Search

Cancer cells, both within a developing tumor and during metastatic spread, encounter many stresses that require adaptive mechanisms to survive and maintain malignant progression. Here we describe a signaling complex involving the small GTPase Cdc42 and Dock7, a Cdc42/Rac GEF and unique Cdc42-effector, that has a previously unappreciated role in regulating AKT, mTOR, and other mTOR signaling and regulatory partners including the TSC1/TCS2 complex and S6K during serum starvation. Dock7 is highly expressed in triple-negative breast cancers and is essential for the malignant properties in nutrient-deprived growth conditions of several cancer cell lines. We find that Dock7 interacts with phosphorylated AKT to maintain a low, but critical activation of a rapamycin-sensitive and Raptor-independent mTORC1-like activity required for survival during nutrient stress. Following the knock-out of Dock7 from cancer cells, interactions between AKT and the phosphatase PHLPP increased while phosphorylation of AKT at Ser473 decreased, suggesting Dock7 protects AKT from dephosphorylation. The DHR1 domain of Dock7, previously of unknown function, is responsible for maintaining AKT Ser473 phosphorylation during serum starvation through an interaction requiring its C2-like motif. Together, these findings indicate that Dock7 protects and maintains the phosphorylation of AKT to sustain a tonic mTOR/S6K activity in cancer cells necessary for their resistance to anoikis and to prevent them from undergoing apoptosis during stressful conditions.

Altered tissue mechanics and metabolism are defining characteristics of cancer that impact not only proliferation but also migration. While migrating through a mechanically and spatially heterogeneous microenvironment, changes in metabolism allow cells to dynamically tune energy generation and bioenergetics in response to fluctuating energy needs. Physical cues from the extracellular matrix influence mechanosignaling pathways, cell mechanics, and cytoskeletal architecture to alter presentation and function of metabolic enzymes. In cancer, altered mechanosensing and metabolic reprogramming supports metabolic plasticity and high energy production while cells migrate and metastasize. Here, we discuss the role of mechanoresponsive metabolism in regulating cell migration and supporting metastasis as well as the potential of therapeutically targeting cancer metabolism to block motility and potentially metastasis.

The tumor microenvironment plays a critical role in tumorigenesis and metastasis. As tightly controlled extracellular matrix homeostasis is lost during tumor progression, a dysregulated extracellular matrix can significantly alter cellular phenotype and drive malignancy. Altered physical properties of the tumor microenvironment alter cancer cell behavior, limit delivery and efficacy of therapies, and correlate with tumorigenesis and patient prognosis. The physical features of the extracellular matrix during tumor progression have been characterized; however, a wide range of methods have been used between studies and cancer types resulting in a large range of reported values. Here, we discuss the significant mechanical and structural properties of the tumor microenvironment, summarizing their reported values and clinical impact across cancer type and grade. We attempt to integrate the values in the literature to identify sources of reported differences and commonalities to better understand how aberrant extracellular matrix dynamics contribute to cancer progression. An intimate understanding of altered matrix properties during malignant transformation will be crucial in effectively detecting, monitoring, and treating cancer.

Altered tissue mechanics and metabolism have gained significant attention as drivers of tumorigenesis, and mechanoresponsive metabolism has been implicated in migration and metastasis. However, heterogeneity in cell populations makes it difficult to link changes in behavior with metabolism, as individual cell behaviors are not necessarily reflected in population-based measurements. As such, the impact of increased collagen deposition, a tumor-associated collagen signature, on metabolism remains ambiguous. Here, we utilize a wide range of collagen densities to alter migration ability and study the bioenergetics of individual cells over time. Sorting cells based on their level of motility revealed energetics are a function of collagen density only for highly motile cells, not the entire population or cells with low motility. Changes in migration with increasing collagen density were correlated with cellular energetics, where matrix conditions most permissive to migration required less energy usage during movement and migrated more efficiently. These findings reveal a link between matrix mechanics, migratory phenotype, and bioenergetics and suggest that energetic costs are determined by the extracellular matrix and influence cell motility.

Introduction Insufficient vascularization of currently available clinical biomaterials has limited their application to optimal wound beds. We designed a hydrogel scaffold with a unique internal microstructure of differential collagen densities to induce cellular invasion and neovascularization. Methods Microsphere scaffolds (MSS) were fabricated by encasing 1% (w/v) type 1 collagen microspheres 50–150 μm in diameter in 0.3% collagen bulk. 1% and 0.3% monophase collagen scaffolds and Integra® disks served as controls. Mechanical characterization as well as in vitro and in vivo invasion assays were performed. Cell number and depth of invasion were analyzed using Imaris™. Cell identity was assessed immunohistochemically. Results In vitro, MSS exhibited significantly greater average depth of cellular invasion than Integra® and monophase collagen controls. MSS also demonstrated significantly higher cell counts than controls. In vivo, MSS revealed significantly more cellular invasion spanning the entire scaffold depth at 14 days than Integra®. CD31+ expressing luminal structures suggestive of neovasculature were seen within MSS at 7 days and were more prevalent after 14 days. Multiphoton microscopy of MSS demonstrated erythrocytes within luminal structures after 14 days. Conclusion By harnessing simple architectural cues to induce cellular migration, MSS holds great potential for clinical translation as the next generation dermal replacement product. Statement of Significance Large skin wounds require tissue engineered dermal substitutes in order to promote healing. Currently available dermal replacement products do not always adequately incorporate into the body, especially in complex wounds, due to poor neovascularization. In this paper, we present a hydrogel with an innovative microarchitecture that is composed of dense type I collagen microspheres suspended in a less-dense collagen bulk. We show that cell invasion into the scaffold is driven solely by mechanical cues inherent within this differential density interface, and that this induces robust vascular cell invasion both in vitro and in a rodent model. Our hydrogel performs favorably compared to the current clinical gold standard, Integra®. We believe this hydrogel scaffold may be the first of the next generation of dermal replacement products.

Akt is a well-known target of mitogenic signaling, which is activated by phosphoinositide 3-kinase, PDK1 and mTORC2, and is commonly regarded as a master regulator of cell survival. However, there is still a good deal to learn regarding how Akt can promote survival when cancer cells are faced by the multitude of challenges they need to overcome during the process of malignant transformation. While investigating the role of Dock7, a Cdc42 and Rac guanine nucleotide exchange factor of the Dock180 family, in tumorigenesis, we discovered an unexpected connection between Dock7, Akt and cell survival. Specifically, we found that Dock7 is necessary for cancer cells to survive under the stressful conditions that arise due to the loss of a substratum or serum deprivation, through its ability to stabilize a low, basal level of Akt kinase activity. Dock7 binds to Akt, as well as associates with the known Akt substrate, TSC2, promoting its phosphorylation. Since the phosphorylation of TSC2 eliminates its ability to work with its partner TSC1 to deactivate the Rheb GTPase, a direct activator of mTORC1, we probed whether mTORC1 function is also necessary for Dock7-dependent cancer cell survival. Indeed, we determined that Dock7, as well as its signaling partner Cdc42, is required to maintain a functional level of S6 kinase activity, a downstream target of mTORC1, which was prevented by the mTORC1 inhibitor rapamycin. We further show an association between mTOR, Rheb and Dock7, but not Raptor, the defining component of mTORC1. Additionally, Raptor is dispensable for the Dock7-dependent S6 kinase activity, despite its rapamycin sensitivity. Together, these findings elucidate a novel Dock7-Akt-mTOR signaling node which promotes cancer cell survival in the absence of classical mTORC1 function to allow cells to overcome stresses faced during the development of cancer. Citation Format: Oriana Y. Teran, Miao-chong J. Lin, Matthew R. Zanotelli, Kristin F. Wilson, Richard A. Cerione. Dock7 regulates the AKT/mTOR pathway to promote survival and sustain the transformative properties of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 139.

The microenvironment provides both active and passive mechanical cues that regulate cell morphology, adhesion, migration, and metabolism. Although the cellular response to those mechanical cues often requires energy-intensive actin cytoskeletal remodeling and actomyosin contractility, it remains unclear how cells dynamically adapt their metabolic activity to altered mechanical cues to support migration. Here, we investigated the changes in cellular metabolic activity in response to different two-dimensional and three-dimensional microenvironmental conditions and how these changes relate to cytoskeletal activity and migration. Utilizing collagen micropatterning on polyacrylamide gels, intracellular energy levels and oxidative phosphorylation were found to be correlated with cell elongation and spreading and necessary for membrane ruffling. To determine whether this relationship holds in more physiological three-dimensional matrices, collagen matrices were used to show that intracellular energy state was also correlated with protrusive activity and increased with matrix density. Pharmacological inhibition of oxidative phosphorylation revealed that cancer cells rely on oxidative phosphorylation to meet the elevated energy requirements for protrusive activity and migration in denser matrices. Together, these findings suggest that mechanical regulation of cytoskeletal activity during spreading and migration by the physical microenvironment is driven by an altered metabolic profile.

Cells expend energy to migrate. Metastatic cancer cell energy levels were investigated as a function of collagen architecture. In more migration-permissive environments or when migration is pharmacologically inhibited, cells reduce ATP:ADP levels. Changes in intracellular ATP:ADP levels during migration were associated with changes in cell speed. The data suggest that cells tune their energy production and utilization relative to their migration., Cell migration in a three-dimensional matrix requires that cells either remodel the surrounding matrix fibers and/or squeeze between the fibers to move. Matrix degradation, matrix remodeling, and changes in cell shape each require cells to expend energy. While significant research has been performed to understand the cellular and molecular mechanisms guiding metastatic migration, less is known about cellular energy regulation and utilization during three-dimensional cancer cell migration. Here we introduce the use of the genetically encoded fluorescent biomarkers, PercevalHR and pHRed, to quantitatively assess ATP, ADP, and pH levels in MDA-MB-231 metastatic cancer cells as a function of the local collagen microenvironment. We find that the use of the probe is an effective tool for exploring the thermodynamics of cancer cell migration and invasion. Specifically, we find that the ATP:ADP ratio increases in cells in denser matrices, where migration is impaired, and it decreases in cells in aligned collagen matrices, where migration is facilitated. When migration is pharmacologically inhibited, the ATP:ADP ratio decreases. Together, our data indicate that matrix architecture alters cellular energetics and that intracellular ATP:ADP ratio is related to the ability of cancer cells to effectively migrate.

Cancer cells exhibit altered biophysical and biomechanical properties that contribute to an overall altered mechanical phenotype. Increasing evidence demonstrates that cancer cells are often associated with decreased bulk stiffness and higher contractility, properties that facilitate increased motility and invasiveness. These changes in cellular properties are most often due to subcellular alterations in the cytoskeletal organization that results from both changes in intracellular regulators as well as changes to the tumor microenvironment. This review will highlight recent key findings describing the role of upregulated cytoskeletal scaffolding proteins and signaling circuits, as well as microenvironmental cues that influence cytoskeletal regulation and epithelial-to-mesenchymal transition to alter cancer cell stiffness and contractility. We also briefly discuss the emerging role of extracellular microvesicles in long-range cell–cell modulation of cytoskeletal mechanics and malignant transformation.

Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer cross-linked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.

Here, we describe an in vitro strategy to model vascular morphogenesis where human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are encapsulated in peptide-functionalized poly(ethylene glycol) (PEG) hydrogels, either on standard well plates or within a passive pumping polydimethylsiloxane (PDMS) tri-channel microfluidic device. PEG hydrogels permissive towards cellular remodeling were fabricated using thiol-ene photopolymerization to incorporate matrix metalloproteinase (MMP)-degradable crosslinks and CRGDS cell adhesion peptide. Time lapse microscopy, immunofluorescence imaging, and RNA sequencing (RNA-Seq) demonstrated that iPSC-ECs formed vascular networks through mechanisms that were consistent with in vivo vasculogenesis and angiogenesis when cultured in PEG hydrogels. Migrating iPSC-ECs condensed into clusters, elongated into tubules, and formed polygonal networks through sprouting. Genes upregulated for iPSC-ECs cultured in PEG hydrogels relative to control cells on tissue culture polystyrene (TCP) surfaces included adhesion, matrix remodeling, and Notch signaling pathway genes relevant to in vivo vascular development. Vascular networks with lumens were stable for at least 14 days when iPSC-ECs were encapsulated in PEG hydrogels that were polymerized within the central channel of the microfluidic device. Therefore, iPSC-ECs cultured in peptide-functionalized PEG hydrogels offer a defined platform for investigating vascular morphogenesis in vitro using both standard and microfluidic formats. Statement of Significance Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) cultured in synthetic hydrogels self-assemble into capillary networks through mechanisms consistent with in vivo vascular morphogenesis.

Aligned collagen architecture is a characteristic feature of the tumor extracellular matrix (ECM) and has been shown to facilitate cancer metastasis using 3D in vitro models. Additional features of the ECM, such as pore size and stiffness, have also been shown to influence cellular behavior and are implicated in cancer progression. While there are several methods to produce aligned matrices to study the effect on cell behavior in vitro, it is unclear how the alignment itself may alter these other important features of the matrix. In this study, we have generated aligned collagen matrices and characterized their pore sizes and mechanical properties at the micro- and macro-scale. Our results indicate that collagen alignment can alter pore-size of matrices depending on the polymerization temperature of the collagen. Furthermore, alignment does not affect the macro-scale stiffness but alters the micro-scale stiffness in a temperature independent manner. Overall, these results describe the manifestation of confounding variables that arise due to alignment and the importance of fully characterizing biomaterials at both micro- and macro-scales.

During metastasis, cancer cells navigate through a spatially heterogeneous extracellular matrix (ECM). Physical properties of ECM, including the degree of confinement, influence cell migration behavior. Here, utilizing in vitro three-dimensional collagen microtracks, we demonstrate that cell-ECM interactions, specifically the degree of spatial confinement, regulate migratory behavior. We found that cells migrate faster when they are fully confined, contacting all four walls (top, bottom, and two sides) of a collagen microtrack, compared with cells that are partially confined, contacting less than four walls. When fully confined, cells exhibit fewer but larger vinculin-containing adhesions and create greater strains in the surrounding matrix directed toward the cell body. In contrast, partially confined cells develop a more elongated morphology with smaller but significantly more vinculin-containing adhesions and displace the surrounding matrix less than fully confined cells. The resulting effect of increasing cell contractility via Rho activation is dependent on the number of walls with which the cell is in contact. Although matrix strains increase in both fully and partially confined cells, cells that are partially confined increase speed, whereas those in full confinement decrease speed. Together, these results suggest that the degree of cell-ECM contact during confined migration is a key determinant of speed, morphology, and cell-generated substrate strains during motility, and these factors may work in tandem to facilitate metastatic cell migration.

Cells receive mechanical cues from their extracellular matrix (ECM), which direct migration, differentiation, apoptosis, and in some cases, the transition to a cancerous phenotype. As a result, there has been significant research to develop methods to tune the mechanical properties of the ECM and understand cell-ECM dynamics more deeply. Here, we show that ionizing radiation can reduce the stiffness of an ex vivo tumor and an in vitro collagen matrix. When non-irradiated cancer cells were seeded in the irradiated matrix, adhesion, spreading, and migration were reduced. These data have ramifications for both in vitro and in vivo systems. In vitro, these data suggest that irradiation may be a method that could be used to create matrices with tailored mechanical properties. In vivo, these suggest that therapeutic doses of radiation may alter tissue mechanics directly.

Vascular endothelial growth factor (VEGF) can mediate endothelial cell migration, proliferation, and angiogenesis. During cancer progression, VEGF production is often increased to stimulate the growth of new blood vessels to supply growing tumors with the additional oxygen and nutrients they require. Extracellular matrix stiffening also occurs during tumor progression, however, the crosstalk between tumor mechanics and VEGF signaling remains poorly understood. Here, we show that matrix stiffness heightens downstream endothelial cell response to VEGF by altering VEGF receptor-2 (VEGFR-2) internalization, and this effect is influenced by cell confluency. In sub-confluent endothelial monolayers, VEGFR-2 levels, but not VEGFR-2 phosphorylation, are influenced by matrix rigidity. Interestingly, more compliant matrices correlated with increased expression and clustering of VEGFR-2; however, stiffer matrices induced increased VEGFR-2 internalization. These effects are most likely due to actin-mediated contractility, as inhibiting ROCK on stiff substrates increased VEGFR-2 clustering and decreased internalization. Additionally, increasing matrix stiffness elevates ERK 1/2 phosphorylation, resulting in increased cell proliferation. Moreover, cells on stiff matrices generate more actin stress fibers than on compliant substrates, and the addition of VEGF stimulates an increase in fiber formation regardless of stiffness. In contrast, once endothelial cells reached confluency, stiffness-enhanced VEGF signaling was no longer observed. Together, these data show a complex effect of VEGF and matrix mechanics on VEGF-induced signaling, receptor dynamics, and cell proliferation that is mediated by cell confluency.

A defining hallmark of cancer and cancer development is upregulated angiogenesis. The vasculature formed in tumors is structurally abnormal, not organized in the conventional hierarchical arrangement, and more permeable than normal vasculature. These features contribute to leaky, tortuous, and dilated blood vessels, which act to create heterogeneous blood flow, compression of vessels, and elevated interstitial fluid pressure. As such, abnormalities in the tumor vasculature not only affect the delivery of nutrients and oxygen to the tumor, but also contribute to creating an abnormal tumor microenvironment that further promotes tumorigenesis. The role of chemical signaling events in mediating tumor angiogenesis have been well researched; however, the relative contribution of physical cues and mechanical regulation of tumor angiogenesis is less understood. Growing research indicates that the physical tumor microenvironment plays a significant role in tumor progression and promoting abnormal tumor vasculature. Here, we review how mechanical cues found in the tumor microenvironment promote aberrant tumor angiogenesis. Specifically, we discuss the influence of matrix stiffness and mechanical stresses in tumor tissue on tumor vasculature, as well as the mechanosensory pathways utilized by endothelial cells to respond to the physical cues found in the tumor microenvironment. We also discuss the impact of the resulting aberrant tumor vasculature on tumor progression and therapeutic treatment.

Background Improved cancer therapeutics and enhanced cancer survivorship have emphasized the severe long-term side effects of chemotherapy. Specifically, studies have linked many chemotherapy agents with primary ovarian insufficiency, although an exact insult model has not yet been determined. To investigate and ultimately solve this problem, a novel device for extended study of mammalian ovaries in vitro was developed. Methods A bioreactor was fabricated for bovine ovarian culture that provides intravascular delivery of media to the ovary through isolation and cannulation of a main ovarian artery branch. Whole ovaries were cultured in vitro using three methods: (1) continuously supplied fresh culture media, (2) recirculated culture media, or (3) continuously supplied fresh culture media supplemented with 500 nM doxorubicin for 24 or 48 h. TUNEL assay was used to assess apoptotic cell percentages in the three groups as compared to uncultured baseline ovaries. Results The ovary culture method was shown to maintain cell viability by effectively delivering nutrient-enriched pH-balanced media at a constant flow rate. Lower apoptosis observed in ovaries cultured in continuously supplied fresh culture media illustrates that this culture device and method are the first to sustain whole bovine ovary viability for 48 h. Meanwhile, the increase in the percentage of cell apoptosis with doxorubicin treatment indicates that the device can provide an alternative model for testing chemotherapy and chemoprotection treatments to prevent primary ovarian insufficiency in cancer patients. Conclusions An ovarian bioreactor with consistent culture media flow through an ovarian vasculature-assisted approach maintains short-term whole bovine ovary viability. Electronic supplementary material The online version of this article (doi:10.1186/s13048-016-0249-4) contains supplementary material, which is available to authorized users.

Arterial stiffening accompanies both aging and atherosclerosis, and age-related stiffening of the arterial intima increases RhoA activity and cell contractility contributing to increased endothelium permeability. Notably, statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors whose pleiotropic effects include disrupting small GTPase activity; therefore, we hypothesized the statin simvastatin could be used to attenuate RhoA activity and inhibit the deleterious effects of increased age-related matrix stiffness on endothelial barrier function. Using polyacrylamide gels with stiffnesses of 2.5, 5, and 10 kPa to mimic the physiological stiffness of young and aged arteries, endothelial cells were grown to confluence and treated with simvastatin. Our data indicate that RhoA and phosphorylated myosin light chain activity increase with matrix stiffness but are attenuated when treated with the statin. Increases in cell contractility, cell-cell junction size, and indirect measurements of intercellular tension that increase with matrix stiffness, and are correlated with matrix stiffness-dependent increases in monolayer permeability, also decrease with statin treatment. Furthermore, we report that simvastatin increases activated Rac1 levels that contribute to endothelial barrier enhancing cytoskeletal reorganization. Simvastatin, which is prescribed clinically due to its ability to lower cholesterol, alters the endothelial cell response to increased matrix stiffness to restore endothelial monolayer barrier function, and therefore, presents a possible therapeutic intervention to prevent atherogenesis initiated by age-related arterial stiffening.

Postsurgical cardiac adhesions increase the number of surgeries as well as patient mortality and morbidity. A fast gelling oxime-crosslinked PEG hydrogel with tunable gelation time, degradation, and mechanical properties is presented. This material is cytocompatible and prevents cellular adhesion. Material retention on different cardiac tissues is demonstrated ex vivo over time and that functional group ratio alters material retention on different cardiac tissues.

Efficient biomaterial screening platforms can test a wide range of extracellular environments that modulate vascular growth. Here, we used synthetic hydrogel arrays to probe the combined effects of Cys-Arg-Gly-Asp-Ser (CRGDS) cell adhesion peptide concentration, shear modulus and vascular endothelial growth factor receptor 2 (VEGFR2) inhibition on human umbilical vein endothelial cell (HUVEC) viability, proliferation and tubulogenesis. HUVECs were encapsulated in degradable poly(ethylene glycol) (PEG) hydrogels with defined CRGDS concentration and shear modulus. VEGFR2 activity was modulated using the VEGFR2 inhibitor SU5416. We demonstrate that synergy exists between VEGFR2 activity and CRGDS ligand presentation in the context of maintaining HUVEC viability. However, excessive CRGDS disrupts this synergy. HUVEC proliferation significantly decreased with VEGFR2 inhibition and increased modulus, but did not vary monotonically with CRGDS concentration. Capillary-like structure (CLS) formation was highly modulated by CRGDS concentration and modulus, but was largely unaffected by VEGFR2 inhibition. We conclude that the characteristics of the ECM surrounding encapsulated HUVECs significantly influence cell viability, proliferation and CLS formation. Additionally, the ECM modulates the effects of VEGFR2 signaling, ranging from changing the effectiveness of synergistic interactions between integrins and VEGFR2 to determining whether VEGFR2 upregulates, downregulates or has no effect on proliferation and CLS formation.

Tumor vasculature is characterized by leaky, tortuous, and dilated blood vessels that create heterogeneous blood flow and diminish the transport of oxygen. Pioneered by Jain, an emerging cancer treatment strategy seeks to normalize tumor vasculature in order to increase the delivery and efficacy of therapeutics. Overexpression of vascular endothelial growth factor (VEGF) and other pro-angiogenic factors within the tumor microenvironment have been considerably investigated as the major contributing factor to abnormal tumor vasculature, but less is understood about the influence of the physical environment and the underlying molecular mechanisms. During tumor progression, increased extracellular matrix (ECM) deposition occurs resulting in mechanically heterogeneous tissue defined by local alterations in density. Such changes to ECM density and composition can significantly disrupt cell behaviors. Our previous work has demonstrated that the presence of an interface produces directional migration and net cell migration is a function of collagen matrix density. Here, we utilize a similar sequential polymerization technique to create a distinct low-to-high collagen density interface, in order to replicate the heterogeneity found in tumor tissue and investigate the role of tumor matrix mechanics in promoting abnormal tumor vasculature. Specifically, Human Umbilical Vein Endothelial Cells (HUVECs) were encapsulated in a low-density collagen matrix, cultured for three days until capillary-like networks were formed, and a second, high-density collagen matrix supplemented with VEGF and basic fibroblast growth factor (bFGF) was added to create a 3D interface. Angiogenesis across and capillary-like network organization at the collagen density interface was then evaluated by time-lapse microscopy and immunofluorescence. Preliminary data indicates that the presence of a low-to-high density interface increased HUVEC capillary-like network branching and length. We demonstrate that the number of branches per network, total number of branches, and total network length all increased as a function of the density interface, where the larger difference in density between the two regions produced more branching points and increased network length. Together, our data demonstrate how ECM heterogeneities, in the form of interfacial boundaries, affect capillary-like network architecture and promote abnormal branching. This work provides further insight into cellular sensing during tumor angiogenesis, as well as helps to identify novel methods for tumor vasculature normalization, as a means to improve the efficacy of many chemotherapeutic and radiation treatments. Citation Format: Matthew R. Zanotelli, Francois Bordeleau, Cynthia A. Reinhart-King. Topographical guidance of angiogenesis at an interface of collagen densities. [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 A65.