Angiogenic Network Research Articles

Abstract We102: Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells by Talin Modulation

Talin is a focal adhesion protein that activates integrins and recruits other focal adhesion proteins. Talin regulates the interactions between integrins and the extracellular matrix, which are critical for endothelial cells during angiogenesis. In this study, we successfully synthesized a novel talin modulator, N-((2-(1H-indol-3-yl)ethyl)carbamoyl)-2-(benzo[d][1,3]dioxol-5-yloxy)acetamide, referred to as KCH-1521. KCH-1521 was determined to bind talin and modulate downstream signaling molecules of talin. After 24 hrs of treatment, KCH-1521 changed the cell morphology of HUVECs and reduced focal adhesion protein expression including vinculin and paxillin. Talin downstream signaling is regulated via FAK, AKT, and ERK pathways, however, treatment with KCH-1521 decreased phosphorylation of FAK, AKT, and ERK, leading to a reduction of cell proliferation, survival, and angiogenesis. Interestingly, the expression of various angiogenic genes was significantly decreased after treatment with KCH-1521. Also, in vitro tube forming assay revealed that KCH-1521 reduced angiogenic networks in a time-dependent manner. To investigate the reversibility of its effects, KCH-1521 was removed after treatment. HUVECs recovered their morphology through rearrangement of the cytoskeleton and the expression of angiogenic genes was also recovered. By further optimization and in vivo studies of KCH-1521, a novel drug of talin modulation could be used to achieve therapeutic anti-angiogenesis for vascular diseases and cancers.

Hemodynamic Characteristics of a Tortuous Microvessel Using High-Fidelity Red Blood Cell Resolved Simulations.

Tortuous microvessels are characteristic of microvascular remodeling associated with numerous physiological and pathological scenarios. Three-dimensional (3D) hemodynamics in tortuous microvessels influenced by red blood cells (RBCs), however, are largely unknown, and important questions remain. Is blood viscosity influenced by vessel tortuosity? How do RBC dynamics affect wall shear stress (WSS) patterns and the near-wall cell-free layer (CFL) over a range of conditions? The objective of this work was to parameterize hemodynamic characteristics unique to a tortuous microvessel. RBC-resolved simulations were performed using an immersed boundary method-based 3D fluid dynamics solver. A representative tortuous microvessel was selected from a stimulated angiogenic network obtained from imaging of the rat mesentery and digitally reconstructed for the simulations. The representative microvessel was a venule with a diameter of approximately 20 μm. The model assumes a constant diameter along the vessel length and does not consider variations due to endothelial cell shapes or the endothelial surface layer. Microvessel tortuosity was observed to increase blood apparent viscosity compared to a straight tube by up to 26%. WSS spatial variations in high curvature regions reached 23.6 dyne/cm2 over the vessel cross-section. The magnitudes of WSS and CFL thickness variations due to tortuosity were strongly influenced by shear rate and negligibly influenced by tube hematocrit levels. New findings from this work reveal unique tortuosity-dependent hemodynamic characteristics over a range of conditions. The results provide new thought-provoking information to better understand the contribution of tortuous vessels in physiological and pathological processes and help improve reduced-order models.

Single-Step 3D Bioprinting of Alginate-Collagen Type I Hydrogel Fiber Rings to Promote Angiogenic Network Formation.

In the advent of tissue engineering and regenerative medicine, the demand for innovative approaches to biofabricate complex vascular structures is increasing. We describe a single-step 3D bioprinting method leveraging Aspect Biosystems RX1 technology, which integrates the crosslinking step at a flow-focusing junction, to biofabricate immortalized adult rat brain endothelial cell (SV-ARBEC)-encapsulated alginate-collagen type I hydrogel rings. This single-step biofabrication process involves the strategic layer-by-layer assembly of hydrogel rings, encapsulating SV-ARBECs in a spatially controlled manner while optimizing access to media and nutrients. The spatial arrangement of the SV-ARBECs within the rings promotes spontaneous angiogenic network formation and the constrained deposition of cells within the hydrogel matrix facilitates tissue-like organized vascular-like network development. This approach provides a platform that can be adapted to many different endothelial cell types and leveraged to better understand the mechanisms driving angiogenesis and vascular-network formation in 3D bioprinted constructs supporting the development of more complex tissue and disease models for advancing drug discovery, tissue engineering, and regenerative medicine applications.

A Molecular Perspective on HIF-1α and Angiogenic Stimulator Networks and Their Role in Solid Tumors: An Update.

Hypoxia-inducible factor-1α (HIF-1α) is a major transcriptional factor, which plays an important role in cellular reprogramming processes under hypoxic conditions, which facilitate solid tumors' progression. HIF-1α is directly involved in the regulation of the angiogenesis, metabolic reprogramming, and extracellular matrix remodeling of the tumor microenvironment. Therefore, an in-depth study on the role of HIF-1α in solid tumor malignancies is required to develop novel anti-cancer therapeutics. HIF-1α also plays a critical role in regulating growth factors, such as the vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor, in a network manner. Additionally, it plays a significant role in tumor progression and chemotherapy resistance by regulating a variety of angiogenic factors, including angiopoietin 1 and angiopoietin 2, matrix metalloproteinase, and erythropoietin, along with energy pathways. Therefore, this review attempts to provide comprehensive insight into the role of HIF-1α in the energy and angiogenesis pathways of solid tumors.

EPAC inhibitor suppresses angiogenesis and tumor growth of triple-negative breast cancer

AimsExchange protein directly activated by cAMP 1 (EPAC1), a major isoform of guanine nucleotide exchange factors, is highly expressed in vascular endothelia cells and regulates angiogenesis in the retina. High intratumor microvascular densities (MVD) resulting from angiogenesis is responsible for breast cancer development. Downregulation of EPAC1 in tumor cell reduces triple-negative breast cancer (TNBC)-induced angiogenesis. However, whether Epac1 expressed in vascular endothelial cells contributes to angiogenesis and tumor development of TNBC remains elusive. Main methodsWe employed NY0123, a previously identified potent EPAC inhibitor, to explore the anti-angiogenic biological role of EPAC1 in vitro and in vivo through vascular endothelial cells, rat aortic ring, Matrigel plug, and chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) assays, as well as the in vivo xenograft tumor models of TNBC in both chick embryo and mice. Key findingsInhibiting EPAC1 in vascular endothelial cells by NY0123 significantly suppresses angiogenesis and tumor growth of TNBC. In addition, NY0123 possesses a better inhibitory efficacy than ESI-09, a reported specific EPAC inhibitor tool compound. Importantly, inhibiting EPAC1 in vascular endothelia cells regulates the typical angiogenic signaling network, which is associated with not only vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR2) signaling, but also PI3K/AKT, MEK/ERK and Notch pathway. ConclusionsOur findings support that EPAC1 may serve as an effective anti-angiogenic therapeutic target of TNBC, and EPAC inhibitor NY0123 has the therapeutic potential to be developed for the treatment of TNBC.

Mesenchymal stem cells and the angiogenic regulatory network with potential incorporation and modification for therapeutic development

Mesenchymal stem cells and the angiogenic regulatory network with potential incorporation and modification for therapeutic development

Abstract 083: Initial Angiogenesis in Vein of Galen Malformations Is Associated with Increased Symptomatic Burden

Introduction Vein of Galen Malformations (VOGM) are congenital brain arteriovenous malformations which often present during the neonatal or infantile periods. While endovascular embolization is the recommended management for this pathology, a subset of VOGM patients present with pathologic angiogenesis which complicates endovascular therapy. Previous studies have shown that these patients require a greater number of embolizations to achieve cure, but it is unclear whether this radiographic finding is associated with differences in clinical presentation. Methods A single‐center retrospective review identified all patients with a Vein of Galen Malformation from January 2004 to May 2023. Patients were stratified into three groups based on the development and timing of angiogenesis during their treatment course: pre‐intervention angiogenesis, post‐intervention angiogenesis, and no angiogenesis. Clinical presentations, relevant imaging, radiographic data, and procedural details were reviewed. Results 178 patients with VOGM were identified. 38 patients were in the pre‐intervention angiogenesis group, 51 patients were in the post‐intervention angiogenesis group, and 89 patients were in the no angiogenesis group. Pre‐intervention angiogenesis patients were significantly older on presentation for initial angiogram than post‐intervention angiogenesis or no angiogenesis patients (p<0.05). At initial clinical presentation, patients with pre‐intervention angiogenesis demonstrated significantly greater developmental delay (p<0.05). This difference persisted after controlling for age at initial presentation. Patients presenting with angiogenesis either pre‐intervention or post‐intervention required more embolizations to achieve cure compared to patients who did not develop angiogenesis during their treatment course (p<0.05). Conclusion Patients with VOGM who present with pre‐intervention angiogenesis demonstrate significantly more serious neurologic symptoms on initial presentation, even after controlling for age at presentation. Given that patients with angiogenesis are often older on initial presentation and require more embolizations to be cured, early and timely treatment is likely necessary to mitigate the further progression of both the angiogenic network and any associated neurological symptoms.

Cortical spheroid on perfusable microvascular network in a microfluidic device.

Human induced pluripotent stem cell (hiPSC)-derived brain spheroids can recapitulate the complex cytoarchitecture of the brain, as well as the genetic/epigenetic footprint of human brain development. However, hiPSC-derived 3D models such as spheroid and organoids does not have a perfusable microvascular network, which plays a vital role in maintaining homeostasis in vivo. With the critical balance of positive and negative angiogenic modulators, 3D microvascular network can be achieved by angiogenesis. This paper reports on a microfluidic-based three-dimensional, cortical spheroid grafted on the vascular-network. Vascular network was formed by inducing angiogenic sprouting using concentration gradient-driven angiogenic factors in the microfluidic device. We investigate critical factors for angiogenic vascular network formation with spheroid placement, including 1) a PKCα activator, phorbol-12-myristate-13-acetate (PMA); 2) orientation of endothelial cells under perfusion and permeability of vascular network; 3) effect of extracellular matrix (ECM) types and their densities on angiogenesis; and 4) integration with cortical spheroid on vascular network. This paper demonstrates proof of concept for the potential utility of a membrane-free in vitro cortical spheroid tissue construct with perfusable microvascular network that can be scaled up to a high throughput platform. It can provide a cost-effective alternative platform to animal testing by modeling brain diseases and disorders, and screening drugs.

Estimation of shear stress heterogeneity along capillary segments in angiogenic rat mesenteric microvascular networks.

Fluid shear stress is thought to be a regulator of endothelial cell behavior during angiogenesis. The link, however, requires an understanding of stress values at the capillary level in angiogenic microvascular networks. Critical questions remain. What are the stresses? Do capillaries experience similar stress magnitudes? Can variations explain vessel-specific behavior? The objective of this study was to estimate segment-specific shear stresses in angiogenic networks. Images of angiogenic networks characterized by increased vascular density were obtained from rat mesenteric tissues stimulated by compound 48/80-induced mast cell degranulation. Vessels were identified by perfusion of a 40 kDa fixable dextran prior to harvesting and immunolabeling for PECAM. Using a network flow-based segment model with physiologically relevant parameters, stresses were computed per vessel for regions across multiple networks. Stresses ranged from 0.003 to 2328.1 dyne/cm2 and varied dramatically at the capillary level. For all regions, the maximum segmental shear stresses were for capillary segments. Stresses along proximal capillaries branching from arteriole inlets were increased compared to stresses along capillaries in more distal regions. The results highlight the variability of shear stresses along angiogenic capillaries and motivate new discussions on how endothelial cells may respond in vivo to segment-specific microenvironment during angiogenesis.

Angiogenic Microvascular Wall Shear Stress Patterns Revealed Through Three-dimensional Red Blood Cell Resolved Modeling.

The wall shear stress (WSS) exerted by blood flowing through microvascular capillaries is an established driver of new blood vessel growth, or angiogenesis. Such adaptations are central to many physiological processes in both health and disease, yet three-dimensional (3D) WSS characteristics in real angiogenic microvascular networks are largely unknown. This marks a major knowledge gap because angiogenesis, naturally, is a 3D process. To advance current understanding, we model 3D red blood cells (RBCs) flowing through rat angiogenic microvascular networks using state-of-the-art simulation. The high-resolution fluid dynamics reveal 3D WSS patterns occurring at sub-endothelial cell (EC) scales that derive from distinct angiogenic morphologies, including microvascular loops and vessel tortuosity. We identify the existence of WSS hot and cold spots caused by angiogenic surface shapes and RBCs, and notably enhancement of low WSS regions by RBCs. Spatiotemporal characteristics further reveal how fluctuations follow timescales of RBC "footprints." Altogether, this work provides a new conceptual framework for understanding how shear stress might regulate EC dynamics in vivo.

Exposure to di-isononyl phthalate during early pregnancy disrupts decidual angiogenesis and placental development in mice

Di-isononyl phthalate (DiNP), an endocrine-disrupting chemical, is found in numerous consumer products and human exposure to this phthalate is becoming inevitable. The impact of DiNP exposure on the establishment and maintenance of pregnancy remains largely unknown. Thus, we conducted studies in which pregnant mice were exposed to an environmentally relevant dose (20 µg/kg BW/day) of DiNP on days 1–7 of gestation, then analyzed the effects of this exposure on pregnancy outcome. Our studies revealed that exposure to DiNP during this window led to fetal loss towards the end of gestation. Further studies showed that, although embryos were able to attach to the uterus, implantation sites in DiNP-exposed uteri exhibited impaired differentiation of stromal cells to decidual cells and an underdeveloped angiogenic network in the decidual bed. We also found that exposure to this phthalate has a significant effect on trophoblast differentiation and causes disorganization of the placental layers. The labyrinth was significantly reduced, resulting in compromised expression of nutrient transporters in the placentas of mice exposed to DiNP. These placental defects in DiNP-exposed females were the cause of fetal loss during the later stages of gestation.

Spheroids of adipose derived stem cells show their potential in differentiating towards the angiogenic lineage

IntroductionAdipose derived stem cells (ASCs) are a mesenchymal stem cell population of great scientific interest due to their abundance and easiness in obtaining them from adipose tissue. Recently, several techniques for three dimensional (3D) ASCs cultivation have been developed to obtain spheroids of adipose stem cells (SASCs). It was already proved that ASCs are able to differentiate towards the endothelial lineage thus, for the first time, we investigated the ability of our 3D SASCs to differentiate endothelially and the effects of not differentiated SASC secreted factors on specific cultured cells. Materials and methodsSASCs were differentiated with a specific medium towards endothelial lineage. Cell viability, gene and protein expression of typical endothelial markers were analysed. Moreover, tube formation, wound healing and migration assays were performed to investigate the ability in migration and angiogenic networks formation of endothelially differentiated cells. SASCs secretome were also tested. ResultsWe showed the ability of SASCs to differentiate towards the endothelial lineage with an increase in cell viability of 15-fold and 8-fold at 14 and 21 days of differentiation respectively. Moreover, we showed the upregulation of VEGF-A and CD31 mRNAs of 9-fold and 1300-fold in SASCs endothelially differentiated cells, whilst protein expression was different. VEGF-A protein expression was upregulated whilst CD31 protein wasn’t translated. In addition, ICAM1, VCAM1, ANGPT1, CD62E protein levels remain unchanged. SASCs were also able to organize themselves into angiogenic networks after 7 days of culturing themon ECMatrix. Secreted factors from undifferentiated 3D SASCs acted in a paracrine way on HUVECs and endothelially differentiated ASCs seeded on ECMatrix to promote angiogenic events. ConclusionsSASCs, thanks to their multilineage differentiation potential, also possess the ability to differentiate towards endothelial lineage and to organize themselves into angiogenic networks. Moreover, they are able to promote angiogenesis through their secreted factors.

Light‐Regulated Pro‐Angiogenic Engineered Living Materials

AbstractRegenerative medicine aims to restore damaged cells, tissues, and organs, for which growth factors are vital to stimulate regenerative cellular transformations. Major advances have been made in growth factor engineering and delivery like the development of robust peptidomimetics and controlled release matrices. However, their clinical applicability remains limited due to their poor stability in the body and need for careful regulation of their local concentration to avoid unwanted side‐effects. In this study, a strategy to overcome these limitations is explored using engineered living materials (ELMs), which contain live microorganisms that can be programmed with stimuli‐responsive functionalities. Specifically, the development of an ELM that releases a pro‐angiogenic protein in a light‐regulated manner is described. This is achieved by optogenetically engineering bacteria to synthesize and secrete a vascular endothelial growth factor peptidomimetic (QK) linked to a collagen‐binding domain. The bacteria are securely encapsulated in bilayer hydrogel constructs that support bacterial functionality but prevent their escape from the ELM. In situ control over the release profiles of the pro‐angiogenic protein using light is demonstrated. Finally, it is shown that the released protein is able to bind collagen and promote angiogenic network formation among vascular endothelial cells, indicating the regenerative potential of these ELMs.

Three-Dimensional Shear Stress Characteristics in Angiogenic Microvascular Networks Revealed Through Red Blood Cell-Resolved Modeling Based on Real Image Data

Introduction: Angiogenesis, the process of growing new blood vessels from existing vessels, generally occurs in response to local stimuli. Predominant among these is the wall shear stress (WSS) exerted by flowing blood. Endothelial cells (ECs) line vessel walls and can sense and respond to external forces1. 3D WSS characteristics experienced in angiogenic microvascular networks with complex surface topologies, however, are unknown. Recent work2 demonstrated significant WSS variations due to both geometric vessel complexity and the presence of red blood cells (RBCs). Such findings drive our hypothesis that WSS behavior unique to angiogenesis may exist but requires high-resolution modeling to reveal. The objective of this work is to determine 3D WSS spatial characteristics in angiogenic microvascular networks by integrating high-fidelity simulation with real image data. Methods: RBC-resolved simulations were performed using a 3D immersed boundary method fluid dynamics solver3 which resolves flow and 3D RBCs with high fidelity. Complex 3D networks were constructed based on image data, with network comprised of 150 vessels. Approximately 1000 RBCs were simulated in a network, with each RBC discretized by 5120 finite elements. Angiogenic microvascular network imaging was performed on rat mesenteric tissues harvested post the stimulation of angiogenesis via 48-80 mast cell degranulation. Data & Results: Output simulation data includes WSS values at 7 million wall points defining the network surfaces, resulting in detailed 3D WSS contours. Significant WSS heterogenity is observed in both space and time. For time-average behavior, WSS values ranged from 0.014 to 225 dyne/cm2 over network vessels. WSS contours over the whole network reveal network-level variations. Within each vessel, the standard deviation of WSS (quantifying spatial variation) averaged 15 dyne/cm2, and reached 55 dyne/cm2 in some vessels. Such variations in local regions occurred over sub-EC length scales. Temporal WSS fluctuations among wall points within vessels and local regions reached 120 dyne/cm2. Conclusions: New findings from this work reveal significant WSS variations can occur in angiogenic microvascular networks at sub-EC length scales. The results provide a new characterization of the EC microenvironment during angiogenesis and provoke consideration of complex temporal and spatial profiles when probing EC responses in experiments.

Vessel-Specific Shear Stress Distribution and Heterogeneity in Angiogenic Rat Mesenteric Microvascular Networks

Fluid shear stress has been identified as an important player in regulating endothelial cell behaviors and angiogenesis, defined as the sprouting of new blood vessels from existing vessels. However, the correlative or functional linking of shear stress to vessel-specific endothelial cell dynamics requires a better understanding of the shear stress values at the capillary level in microvascular networks. Critical questions remain unanswered. What is the shear stress experienced by endothelial cells along capillaries? Do all capillaries experience similar shear stress magnitudes? Can variations in shear stress along different capillary segments explain vessel-specific responses? The objective of this project was to estimate shear stress values in regions of angiogenic microvascular networks. Rat mesenteric tissues were harvested from adult male Wistar rats post the stimulation of angiogenesis via 48-80 mast cell degranulation. Microvascular networks were identified by perfusion of a 40kDa fixable dextran prior to harvesting and immunolabelling for PECAM. Dextran presence identified perfused segments along the hierarchy of microvascular networks, which were characterized by increased vessel density indicative of angiogenesis. Representative network regions with arteriole input and venule output vessels were selected for analysis. Vessel segments were defined by adjacent nodes, and the measurements of lengths and diameter. Assuming an inlet pressure of 75 mmHg and outlet pressure of 10 mmHg, a network segmental Newtonian flow model was used to computationally estimate vessel-specific shear stresses and velocities. Viscosity was set to 4 cP and assumed to be constant. In the results, shear stresses along vascular segments in 3 medium microvascular regions ranged from 0.1 to 1114.3 dyne/cm2 (average = 81.3 ± 3.5 dyne/cm2), in 4 small regions ranged from 0.1 to 518.7 dyne/cm2 (average = 61 ± 3.4 dyne/cm2), and in 1 large region ranged from 0.003 to 2328.1 dyne/cm2 (average = 39.8 ± 1.6 dyne/cm2). For all regions, the highest shear stress values located at capillary segments, defined as vascular segments with diameters lower than 10 μm. As segment diameters increased, the matched maximum shear stresses decreased accordingly. Also, the high heterogeneity of shear stresses in capillaries indicates the possibility for segment-specific regulation. For 3 medium regions, shear stresses along proximal capillaries branching from arteriolar inlets to venular outlets were higher than stresses along capillaries in regions distal to inlets and outlets. The results highlight the variability of segment-specific shear stresses along capillaries in microvascular regions and motivate research looking into how endothelial cells respond to segment-specific microenvironment and regulation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Mathematical modeling of Cancer growth and angiogenesis by an individual based interacting system

We present a mathematical model for the complex system for the growth of a solid tumor. The system embeds proliferation of cells depending on the surrounding oxygen field, hypoxia caused by insufficient oxygen when the tumor reaches a certain size, consequent VEGF release and angiogenic new vasculature growth, re-oxygenation of the tumor and subsequent tumor growth restart. Specifically cancerous cells are represented by individual units, interacting as proliferating particles of a solid body, oxygen, and VEGF are fields with a source and a sink, and new angiogenic vasculature is described by a network of growing curves. The model, as shown by numerical simulations, captures both the time-evolution of the tumor growth before and after angiogenesis and its spatial properties, with different distribution of proliferating and hypoxic cells in the external and deep layers of the tumor, and the spatial structure of the angiogenic network. The microscopic description of the growth opens the possibility of tuning the model to patient-specific scenarios.

Angiogenesis driven extracellular matrix remodeling of 3D bioprinted vascular networks

Angiogenesis plays a pivotal role in development and tissue growth, as well as in pathological conditions such as cancer. Being able to understand the basic mechanisms involved in the vascularization of tissues and angiogenic network formation provides a window to advance the development of in vitro tissue models and enhance tissue engineering applications. In this study, we leveraged a novel microfluidic-based three dimensional (3D) bioprinting technology and alginate-collagen type I (AGC) bioink, to develop a 3D bioprinting strategy to enable the biofabrication of complex angiogenic networks within the 3D structure. These networks were comprised of simian vacuolating virus 40 (SV40) transformed adult rat brain endothelial cell (SV-ARBEC)-laden hydrogel rings. With mechanical properties relevant for vascular tissue engineering applications, these bioprinted constructs formed spontaneous vascular networks, reminiscent of anisotropic tissue-like structures, while retaining high cellular viability. The vascular network formation was accompanied by extracellular matrix (ECM) remodeling, confirming sequential SV-ARBEC mediated collagen type I fiber deposition and reorganization. Treatment with broad spectrum matrix metalloproteinase (MMP) inhibitor supressed SV-ARBEC angiogenic sprouting, highlighting requirements of ECM remodeling in angiogenic network formation. This novel 3D microfluidic bioprinting technology and biocompatible AGC hydrogel fiber rings supported robust SV-ARBEC angiogenesis and corresponding ECM remodeling, allowing us to present a strategy suitable to advancing applications in vascular research and supporting the further development of disease models, novel testing beds for drug discovery and tissue engineering applications.

The Tumor Microenvironment in Tumorigenesis and Therapy Resistance Revisited.

Tumorigenesis is a complex and dynamic process involving cell-cell and cell-extracellular matrix (ECM) interactions that allow tumor cell growth, drug resistance and metastasis. This review provides an updated summary of the role played by the tumor microenvironment (TME) components and hypoxia in tumorigenesis, and highlight various ways through which tumor cells reprogram normal cells into phenotypes that are pro-tumorigenic, including cancer associated- fibroblasts, -macrophages and -endothelial cells. Tumor cells secrete numerous factors leading to the transformation of a previously anti-tumorigenic environment into a pro-tumorigenic environment. Once formed, solid tumors continue to interact with various stromal cells, including local and infiltrating fibroblasts, macrophages, mesenchymal stem cells, endothelial cells, pericytes, and secreted factors and the ECM within the tumor microenvironment (TME). The TME is key to tumorigenesis, drug response and treatment outcome. Importantly, stromal cells and secreted factors can initially be anti-tumorigenic, but over time promote tumorigenesis and induce therapy resistance. To counter hypoxia, increased angiogenesis leads to the formation of new vascular networks in order to actively promote and sustain tumor growth via the supply of oxygen and nutrients, whilst removing metabolic waste. Angiogenic vascular network formation aid in tumor cell metastatic dissemination. Successful tumor treatment and novel drug development require the identification and therapeutic targeting of pro-tumorigenic components of the TME including cancer-associated- fibroblasts (CAFs) and -macrophages (CAMs), hypoxia, blocking ECM-receptor interactions, in addition to the targeting of tumor cells. The reprogramming of stromal cells and the immune response to be anti-tumorigenic is key to therapeutic success. Lastly, this review highlights potential TME- and hypoxia-centered therapies under investigation.

Abstract 6383: Paracrine signaling between differentiated tumor cell-secreted fibromodulin and the stroma promotes tumor growth by enhancing angiogenesis

Abstract Cancer stem-like cells (CSCs) and the differentiated bulk cells mainly contribute to cellular heterogeneity within a tumor. The dichotomous division pattern of the CSCs sitting atop the tumor hierarchy allow them to self-renew, and differentiate to initiate and repopulate aggressive tumors. These CSC-differentiation-derived bulk tumor cells reprogram to stem-like states, accounting for high plasticity within a tumor; a phenomenon governed by the distinct epigenetic profiles of the two cell populations. Reciprocal signaling between the tumor and its complex microenvironment is a vital driver of plasticity and heterogeneity in cancer. The indispensable role of CSCs in cellular cross-talk in the tumor niche as critical drivers of tumorigenesis is well-studied. However, the massive outnumbering of the CSCs by the differentiated cells raises several vital questions regarding the role of the latter in tumor growth; whether they contribute to the complex signaling within the tumor microenvironment (TME) to drive tumor growth remains unexplored. In this study, we aimed to mechanistically delineate the distinct roles of the cancer stem-like and differentiated cells by investigating their interactions with other stromal cells in the TME.In our model system, Glioblastoma (GBM), a highly aggressive brain cancer, we adopted a quantitative, label-free, high-throughput proteomics-based approach to identify the differentially abundant secreted proteins between the glioma stem-like cell (GSC) and differentiated glioma cell (DGC). This analysis showed a significantly higher abundance of the Fibromodulin (FMOD), a collagen-binding proteoglycan, in the conditioned medium of the DGCs compared with that of the GSCs. Our investigation revealed that an activated TGF-β signaling leads to p-SMAD2 occupancy on the FMOD promoter region, leading to increased FMOD secretion by the DGCs. DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth in a subcutaneous mouse model. We also observed that FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. Moreover, DGC-secreted FMOD activates integrin-dependent Notch signaling in endothelial cells, promoting angiogenesis. FMOD-silencing also inhibits the capability of the transdifferentiated endothelial cells (derived from the DGCs) to form angiogenic networks. Conditional silencing of FMOD in the de novo formed DGCs in an orthotopic intracranial C57BL/6 mouse model inhibited the growth of GSC-initiated tumors due to poorly developed vasculature, thereby increasing mouse survival. The same observation was recapitulated using patient-derived GSCs in an immunocompromised intracranial mouse model, thereby highlighting the human relevance of our findings. Collectively, our findings demonstrate how paracrine signaling between DGC-secreted FMOD and the stromal cells promotes tumor growth by enhancing angiogenesis, thereby highlighting a previously less studied supportive role of DGCs in tumor growth, albeit the GSCs retaining the tumor-initiating capacity. Thus our study identifies DGC-specific FMOD as a potential therapeutic target and emphasizes that targeting both cancer stem cells and differentiated cancer bulk cells is vital to achieving a durable response in GBM. Citation Format: Shreoshi Sengupta, Kumaravel Somasundaram, Dinorah Friedmann Morvinski, Philippe Marin, Prasasvi Reddy, Arani Mukherjee, Prerna Magod, Mainak Mondal, Serge Urbach. Paracrine signaling between differentiated tumor cell-secreted fibromodulin and the stroma promotes tumor growth by enhancing angiogenesis [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 6383.

Estimation of Shear Stress Heterogeneity along Capillary Segments in Angiogenic Rat Mesenteric Microvascular Networks

Fluid shear stress has been identified as an important player in regulating endothelial cell behavior and angiogenesis, defined as the sprouting of new blood vessels from existing vessels. However, the correlative or functional linking of shear stress to vessel‐specific endothelial cell dynamics requires a better understanding of the shear stress values at the capillary level in microvascular networks. Critical questions remain unanswered. What is the shear stress experienced by endothelial cells along capillaries? Do all capillaries experience similar shear stress magnitudes? Can variations in shear stress along different capillary segments explain vessel‐specific responses? The objective of this project was to estimate shear stress values in regions of angiogenic microvascular networks. Rat mesenteric tissues were harvested from adult male Wistar rats post the stimulation of angiogenesis via 48‐80 mast cell degranulation. Microvascular networks were identified by perfusion of a 40kDa fixable dextran prior to harvesting and immunolabelling for PECAM. Dextran presence identified perfused segments along the hierarchy of microvascular networks, which were characterized by increased vessel density indicative of angiogenesis. Two representative network regions with arteriole input and venule output vessels were selected for analysis. Vessel segments were defined by nodes, branch order position, and the measurements of lengths and diameter. Assuming an inlet pressure of 75 mmHg and outlet pressure of 10 mmHg, a network segmental Newtonian flow model was used to computationally estimate vessel‐specific shear stresses and velocities. Viscosity was set to 4 cP and assumed to be constant. For region 1, shear stresses along capillaries (diameters less than 10 microns) ranged from 0.7 to 479 dyne/cm2 (average = 57± 69 dyne/cm2). For region 2, shear stresses along capillaries ranged 0.1 to 1032 dyne/cm2 (average = 75± 94 dyne/cm2). For both regions, the maximum shear stress along capillary segments was greater than the maximum shear stress in the initial arterial branches. The high variability at the capillary level in both network regions suggests the potential for vessel‐specific stimuli. The maximum shear stresses in proximal capillary segments connecting the high‐pressure arterial sides to the lower pressure venous sides in a network were increased compared to stresses for segments in more distal network regions. The results of this study highlight the heterogeneity of shear stress experienced by endothelial cells at the capillary level in angiogenic microvascular networks and motivate questions related to how vessel‐specific hemodynamic environments regulate endothelial cell function.