Cell-extracellular Matrix Contacts Research Articles

The cytoskeleton adaptor protein Sorbs1 controls the development of lymphatic and venous vessels in zebrafish.

Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish. We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.

An overview of glioblastoma multiforme <i>in vitro</i> experimental models

Glioblastoma multiforme (GBM) is the most common primary brain tumor, characterized by a remarkable inner complexity and inter-tumor variability. Moreover, it is very aggressive and resistant to conventional treatments, so that it rapidly relapse. Therefore, there is an immediate need for experimental strategies to enhance our comprehension of GBM, aiming to mitigate its economic and social impact. Here, we described different in vivo and in vitro strategies currently used for the study of GBM. First, we gave a brief and general overview of the classical in vivo models, including xenograft mouse and zebrafish models and canine models, offering a wide range of advantages but also presenting a series of strong limitations. Thus, we described in vitro models, starting from more traditional 2D culture models, comparing different approaches and critically exposing the advantages and disadvantages of using one or the other methods. We also briefly described GBM 2D culture systems that allow recreating multiple cell-cell and cell-extracellular matrix contacts but still do not reflect the complexity of in vivo tumors. We finally described the intricacies of the more novel 3D in vitro models, e.g., spheroids and organoids. These sophisticated models have demonstrated exceptional suitability across a wide spectrum of applications in cancer research, ranging from fundamental scientific inquiries to applications in translational research. Their adaptability and three-dimensional architecture render them invaluable tools, offering new insights and paving the way for advancements in both basic and applied research.

Hippo signaling in cancer: regulatory mechanisms and therapeutic strategies

As an evolutionarily conserved pathway, Hippo signaling not only plays a key role in embryonic development, but also regulates the initiation and progression of cancer. The upstream factors regulating the Hippo pathway are complex, including cell–cell contact, cell–extracellular matrix contact, membrane receptor–ligand binding, and cytoskeletal tension. In response to these mechanical or soluble cues, the Hippo core kinases are activated or inactivated, regulating the activity of key transcription co-factor YAP/TAZ thus yielding biological consequences. In the context of neoplasm, dysregulation of Hippo signaling contributes to cancer hallmarks such as sustained proliferation, stem-like properties, and metastasis. Importantly, targeting Hippo signaling by chemicals is emerging as a promising anticancer strategy. This article briefly introduces the discovery process of the Hippo pathway, summarizes the upstream signals regulating the Hippo pathway, discusses the relationship between Hippo inactivation and cancer development, and highlights the potential use of chemicals targeting Hippo signaling in cancer treatment.

WITHDRAWN: Computational modeling of multiple myeloma growth and tumor aggregate formation

The mechanical signals received from the multiple myeloma microenvironment play a key role in bone marrow cancer development. However, the complexity of the cues received hinders the development of a novel and effective treatment. As such, agent-based computational models that consider coupled fluid and mechanical dynamics for every single cell may provide distinct perspectives and key information to adequately identify the specific tumor microenvironment. We have developed a novel 3D agent-based computational model that considers coupled fluid and particle dynamics to study multiple myeloma cell growth in response to the stimuli received from the cell microenvironment. Cell processes such as cell–cell interactions, cell maturation, and cell proliferation were considered by implementing user-defined functions in the commercial software Fluent. To calibrate and validate the developed model, we performed two experiments comparing the results of cell sedimentation velocity and cell proliferation with in vitro results. The results of the model were also compared with a finite element method model previously developed in-house. As the cells proliferated, the number of cell–cell and cell–extracellular matrix contacts increased ( ∼ 6.1% per hour), which reduced the cell maturation time ( ∼ 69.7%). Once the cells started to form aggregates, the proliferation process speeded up ( ∼ 9.9% per hour). Saturation of cell proliferation was observed after long-term simulation. Our results are qualitatively consistent with the in vitro results. In addition, they show that the cell proliferation rate increased as the number of cells considered increased. Furthermore, in a very dense aggregation, the lack of space prevented proliferation of internal cells. The model developed herein has significant advantages compared to the previous model in terms of computation, as it results in a significant reduction (84%–91%) in computational costs, thus allowing a more realistic simulation. • Agent-based model coupled with a continuous fluid model for Multiple Myeloma cell simulation. • Based on specific cell conditions, the model has been applied to study Multiple Myeloma cells’ growth and tumor aggregation formation. • Results have been validated with the literature, in-vitro experiments developed by the authors, and a previous in-house Finite Element model. • Cell maturation is stimulated by the increase in cell–cell and cell-ECM interactions, but it is inhibited by a lack of space in the inner parts of cell aggregations. • Computational costs have been reduced when compared to the previous Finite Element model.

The deadly cross-talk between Hippo pathway and epithelial-mesenchymal transition (EMT) in cancer.

Hippo signaling pathway is an evolutionarily conserved network that regulates organ size growth and tissue regeneration. Hippo signaling dysfunction results in uncontrolled cell proliferation and influences cell differentiation. Aberrant Hippo pathway signaling is implicated in cancer progression, by promoting cell proliferation, cancer stem cell properties, chemoresistance and metastatic capacity. Epithelial-mesenchymal transition (EMT) is also well known to be implicated in carcinogenesis. Loss of cell polarity, disruption of cell-cell junctions and cytoskeletal remodeling are essential during EMT. At the same time, signals related to intercellular contact, cell-extracellular matrix contact, polarity and mechanotransduction are included in the list of regulatory inputs into Hippo pathway. Therefore, the emerging association between Hippo pathway and EMT in cancer is not surprising. Recent studies have begun to unravel the mechanisms of interaction between Hippo signaling pathway and EMT. In this review, we describe the existing evidence of cross talk between Hippo signaling pathway key molecules and the process of EMT, with emphasis on the role of Hippo-EMT interplay in cancer.

DNA methylation in relation to gestational age and brain dysmaturation in preterm infants.

Preterm birth is associated with dysconnectivity of structural brain networks and is a leading cause of neurocognitive impairment in childhood. Variation in DNA methylation is associated with early exposure to extrauterine life but there has been little research exploring its relationship with brain development. Using genome-wide DNA methylation data from the saliva of 258 neonates, we investigated the impact of gestational age on the methylome and performed functional analysis to identify enriched gene sets from probes that contributed to differentially methylated probes or regions. We tested the hypothesis that variation in DNA methylation could underpin the association between low gestational age at birth and atypical brain development by linking differentially methylated probes with measures of white matter connectivity derived from diffusion MRI metrics: peak width skeletonized mean diffusivity, peak width skeletonized fractional anisotropy and peak width skeletonized neurite density index. Gestational age at birth was associated with widespread differential methylation at term equivalent age, with genome-wide significant associations observed for 8870 CpG probes (P < 3.6 × 10−8) and 1767 differentially methylated regions. Functional analysis identified 14 enriched gene ontology terms pertaining to cell–cell contacts and cell–extracellular matrix contacts. Principal component analysis of probes with genome-wide significance revealed a first principal component that explained 23.5% of the variance in DNA methylation, and this was negatively associated with gestational age at birth. The first principal component was associated with peak width of skeletonized mean diffusivity (β = 0.349, P = 8.37 × 10−10) and peak width skeletonized neurite density index (β = 0.364, P = 4.15 × 10−5), but not with peak width skeletonized fraction anisotropy (β = −0.035, P = 0.510); these relationships mirrored the imaging metrics’ associations with gestational age at birth. Low gestational age at birth has a profound and widely distributed effect on the neonatal saliva methylome that is apparent at term equivalent age. Enriched gene ontology terms related to cell–cell contacts reveal pathways that could mediate the effect of early life environmental exposures on development. Finally, associations between differential DNA methylation and image markers of white matter tract microstructure suggest that variation in DNA methylation may provide a link between preterm birth and the dysconnectivity of developing brain networks that characterizes atypical brain development in preterm infants.

Insights Into Bone Marrow Niche Stability: An Adhesion and Metabolism Route.

The bone marrow microenvironment provides critical cues for hematopoietic stem cell (HSC) self-renewal and differentiation and contributes to their malignant conversion. The microenvironment comprises a complex mixture of multiple cell types, soluble factors, and extracellular matrix in specialized regions termed ‘niches.’ Positioning of the various cellular players within these niches depends on their repertoire of adhesion molecules and chemotactic signaling, involving integrins and chemokine receptors and the corresponding intracellular players such as kinases and GTPases. The mechanical role of adhesion is to control the strength and morphology of the cell-cell and cell-extracellular matrix contacts and thereby the energy needed for the optimal localization of cells to their surroundings. While it is clear that biomechanical adhesive bonds are energetically expensive, the crosstalk between cell adhesion and metabolic pathways in the normal and malignant microenvironment is far from understood. The metabolic profile of the various cell types within the niche includes key molecules such as AMPK, glucose, mTOR, and HIF-1α. Here, we describe our most recent understanding of how the interplay between adhesion and these metabolic components is indispensable for bone marrow niche stability. In parallel, we compare the altered crosstalk of different cell types within the bone marrow niches in hematological malignancies and propose potential therapeutic associations.

Rhenium Perrhenate (188ReO4) Induced Apoptosis and Reduced Cancerous Phenotype in Liver Cancer Cells.

Recurrence in hepatocellular carcinoma (HCC) after conventional treatments is a crucial challenge. Despite the promising progress in advanced targeted therapies, HCC is the fourth leading cause of cancer death worldwide. Radionuclide therapy can potentially be a practical targeted approach to address this concern. Rhenium-188 (188Re) is a β-emitting radionuclide used in the clinic to induce apoptosis and inhibit cell proliferation. Although adherent cell cultures are efficient and reliable, appropriate cell-cell and cell-extracellular matrix (ECM) contact is still lacking. Thus, we herein aimed to assess 188Re as a potential therapeutic component for HCC in 2D and 3D models. The death rate in treated Huh7 and HepG2 lines was significantly higher than in untreated control groups using viability assay. After treatment with 188ReO4, Annexin/PI data indicated considerable apoptosis induction in HepG2 cells after 48 h but not Huh7 cells. Quantitative RT-PCR and western blotting data also showed increased apoptosis in response to 188ReO4 treatment. In Huh7 cells, exposure to an effective dose of 188ReO4 led to cell cycle arrest in the G2 phase. Moreover, colony formation assay confirmed post-exposure growth suppression in Huh7 and HepG2 cells. Then, the immunostaining displayed proliferation inhibition in the 188ReO4-treated cells on 3D scaffolds of liver ECM. The PI3-AKT signaling pathway was activated in 3D culture but not in 2D culture. In nude mice, Huh7 cells treated with an effective dose of 188ReO4 lost their tumor formation ability compared to the control group. These findings suggest that 188ReO4 can be a potential new therapeutic agent against HCC through induction of apoptosis and cell cycle arrest and inhibition of tumor formation. This approach can be effectively combined with antibodies and peptides for more selective and personalized therapy.

Tissue remodeling by invadosomes.

One of the strategies used by cells to degrade and remodel the extracellular matrix (ECM) is based on invadosomes, actin-based force-producing cell–ECM contacts that function in adhesion and migration and are characterized by their capacity to mediate pericellular proteolysis of ECM components. Invadosomes found in normal cells are called podosomes, whereas invadosomes of invading cancer cells are named invadopodia. Despite their broad involvement in cell migration and in protease-dependent ECM remodeling and their detection in living organisms and in fresh tumor tissue specimens, the specific composition and dynamic behavior of podosomes and invadopodia and their functional relevance in vivo remain poorly understood. Here, we discuss recent findings that underline commonalities and peculiarities of podosome and invadopodia in terms of organization and function and propose an updated definition of these cellular protrusions, which are increasingly relevant in patho-physiological tissue remodeling.

Human Platelet Lysate Acts Synergistically With Laminin to Improve the Neurotrophic Effect of Human Adipose-Derived Stem Cells on Primary Neurons in vitro.

BackgroundDespite the advancements in microsurgical techniques and noteworthy research in the last decade, peripheral nerve lesions have still weak functional outcomes in current clinical practice. However, cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit has shown promising results in animal studies. Human platelet lysate (hPL) has been adopted to avoid fetal bovine serum (FBS) in consideration of the biosafety concerns inherent with the use of animal-derived products in tissue processing and cell culture steps for translational purposes. In this work, we investigate how the interplay between hPL-expanded hADSC (hADSChPL) and extracellular matrix (ECM) proteins influences key elements of nerve regeneration.MethodshADSC were seeded on different ECM coatings (laminin, LN; fibronectin, FN) in hPL (or FBS)-supplemented medium and co-cultured with primary dorsal root ganglion (DRG) to establish the intrinsic effects of cell–ECM contact on neural outgrowth. Co-cultures were performed “direct,” where neural cells were seeded in contact with hADSC expanded on ECM-coated substrates (contact effect), or “indirect,” where DRG was treated with their conditioned medium (secretome effect). Brain-derived nerve factor (BDNF) levels were quantified. Tissue culture plastic (TCPS) was used as the control substrate in all the experiments.ResultshPL as supplement alone did not promote higher neurite elongation than FBS when combined with DRG on ECM substrates. However, in the presence of hADSC, hPL could dramatically enhance the stem cell effect with increased DRG neurite outgrowth when compared with FBS conditions, regardless of the ECM coating (in both indirect and direct co-cultures). The role of ECM substrates in influencing neurite outgrowth was less evident in the FBS conditions, while it was significantly amplified in the presence of hPL, showing better neural elongation in LN conditions when compared with FN and TCPS. Concerning hADSC growth factor secretion, ELISA showed significantly higher concentrations of BDNF when cells were expanded in hPL compared with FBS-added medium, without significant differences between cells cultured on the different ECM substrates.ConclusionThe data suggest how hADSC grown on LN and supplemented with hPL could be active and prone to support neuron–matrix interactions. hPL enhanced hADSC effects by increasing both proliferation and neurotrophic properties, including BDNF release.

Piezo1 channels mediate trabecular meshwork mechanotransduction and promote aqueous fluid outflow.

Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye. Chronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure-induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1-deficient cells. The majority of TM cells exhibited a delayed stretch-activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell-matrix contacts. Analysis of TM-dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.

Dynamics and Physics of Integrin Activation in Tumor Cells by Nano-Sized Extracellular Ligands and Electromagnetic Fields.

Integrins are stress-sensing proteins expressed on the surface of cells. They regulate bidirectional signal transduction during cell-cell or cell-extracellular matrix (ECM) contacts. Integrins link the ECM with the cytoplasm through interaction with their ligands. Biophysically, such interactions can be understood as changes in stress fields at specific integrin stress-sensing domains, such as the MIDAS and ADMIDAS domains. Stress changes between ligands and cytoskeletal structures are involved in cancer cell growth by altering signal transduction pathways dependent on integrin activation. In this chapter, previous results regarding integrin activation and tumor cell growth using nanoparticles (NPs) of different materials, sizes and shapes are placed within a framework of polarized NPs in the ECM by external electromagnetic fields, in which the synergic action between polarized NPs and electromagnetic fields activates the integrins. Small size NPs activate integrins via the polar component of the dipole force between NPs and integrin sensing stress sites, while large size NPs exercise a similar action via the radial component. A quantum electrodynamic model also accounts for ECM overstressing by electromagnetic mode trapping between coherent symmetric and antisymmetric quantum states.

Invadopodia: clearing the way for cancer cell invasion.

The invasive nature of many cancer cells involves the formation of F-actin-based, lipid-raft-enriched membrane protrusions known as invadopodia or, more broadly, invadosomes. Invadopodia are specialized adhesive structures arising from ventral cell surface within cell-extracellular matrix (ECM) contacts and concentrate high proteolytic activities that allow cells to overcome the dense scaffold of local microenvironment, comprising a natural barrier to cell spreading. This degradative activity distinguishes invadopodia from other adhesive structures like focal adhesions, lamellipodia or filopodia, and is believed to drive cancer progression.

Maximizing the Value of Cancer Drug Screening in Multicellular Tumor Spheroid Cultures: A Case Study in Five Head and Neck Squamous Cell Carcinoma Cell Lines.

With approval rates <5% and the probability of success in oncology clinical trials of 3.4%, more physiologically relevant in vitro three-dimensional models are being deployed during lead generation to select better drug candidates for solid tumors. Multicellular tumor spheroids (MCTSs) resemble avascular tumor nodules, micrometastases, or the intervascular regions of large solid tumors with respect to morphology, cell-cell and cell-extracellular matrix contacts, and volume growth kinetics. MCTSs develop gradients of nutrient and oxygen concentration resulting in diverse microenvironments with differential proliferation and drug distribution zones. We produced head and neck squamous cell carcinoma (HNSCC) MCTSs in 384-well U-bottom ultra-low-attachment microtiter plates and used metabolic viability and imaging methods to measure morphologies, growth phenotypes and the effects of 19 anticancer drugs. We showed that cell viability measurements underestimated the impact of drug exposure in HNSCC MCTS cultures, but that incorporating morphology and dead-cell staining analyses increased the number of drugs judged to have substantially impacted MCTS cultures. A cumulative multiparameter drug impact score enabled us to stratify MCTS drug responses into high-, intermediate-, and low-impact tiers, and maximized the value of these more physiologically relevant tumor cultures. It is conceivable that the viable cells present in MCTS cultures after drug exposure arise from drug-resistant populations that could represent a source of drug failure and recurrence. Long-term monitoring of treated MCTS cultures could provide a strategy to determine whether these drug-resistant populations represent circumstances where tumor growth is delayed and may ultimately give rise to regrowth.

Abstract LB-127: Loss of function of GAS7 promotes neuroblastoma metastasis in the context of MYCN overexpression

Abstract Neuroblastoma is an embryonal tumor arising in the peripheral sympathetic nervous system (PSNS), and often presents with widespread metastasis at diagnosis. We recently demonstrated that overexpression of LMO1 can synergize with MYCN to promote tumor metastasis in our zebrafish model system. Here, we identify a “metastasis suppressor” gene – growth arrest specific 7 (GAS7) – whose loss of function or reduced expression can promote dissemination and migration of neuroblastoma cells with MYCN overexpression, both in vitro and in vivo. GAS7 resides on chromosome 17p13.1, a region that is deleted in high-risk neuroblastomas with higher frequency in MYCN-amplified cases. Expression levels of GAS7 are also significantly downregulated in MYCN-amplified or otherwise MYCN-overexpressing human neuroblastoma cells, as well as MYCN-overexpressing zebrafish PSNS cells, suggesting an inverse relationship between the oncogenic activities of MYCN and GAS7downregulation. To determine whether loss of function or reduced expression of GAS7 could contribute to neuroblastoma pathogenesis in the context of MYCN overexpression, we developed a gas7 knockout zebrafish line using a transcription activator-like effector nucleases (TALEN) method. Loss of function of gas7 promoted hematogenous metastasis of MYCN-overexpressing tumor cells to tissues and organs that paralleled the common sites of metastasis in neuroblastoma patients. Further RNA-sequencing and qRT-PCR analyses showed that representative signature genes encoding proteins involved in tumor cell-cell or cell-extracellular matrix interactions were significantly downregulated in fish tumors with loss of function of gas7, consistent with our electron microscopic finding in the fish model showing a loss of contact among tumor cells in fish lacking functional gas7, suggesting that GAS7 may physically act as a metastasis suppressor. Thus, in high-risk neuroblastoma with MYCNamplification or overexpression, we propose that deletion or reduced expression of GAS7 cooperates with MYCN to promote neuroblastoma cell dissemination. Although the precise mechanism of this interaction remains unclear, we suggest that it relies, in part, on the downregulation of pivotal genes involved in tumor cell-cell or cell-extracellular matrix contacts. Citation Format: Zhiwei Dong, Gonzalo Lepez Garcia, Cheng Zhang, Erin N. Dankert, Jo Lynne Harenza, Xiaonan Hou, Hu Li, John M. Maris, Shizhen (Jane) Zhu. Loss of function of GAS7 promotes neuroblastoma metastasis in the context of MYCN overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-127.

Integrins and Exosomes, a Dangerous Liaison in Cancer Progression.

Integrin activity and function is classically related to the bi-directional regulation of cell-extracellular matrix (ECM) contacts that regulate a number of cell pathways linked to cell adhesion, cell detachment from ECM, cell migration, and anoikis. Interestingly, emerging data continue to uncover new roles for integrins in cancer-relevant pathways, particularly concerning the regulation of immune cell activity in the tumor niche, like myeloid cell differentiation and function and, very recently, the regulation of metastatic processes by exosomes. Exosomes are deeply involved in cell-cell communication processes and several studies have shown that integrins found in tumor-associated exosomes can promote cancer progression by two novel cooperative mechanisms: horizontal transfer of integrin transcripts as vescicle cargo, and selection of target tissues to form new tumor niches during metastatic spread by integrins carried on the exosome’s surface. In this review we will discuss mounting evidence that contribute to the development of a new picture for integrins in cancer, highlighting the role of integrins in the processes that leads to tumor niche formation. In particular, the role of the periostin pathway in the recruitment of tumor-associated macrophages, and the proposed contribution of exosome-derived integrins in the metastatic spread will be discussed. Finally, in light of the above considerations, an evaluation of integrins as possible therapeutic targets will be conducted.

A Vascular Permeability Assay Using an In Vitro Human Microvessel Model Mimicking the Inflammatory Condition.

The vascular barrier is an important function of the endothelium and its dysfunction is involved in several diseases. The barrier function of the endothelial cell monolayer is governed by cell-cell, cell-extracellular matrix (cell-ECM) contacts, and inflammatory factors such as thrombin, histamine or vascular endothelial growth factor. Several in vivo and in vitro assays that measure the vascular permeability induced by these factors have been developed. However, they suffer limitations such as being challenging for assessing details of biological processes at a cellular level or lacking the architecture of a vessel, that raise the need for new methods. In vitro 3D model-based assays have thus been developed but assays for investigating compounds that protects the barrier function are lacking. Here we describe the development of an in vitro three-dimensional (3D) vascular endothelium model in which we can manipulate the endothelial barrier function and permeability to molecules, which have a molecular weight similar to human serum albumin, allowing to assess the protective effect of compounds. A microvessel was prepared by culturing human umbilical vein endothelial cells (HUVECs) within a collagen gel on a polydimethylsiloxane (PDMS) chip. Using fluorescein isothiocyanate (FITC)-conjugated dextran (70 kDa, FITC-dextran) and confocal fluorescence microscopy, we showed that the microvessel presented an effective barrier function. We were then able to induce the loss of this barrier function by treatment with the inflammatory factor thrombin. The loss of barrier function was quantified by the extravasation of FITC-dextran into collagen matrix. Furthermore, we were able to analyze the protective effect on the endothelial barrier function of the cyclic adenosine monophosphate (cAMP) analog, 8-pCPT-2'-O-Me-cAMP (also called 007). In an attempt to understand the effects of thrombin and 007 in our model, we analyzed the adherens junctions and cytoskeleton through immunostaining of the vascular endothelial cadherin and actin, respectively. Our assay method could be used to screen for compounds modulating the barrier function of endothelial cells, as well as investigating mechanistic aspects of barrier dysfunction.

Mining the epigenetic landscape of tissue polarity in search of new targets for cancer therapy.

The epigenetic nature of cancer encourages the development of inhibitors of epigenetic pathways. Yet, the clinical use for solid tumors of approved epigenetic drugs is meager. We argue that this situation might improve upon understanding the coinfluence between epigenetic pathways and tissue architecture. We present emerging information on the epigenetic control of the polarity axis, a central feature of epithelial architecture created by the orderly distribution of multiprotein complexes at cell-cell and cell-extracellular matrix contacts and altered upon cancer onset (with apical polarity loss), invasive progression (with basolateral polarity loss) and metastatic development (with basoapical polarity imbalance). This information combined with the impact of polarity-related proteins on epigenetic mechanisms of cancer enables us to envision how to guide the choice of drugs specific for distinct epigenetic modifiers, in order to halt cancer development and counter the consequences of polarity alterations.

Abstract A78: The role of tumor microenvironment in prostate cancer of African Americans

Abstract Background: The incidence of prostate cancer (PCa) is approximately 60% higher and the mortality rate 2 to 3 times greater among African American men (AA) in the U.S. compared to men with a European background, European Americans (EA). Men of West African ancestry from the Caribbean and South America share a similar increase in incidence and mortality to AA suggesting a genetic contribution to these outcomes. We have observed that expression of hundreds of genes in the tumor microenvironment is strikingly different for AA and EA patients1. The majority of significant differences in the stroma of AA patients were down-regulated compared to EA patients. These expression alterations could be related to somatically acquired DNA changes in the stroma but non-tumor tissue in cancer usually exhibits far fewer genetic changes than tumors. Thus, it may be that inherited or distinct environmental changes are responsible for the differences seen between AA and EA stroma. Whatever the basis of the large number of differences observed, the distinct patterns for AA and EA argue strongly, and for the first time, that there are many molecular distinctions between PCa in these races that are resident in the tumor-adjacent microenvironment. We hypothesize that the stroma of AA PCa exhibits a large-scale lower expression of protein mediators of cellular antitumor immunity and reduction in factors involved in cell-cell and cell-extracellular matrix (ECM) contacts. Such differences may, thereby, favor aggressive cancer including early metastases in AA. Methods: In order to test the basis of the relative decreased expression in AA stroma, ChIP methods for isolating methylated and unmethylated DNA combined with NGS have been used to define methylated sites in two PCa EA cells lines as well as FFPE samples of stroma from EA and AA PCa. To validate the microarray data, we are using Tumor Microarrays from both CA (n=443) and AA (n=105) patients to confirm in immunohistochemistry (IHC) the differential protein expression of many immune and ECM mediators identified in our initial studies. Results: We validated the ChIP-NGS methods by quantitatively confirming significant methylation within the promoter regions of LnCAP and DU135 cells compared to immortalized normal p69 cells for 35 reported methylated genes including GSTPi. Of the 965 largest down-regulated transcripts observed in AA stroma (1), 237 were observed to exhibit increased promoter methylation in AA stroma tissue compared to EA stroma. These 237 genes identified four main pathways (WNT, TGFβ, integrin mediated cell matrix adhesion, and EMT, all with p&amp;lt;0.00001) that are deregulated in AA stroma compared to CA stroma. The IHC studies have shown that CD8+ cell infiltration is significantly greater into the tumors of EA patients than those from AA. Immune mediators of antigen presentation are greater expressed in EA tumors, strongly suggesting immune processes involved in anti-tumor immunity may be more efficient in EA than AA patients. In addition, factors mediating interaction with the ECM such as ITGA5 and ECM formation such as COL4α1 also are increased in EA. Conclusions: These preliminary studies support the hypothesis that the stroma of AA PCa exhibits a large-scale lower expression of protein mediators of cellular antitumor immunity and reduction in factors involved in cell-cell and cell-ECM contacts. The results suggest a role for the microenvironment in the aggressive features of PCa of AA. Acknowledgements: This study was funded by the NCI Comprehensive Partnerships to Reduce Cancer Health Disparities (CPRCHD) grants (U54CA132384 and U54CA132379) and by 1U01CA162147 (KLM), NCI 3UL1TR000004-07S2 (DAM) and NCI UO1CA152738A, P30CA62203 and the Congressionally Directed Medical Research Programs W81XWH-12-PCRP-IDA (DAM, MMCC). 1. Kinseth M, A., Z. Jia, et al., 2014. Int. J. Cancer 134: 81-91. DOI: 10.1002/ijc.28326. Citation Format: Farah Rahmatpanah, Tracy Luu, Pardis Zaeri, Xin Chen, Yuanjie Hu, Joe Adams, Harmony Saunders, Sam Takahashi, Michael McClelland, Kathleen L. McGuire, Dan Mercola. The role of tumor microenvironment in prostate cancer of African Americans. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A78.

Generic Role of Polymer Supports in the Fine Adjustment of Interfacial Interactions between Solid Substrates and Model Cell Membranes.

To understand the generic role of soft, hydrated biopolymers in adjusting interfacial interactions at biological interfaces, we designed a defined model of the cell-extracellular matrix contacts based on planar lipid membranes deposited on polymer supports (polymer-supported membranes). Highly uniform polymer supports made out of regenerated cellulose allow for the control of film thickness without changing the surface roughness and without osmotic dehydration. The complementary combination of specular neutron reflectivity and high-energy specular X-ray reflectivity yields the equilibrium membrane-substrate distances, which can quantitatively be modeled by computing the interplay of van der Waals interaction, hydration repulsion, and repulsion caused by the thermal undulation of membranes. The obtained results help to understand the role of a biopolymer in the interfacial interactions of cell membranes from a physical point of view and also open a large potential to generally bridge soft, biological matter and hard inorganic materials.