple levels of strain to the cancer cell lines (0, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5% strain). We then trypsinized the cells and measured their invasion in a Transwell assay with a confluent layer of endothelial cells cultured on a porous membrane. We found that the mid-level strains ranging from 7.5-15% strain decreased the invasiveness of the MDA-MB-231 cells compared to the non-strained control cells. For the MCF-7 cells, mechanical strain of 5% or higher led to decreased invasion through the endothelial layer. Mechanical forces alter signaling through the TGF-β and Yap/Taz pathways as well the expression of markers of epithelial-to-mesenchymal transition (EMT). We applied cyclic strain at multiple levels (2.5-17.5% strain) to breast cancer cells for 24 hours then immunostained for various signaling proteins. In MDA-MB-231 cells, we saw a significant decrease in the intensity of Smad2/3 and nuclear phospho-Smad2/3 at mid-range strains (5-12.5% strain) as well as an increase in the nuclear localization of Yap/Taz using fluorescent immunostaining. At high levels of strain (15 and 17.5% strain), there was increased nuclear p-Smad2/3 intensity. Consistent with these findings, we found increased Smad activity in the cells using a luciferase reporter assay. An analysis of EMT markers using PCR, western blotting and immunostaining showed a reduction in mesenchymal markers including α−SMA, Slug, ZEB, fibronectin and vimentin with mid-range mechanical strain levels. Conclusion: Together, our studies demonstrate that mechanical forces can profoundly alter the propensity of cancer cells to adhere and invade during metastasis. Moreover, the response to mechanical strain is dependent on the magnitude of the strain and cancer cell type. These effects are mediated, in part, through integrin interactions and the differential regulation of the TGF-β and Hippo pathways by biophysical forces. Citation Format: Adrianne Spencer, Jason Lee, Katerina Lee, Darshil Choksi, Jerry Wang, Christopher Spruell, Aaron Baker. Biophysical Regulation of Breast Cancer Metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A38.

A fundamental limitation in the development of new therapies to prevent metastatic cancer is a lack of in vitro systems that can accurately recapitulate the steps of cancer cell metastasis. Currently, most assays for examining the steps of metastasis fail to incorporate the biophysical forces experienced by tumor cells due to blood flow, or are low throughput and thereby not amenable to drug screens or high throughput experimentation. We have developed a novel high throughput mesofluidic platform for assaying cell adhesion under flow in a 96-well format. This device functions like a cone and plate viscometer in each well by inducing shear stress on cells cultured in a standard 96-well plate. We validated the fluid flow and alignment of the device and studied the adhesion of cultured leukocytic monocytes (THP-1 cells) and multiple cancer cell lines (MDA-MB-231 and MCF-7 breast cancer cell lines) to purified extracellular matrix molecules (ECM), endothelial cells and immobilized platelets. Assays were carried out under flow (0.5 dynes/cm2 shear stress) and static conditions. Our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays. Treatment of breast cancer cells with a small library of integrin inhibitors demonstrated that these compounds had minimal effect on cancer cell adhesion to endothelial cells or immobilized platelets under static conditions, whereas under shear conditions many of these compounds significantly reduced adhesion of cancer cells. As well, this experiment elucidated integrins important for breast cancer adhesion to endothelial cells and platelets. A static adhesion assay of breast cancer cells to various types of ECM showed greater adhesion of the less aggressive MCF-7 cell line in comparison to the more aggressive MDA-MB-231 cell line. In contrast, flow incorporating assays showed increased adhesion of the more aggressive MDA-MB-231 breast cancer cell line. Specifically, the shear assay saw a significant increase in adhesion for multiple ECM as well as an increase in the strength of adhesion to laminin. Finally, we performed a high throughput screening experiment using a kinase inhibitor library of 80 compounds and found that the shear based assay yielded notably different results from a similar screen under static conditions for breast cancer cell adhesion to endothelial cells, immune cell adhesion to endothelial cells and breast cancer cell adhesion to platelets. This shear experiment yielded seven "hits", many of which match targets of drugs in clinical trials. In conclusion, our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays, and are better at identifying both aggressive cancer cells lines and known pathways for circulating cancer and immune cell adhesion. Thus, this high-throughput screening platform may enable the development of novel compounds to inhibit cancer metastasis and facilitate the study of the systems level behavior of cancer-endothelium adhesion. Citation Format: Spencer A, Spruell C, Nandi S, Le V, Crexiell M, Dunn AK, Baker AB. Mesofluidic platform for high throughput screening of inhibitors of metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-18.

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