Enriched Extracellular Matrix Research Articles

Delineating three-dimensional behavior of uveal melanoma cells under anchorage independent or dependent conditions

BackgroundAlthough rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention.MethodsSix UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis.ResultsUM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions.ConclusionsUnder an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

Abstract 4271: The ITGA2 (CD49b) collagen receptor excludes the CD8+ T cell infiltration in pancreatic ductal adenocarcinoma

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma cancer (PDAC) is characterized by an enriched extracellular matrix and limited T cell infiltration, which poses challenges for immunotherapy. The significant accumulation of collagen type I (COL1) in PDAC tumor microenvironment raises questions about its functional role in PDAC progression and its interactions with the cellular compartments. OBJECTIVE: The objective of this study was to investigate whether the collagen receptors expressed by cancer cells modulate the anti-tumor activity of CD8+ tumor-infiltrated lymphocytes (CD8+ TILs).METHODS: Collagen receptor expression patterns in PDAC were examined using flow cytometry, western blot, multiplex immunofluorescence, RNA seq, and scRNA seq databases in KPC and treatment-naïve PDAC that were resected from patients. The KPC cell line, known as Hy15549, was established from KPC mouse model (Ptf1-Cre; LSL-KRAS-G12D; Trp53 Lox/+). Using CRISPR-Cas9, we conducted targeted deletions for either ITGA2(CD49b) or ITGB1(CD29), as well as generating double knockouts for both CD49b and CD29. Growth of these cell lines implanted into the flanks of immunocompetent, immunodeficient mice was measured, as well as after CD8+ T cell depletion in immunocompetent mice. We analyzed TILs using FACs. We used multiplex immunofluorescence to analyze tumors microenvironment including collagen fiber alignment. Downstream signaling pathways were assessed using RNA seq and western blot. RESULTS: CD49b is highly expressed specifically in PDAC cancer cells, as assessed in both KPC cell flank tumors and resected human PDAC. Using adhesion assay, we found that CD49b specifically binds to Col1 rather than ColIV. KPC CD29b KO exhibited a complete loss of adhesion to both Col1 and ColIV. Tumor growth of the KPC CD49b KO, KPC CD29 KO, KPC CD49b CD29 KO was observed to be significantly slower than KPC WT despite identical in vitro growth kinetics. Knockout of CD49b in tumors promoted intratumoral infiltration of CD8+ T cells and significantly impeded tumor growth. Tumor growth by KPCCD49bKO was rescued by depletion of CD8+ T cells. In contrast to observations in immune-competent mice, KPCCD49bKO tumor growth was similar to KPCwt in immunodeficient nude mice. Analysis of downstream signaling in KPCCD49bKO tumors revealed a decrease of FAK as well as pFAk expression pathway. The proteomic profiling of KPCCD49bKO revealed an increase of CXCL10 chemokine. Moreover, In the absence of CD49b, the orientation of collagen fiber alignment appeared more scattered, with thinner fibers. This altered alignment could potentially contribute to immune cell infiltration.Conclusion:These findings reveal a previously unreported function of CD49b in PDAC cancer progression and the immune response within the TME. Together, our study reveals a mechanism underlying immune exclusion and suggests a novel immunotherapeutic target in PDAC. Citation Format: Ibrahim Ragab Eissa, Eric D. Abston, Darshini Kuruppu, Yongtao Wang, Guoliang Qiao, Motaz Qadan, Michael Lanuti, Kenneth K. Tanabe. The ITGA2 (CD49b) collagen receptor excludes the CD8+ T cell infiltration in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4271.

Abstract 5482: Spatial and multi-omic interrogation of the primary and recurrent glioblastoma microenvironment

Abstract Glioblastoma remains as one of the deadliest malignancies with most cases likely to recur. Recent work has focused on characterizing not only glioma cells, but the tumor microenvironment via single-cell and spatial transcriptomic technologies. This has improved our understanding of the high heterogeneity of this disease and described potential tumor domains. However, there is still a need to further understand the changes that occur between the primary and the recurrent lesion. We propose that a spatial, multi-omic characterization of matched primary and recurrent glioblastoma can yield insights into recurrence mechanisms occurring throughout the tumor microenvironment. We collected 22 IDH-WT glioblastoma samples from 12 patients (11 primary, 11 recurrent, 10 matched pairs) from the Mayo Clinic Neuroscience Biobank. We performed single-nucleus RNA sequencing (15 samples, 10 patients, 5 matched pairs) and proteomics (16 samples, 6 matched pairs) from fresh-frozen tissue, as well as spatial transcriptomics with 10x Visium (14 samples, 8 patients, 6 matched pairs) and 10x Xenium in-situ molecular imaging (6 samples, 2 matched pairs). Using spatial transcriptomics data, we identified regional subsets present in different proportions and distributions between primary and recurrent glioblastoma. These clusters were characterized with gene set and gene ontology analysis, identifying regions enriched with known glioma subtypes and cellular activity. Spatial-based cluster genes were consistent with significantly differentially expressed proteins. We used single-nucleus RNAseq and in-situ molecular imaging data from matched tissue samples to further interrogate relevant cell populations and location in the tumor micro-environment. We identified regions with enriched extra cellular matrix interaction and remodeling signatures to be highly expressed in recurrent glioblastoma. Primary glioblastoma was enriched with oligodendrocyte-like regions with high cell communication and extracellular vesicle signatures. This study will further our understanding of the changes in the mechanisms at play in the complex ecosystem of glioblastoma as it recurs. Citation Format: Jean R. Clemenceau, Paola Suarez-Meade, Alfredo Quiñones-Hinojosa, Tae Hyun Hwang. Spatial and multi-omic interrogation of the primary and recurrent glioblastoma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5482.

Single-cell transcriptome analysis of epithelial, immune, and stromal signatures and interactions in human ovarian cancer

A comprehensive investigation of ovarian cancer (OC) progression at the single-cell level is crucial for enhancing our understanding of the disease, as well as for the development of better diagnoses and treatments. Here, over half a million single-cell transcriptome data were collected from 84 OC patients across all clinical stages. Through integrative analysis, we identified heterogeneous epithelial-immune-stromal cellular compartments and their interactions in the OC microenvironment. The epithelial cells displayed clinical subtype features with functional variance. A significant increase in distinct T cell subtypes was identified including Tregs and CD8+ exhausted T cells from stage IC2. Additionally, we discovered antigen-presenting cancer-associated fibroblasts (CAFs), with myofibroblastic CAFs (myCAFs) exhibiting enriched extracellular matrix (ECM) functionality linked to tumor progression at stage IC2. Furthermore, the NECTIN2-TIGIT ligand-receptor pair was identified to mediate T cells communicating with epithelial, fibroblast, endothelial, and other cell types. Knock-out of NECTIN2 using CRISPR/Cas9 inhibited ovarian cancer cell (SKOV3) proliferation, and increased T cell proliferation when co-cultured. These findings shed light on the cellular compartments and functional aspects of OC, providing insights into the molecular mechanisms underlying stage IC2 and potential therapeutic strategies for OC.

Profibrotic Subsets of SPP1+ Macrophages and POSTN+ Fibroblasts Contribute to Fibrotic Scarring in Acne Keloidalis

Acne keloidalis (AK) is a primary scarring alopecia characterized by longstanding inflammation in the scalp causing keloid-like scar formation and hair loss. Histologically, AK is characterized by mixed leukocytic infiltrates in the acute stage followed by a granulomatous reaction and extensive fibrosis in the later stages. To further explore its pathogenesis, bulk RNA-sequencing, single-cell RNA-sequencing, and spatial transcriptomics were applied to occipital scalp biopsy specimens of lesional and adjacent non-lesional skin in patients with clinically active disease. Unbiased clustering revealed 19 distinct cell populations, including two notable populations, POSTN+ fibroblasts with enriched extracellular matrix signatures and SPP1+ myeloid cells with a M2 macrophage phenotype. Cell communication analyses indicated that fibroblasts and myeloid cells communicated by SPP1 signaling networks in lesional skin. A reverse transcriptomics in-silico approach identified corticosteroids as possessing the capability to reverse the gene expression signatures of SPP1+ myeloid cells and POSTN+ fibroblasts. Intralesional corticosteroid injection greatly reduced SPP1 and POSTN gene expression, as well as AK disease activity. Spatial transcriptomics and immunofluorescence staining verified micro-anatomic specificity of SPP1+ myeloid cells and POSTN+ fibroblasts with disease activity. In summary, the communication between POSTN+ fibroblasts and SPP1+ myeloid cells by SPP1 axis may contribute to the pathogenesis of AK.

Early clinical outcomes and molecular smooth muscle cell phenotyping using a prophylactic aortic arch replacement strategy in Loeys-Dietz syndrome

ObjectivesPatients with Loeys-Dietz syndrome demonstrate a heightened risk of distal thoracic aortic events after valve-sparing aortic root replacement. This study assesses the clinical risks and hemodynamic consequences of a prophylactic aortic arch replacement strategy in Loeys-Dietz syndrome and characterizes smooth muscle cell phenotype in Loeys-Dietz syndrome aneurysmal and normal-sized downstream aorta. MethodsPatients with genetically confirmed Loeys-Dietz syndrome (n = 8) underwent prophylactic aortic arch replacement during valve-sparing aortic root replacement. Four-dimensional flow magnetic resonance imaging studies were performed in 4 patients with Loeys-Dietz syndrome (valve-sparing aortic root replacement + arch) and compared with patients with contemporary Marfan syndrome (valve-sparing aortic root replacement only, n = 5) and control patients (without aortopathy, n = 5). Aortic tissues from 4 patients with Loeys-Dietz syndrome and 2 organ donors were processed for anatomically segmented single-cell RNA sequencing and histologic assessment. ResultsPatients with Loeys-Dietz syndrome valve-sparing aortic root replacement + arch had no deaths, major morbidity, or aortic events in a median of 2 years follow-up. Four-dimensional magnetic resonance imaging demonstrated altered flow parameters in patients with postoperative aortopathy relative to controls, but no clear deleterious changes due to arch replacement. Integrated analysis of aortic single-cell RNA sequencing data (>49,000 cells) identified a continuum of abnormal smooth muscle cell phenotypic modulation in Loeys-Dietz syndrome defined by reduced contractility and enriched extracellular matrix synthesis, adhesion receptors, and transforming growth factor-beta signaling. These modulated smooth muscle cells populated the Loeys-Dietz syndrome tunica media with gradually reduced density from the overtly aneurysmal root to the nondilated arch. ConclusionsPatients with Loeys-Dietz syndrome demonstrated excellent surgical outcomes without overt downstream flow or shear stress disturbances after concomitant valve-sparing aortic root replacement + arch operations. Abnormal smooth muscle cell–mediated aortic remodeling occurs within the normal diameter, clinically at-risk Loeys-Dietz syndrome arch segment. These initial clinical and pathophysiologic findings support concomitant arch replacement in Loeys-Dietz syndrome.

Suppression of pancreatic ductal adenocarcinoma growth and metastasis by fibrillar collagens produced selectively by tumor cells

Pancreatic ductal adenocarcinoma (PDAC) has a collagen-rich dense extracellular matrix (ECM) that promotes malignancy of cancer cells and presents a barrier for drug delivery. Data analysis of our published mass spectrometry (MS)-based studies on enriched ECM from samples of progressive PDAC stages reveal that the C-terminal prodomains of fibrillar collagens are partially uncleaved in PDAC ECM, suggesting reduced procollagen C-proteinase activity. We further show that the enzyme responsible for procollagen C-proteinase activity, bone morphogenetic protein1 (BMP1), selectively suppresses tumor growth and metastasis in cells expressing high levels of COL1A1. Although BMP1, as a secreted proteinase, promotes fibrillar collagen deposition from both cancer cells and stromal cells, only cancer-cell-derived procollagen cleavage and deposition suppresses tumor malignancy. These studies reveal a role for cancer-cell-derived fibrillar collagen in selectively restraining tumor growth and suggest stratification of patients based on their tumor epithelial collagen I expression when considering treatments related to perturbation of fibrillar collagens.

Integrated bioinformatics analysis identified COL11A1 as an immune infiltrates correlated prognosticator in pancreatic adenocarcinoma

Pancreatic adenocarcinoma (PAAD) is the most common pancreatic cancer, with high mortality rate and limited treatment options. Tumor infiltrating cells and genes in microenvironment are emerging as pivotal players in PAAD progression and prognosis. In this study, we obtained genes expression data set GSE119794 of PAAD, which contains data from 10 tumor and 10 normal samples. A total of 262 differentially expressed genes (DEGs), including 169 up-regulated and 93 down-regulated genes, were obtained based on expression fold change and significance. Combining the pathway analysis of DEGs and GSEA analysis of all genes, four KEGG pathways were enriched. The 4 pathways include pancreatic secretion, protein digestion and absorption, fat digestion and absorption, and PPAR signaling pathways. Functional enrichment of Gene Ontology significantly enriched extracellular matrix, an important component in microenvironment. In the Protein-protein interaction (PPI) network, we screened out 3 hub genes of COL11A1, KRT19 and CXCL5 by CytoHubba. At last, the expression level, prognostic significance and correlation with tumor infiltrates were validated in TCGA database, with GEPIA and TIMER. The validation identified Collagen Type XI Alpha 1 Chain (COL11A1), an extracellular matrix structural constituent, as a hazardous prognosticator with significant correlation with macrophage, neutrophil and dendritic cells. In sum, we identified COL11A1 as an immune infiltrates correlated prognosticator in pancreatic adenocarcinoma.

Alveolar Extracellular Matrix Hydrogels Facilitate Proliferation of Induced Pluripotent Stem Cell-Derived Alveolar Epithelial Spheroids

Background & Aim Type 2 alveolar epithelial cells (AEC2s) are adult cells in the human lung that have a critical role in lung homeostasis. In addition to secreting surfactant that is necessary to prevent alveolar collapse, AEC2s are the facultative progenitors of the alveolus. Specifically, in response to injury AEC2s proliferate to replenish AEC2s and type 1 alveolar epithelial cells (AEC1s). However, despite the critical role of AEC2s in lung physiology and injury response, an inability to isolate an expandable culture of AEC2 in vitro has impeded a full understanding of AEC2 biology. Recently, a pure and expandable culture of induced-AEC2s (iAEC2s) spheroids (i.e. alveolospheres) has been derived using a directed differentiation method from induced pluripotent stem cells (iPSCs). These iAEC2s spheroids are amenable to prolonged culture periods, possess mature markers and functionality (e.g. surfactant secretion) of in vivo AEC2s, and can recapitulate AEC2 dysfunction. However, initial studies of iAEC2s were performed in Matrigel, an extracellular matrix (ECM) derived from a tumorigenic mouse cell line, and it is unknown how iAEC2s respond to a physiologically relevant lung matrix. Methods, Results & Conclusion Here, we employ a novel and innovative culture method to determine how iAEC2s respond to human alveolar ECM. Decellularized lungs from adult patients with a healthy lung history were used to produce 3-dimensional hydrogels from alveolar enriched ECM (aECM) for the culture of iAEC2s. Our preliminary results provide evidence that iAEC2s maintain alveolosphere formation and efficiently proliferate in aECM hydrogels in an aECM protein concentration dependent manner. Furthermore, gene expression analysis suggests that aECM promotes lamellar body associated AEC2 maturation markers (i.e. SFTPC, LAMP3, ABCA3) in iAEC2s. Importantly, the ability to culture iAEC2s in a physiologically relevant lung matrix could provide a unique model to determine the effects of normal and diseased lung ECM (e.g. idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease) on AEC2 behavior.

Increased Baseline YAP/TAZ Nuclear Localization and Altered Transcriptional Activity in Uterine Fibroids

BackgroundUterine leiomyomas, also known as fibroids (UF), are benign neoplasms of the uterus. UF consist of smooth muscle cells and fibroblasts, and display enriched extracellular matrix (ECM). Reports estimate a cumulative incidence of 70% in premenopausal women, with annual total health care costs attributed to UF exceeding 34 billion dollars in the United States alone. In spite of their prevalence, the underlying processes that drive UF development and growth are still poorly understood. Accumulating evidence implicates the canonical Hippo signaling pathway effectors Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding domain (TAZ) as key drivers of fibroblast biology. Thus, we seek to assess the differences in YAP/TAZ localization, ECM deposition, and gene expression between UF and normal myometrium (Myo).MethodsMatched samples of UF and Myo were obtained from premenopausal women with IRB approval. Immunohistochemistry (N=5) was performed on OCT embedded tissue with YAP/TAZ antibody. For cell culture, UF and Myo were minced and digested with collagenase type 4. Cells (passages 1–4) were plated on plastic at confluence (N=4 for both). Immunostaining for YAP/TAZ, Fibronectin (FN), Collagen Type I (COL1), and Collagen Type III (COL3) were performed (N=4–5). Transcript levels were analyzed by treating cells with control or YAP/TAZ siRNA, and performing subsequent cDNA synthesis and qPCR reactions (N=4). Kruskall‐Wallis and Mann Whitney tests were used as appropriate.ResultsYAP/TAZ nuclear localization was upregulated in UF when compared to Myo tissue by immunofluorescence imaging. In freshly isolated cells cultured at confluence on tissue culture plastic, baseline YAP/TAZ nuclear localization was increased in UF when compared to Myo (p=0.03). UFs also exhibited a trend toward increased FN, COL1, and COL3 deposition when compared to Myo. UF displayed increased transcript levels for several matrix and pro‐fibrotic signaling genes (COL1A1, COL3A1, CTGF, EDN1, FN1) compared to the matched Myo. Following siRNA treatment for YAP/TAZ gene expression was reduced 40% and 50% for EDN1 (encoding endothelin‐1), as well as 70% and 65% for CTGF (encoding connective tissue growth factor), in UF and Myo respectively.ConclusionsUF nuclear YAP/TAZ is increased at baseline in vivo as well as in vitro. UF also display upregulated deposition of the ECM proteins. Additionally, there are increased transcript levels of profibrotic genes in UF compared to Myo. Moreover, the observed decreases in gene expression following siRNA knockdown of YAP/TAZ suggests a role for the Hippo signaling pathway in regulation of certain profibrotic genes in UF. Together these results suggest that enhanced baseline YAP/TAZ localization in UF may drive upregulated expression of pro‐fibrotic genes promoting UF progression.Support or Funding InformationCenter for Biomedical Discovery‐Department of Obstetrics and Gynecology Team Science Pilot Award ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Defining the Role of Type I Interferon Signaling in the Increased Accumulation of Versican in Lungs of Mice Exposed to Influenza Infection

The accumulation of versican in the extracellular matrix (ECM) has been described as part of the innate immune response to viral lung infection. However, the mechanism(s) regulating versican expression during influenza infection are not understood. Previous work has identified both the β‐catenin/T‐cell factor pathway and the TLR/Trif/Type I interferon (IFN) receptor pathway as controlling versican expression in stromal cells and macrophages respectively. The goal of this study, is to investigate the role of Type I IFN receptor signaling in directing versican accumulation in the lungs during influenza infection.Versican is a proteoglycan that plays an immunomodulatory role in the innate immune response. During influenza infection, creation of a versican enriched ECM coincides with retention of leukocytes in lungs. Our published studies show that versican impacts leukocyte phenotype; macrophage‐derived versican has anti‐inflammatory properties; Type I IFN receptor signaling pathway directs versican synthesis in macrophages; and versican is necessary for Type I IFN production by macrophages. Based on these findings, we hypothesized that IFNβ expression levels should increase prior to versican expression and accumulation and coincide with significant influenza disease burden.We used qPCR, immunohistochemistry, and quantitative digital pathology of whole lung tissue to evaluate the lungs of C57Bl6/J mice infected with H1N1 A/PR/8/34 influenza. Tissues were harvested on days 3, 6, 9 and 12 post instillation to determine changes in cytokine and versican expression and versican accumulation overtime. Analysis showed that versican mRNA expression was bimodal with significant increases on days 3 and 9. However, significant increases in immunostaining for the versican β‐glycosaminoglycan domain was seen on days 6, 9, and 12 and corresponded to significant weight loss in mice on days 6 and 9. To better understand the involvement of Type I IFN directed expression of versican, we measured IFNβ expression levels and found that they were significantly increased on day 6, after the initial increase in versican mRNA expression on day 3 and coinciding with the primary increase in versican tissue accumulation. These studies suggest that Type I IFN signaling plays an important role in the expression and accumulation of versican on day 9 after influenza infection.Taken together with what is known about versican regulation by Type I IFNs, these findings suggest that the Type I IFN pathway may be important in the regulation of versican expression late in influenza disease progression. Studies in conditional cell specific versican deficient mice will clarify the role of Type I IFNs in regulating versican expression; the cellular source of versican expression following activation of Type I IFN receptors; and the role of versican in modulating the immune response to influenza infection. Additional studies are needed to determine whether β‐catenin signaling also contributes to pulmonary versican accumulation following influenza infection.Support or Funding InformationNIH/NIAID 1R01AI136468‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Basement membrane extract attenuates the more malignant gene expression profile accentuated by fibronectin in prostate cancer cells.

Prostate cancer (PCa) has high mortality rates, with most of the deaths resulting from the development of metastasis. Fibronectin (FN) plays key roles in cell adhesion and affects the migratory behavior of cells. In the tumor microenvironment and also in the blood plasma during metastasis, FN displays increased expression, however its role in prostate cancer remains poorly understood. This study aimed to unveil the specific roles of FN as a soluble component, alone or in combination with a complex basement membrane. To investigate the impact of FN in neoplastic prostate cells, we evaluated the gene expression of LNCaP cells by RT-qPCR after exposure to soluble FN (25µg/mL) either alone or in combination with a basement membrane. When FN was the predominant matrix element, such as in blood plasma, PCa tumor cells increased their expression of genes related to an invasive behavior and resistance to apoptosis, including CDH2, ITGA5, AKT1, and BCL2. However, the combined presence of FN and a complex basement membrane had the opposite effect on LNCaP cells, in which the expression levels of CDH2, ITGA5, AKT1, and BCL2 were reduced. Hierarchical clustering analysis with LNCaP and RWPE-1 cells showed that LNCaP cells exposed to an enriched extracellular matrix displayed an expression pattern more similar to that shown by RWPE-1 cells, a cell line that illustrates characteristics of the normal prostate epithelium. These findings provide the groundwork for future studies addressing the role of FN in tumor growth, particularly in the context of cancer evolution/progression from a solid primary tumor to a transitory circulating state.

Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization.

The extracellular matrix (ECM) is readily enriched by decellularizing tissues with nondenaturing detergents to solubilize and deplete the vast majority of cellular components. This approach has been used extensively to generate ECM scaffolds for regenerative medicine technologies and in 3D cell culture to model how the ECM contributes to disease progression. A highly enriched ECM fraction can then be generated using a strong chaotrope buffer that is compatible with downstream bottom-up proteomic analysis or 3D cell culture experiments after extensive dialysis. With most tissues, an insoluble pellet remains after chaotrope extraction that is rich in structural ECM components. Previously, we showed that this understudied fraction represented approximately 80% of total fibrillar collagen from the lung and other ECM fiber components that are known to be covalently cross-linked. Here, we present a hydroxylamine digestion approach for chaotrope-insoluble ECM analysis with comparison to an established CNBr method for matrisome characterization. Because ECM characteristics vary widely among tissues, we chose five tissues that represent unique and diverse ECM abundances, composition, and biomechanical properties. Hydroxylamine digestion is compatible with downstream proteomic workflows, yields high levels of ECM peptides from the insoluble ECM fraction, and reduces analytical variability when compared to CNBr digestion. Data are available via ProteomeXchange with identifier PXD006428.

Fabrication of a matrigel–collagen semi-interpenetrating scaffold for use in dynamic valve interstitial cell culture

The study of heart valve homeostatic and disease mechanisms are often limited by the challenges in simulating the in vivo milieu, where valve cells are surrounded by the extracellular matrix in a three-dimensional (3D) environment and experience multiple dynamic mechanical forces. Type I collagen is typically the most common 3D matrix used to culture valve cells in vitro. Unfortunately, this material has poor mechanical behavior due to an inherent propensity to compact significantly, unlike native valve leaflets. We hypothesized that incorporation of matrigel, which contains other heart valve-relevant matrix components such as type IV collagen and sulfated proteoglycans, to type I collagen would provide an appropriate physiological milieu for in vitro valve interstitial cell culture. Different semi-interpenetrating mixtures of collagen type I and matrigel were prepared and a thorough characterization of their physical, mechanical and biocompatibility properties was performed. We observed that the matrigel–collagen hydrogel was porous and degradable with tunable swelling behavior. Incorporation of matrigel not only enhanced the mechanical behavior of the composite hydrogel but also presented the cultured valve interstitial cells with a more enriched extracellular matrix network for in vitro culture. We showed that cells cultured in the composite hydrogel had comparable viability, proliferation and cell phenotype as compared with those in a collagen only gel. Importantly, the composite hydrogel was also amenable to in vitro cyclic stretching culture for 48 h. Overall, we report here the potential use of the matrigel–collagen hydrogel as a three dimensional scaffold for the dynamic mechanical culture of valve interstitial cells in vitro.

The Sec domain protein Scfd1 facilitates trafficking of ECM components during chondrogenesis

Chondrogenesis in the developing skeleton requires transformation of chondrocytes from a simple mesenchymal condensation to cells with a highly enriched extracellular matrix (ECM). This transition is in part accomplished by alterations in the chondrocyte protein transport machinery to cope with both the increased amount and large size of ECM components. In a zebrafish mutagenesis screen to identify genes essential for cartilage development, we uncovered a mutant that disrupts the gene encoding Sec1 family domain containing 1 (scfd1). Homozygous scfd1 mutant embryos exhibit a profound craniofacial abnormality caused by a failure of chondrogenesis. Loss of scfd1 was found to hinder ER to Golgi transport of ECM proteins and is accompanied with activation of the unfolded protein response in chondrocytes. We further demonstrate a conserved role for Scfd1 in differentiation of mammalian chondrocytes, in which loss of either SCFD1 or STX18, a SLY1 interacting t-SNARE, severely impair transport of type II collagen. These results show that the existence of a specific export pathway, mediated by a complex containing SCFD1 and STX18 that plays an essential role in secretion of large ECM proteins during chondrogenesis.

Abstract 1974: Ovulatory wound: the site of origin for ovarian serous carcinoma

Abstract Background: New clinical and molecular studies suggest that HGSOC originates from the neighboring fallopian tubes, rather than the surface epithelial layer of the ovary. However, very little is known about the process of tubal malignant cells migrating and implanting into ovary. Our research focuses on understanding how the extraovarian malignant cells implant into the ovary and how ovulation affects the process of their migration and implantation. Method: Tumor initiating cells (TICs) isolated from human HGSOC were labeled by red fluorescent protein (RFP)-expressing lentivirus and subcutaneously or intrauterine injected to immunocompromised mice. Hormones (PMS and HCG) were interperitoneally injected to induce superovulation in mice. In the in vitro models, TICs were cultured with the granulosa cell conditional medium and the extra cellular matrix (ECM) extracted from ovarian stroma cells to evaluate their effects on the migration and attachment of TICs Result: Superovulation increased the ovarian tumor-forming rate from 38% (15/39 in control group) to 100% (24/24 in supervolation group). The increased frequency of ovulation enhances the formation of ovarian tumors in mouse models. In the intrauterine injection model, we were able to trace the migration of TICs towards ovaries and capture the early implantation of TICs inside ovaries in 5-10 days after the injection. We detected stage I ovarian tumors in mice. The tumors are encapsulated in the ovary and covered by the intact surface epithelial layer. These tumor cells closely interacted with the stroma of ovary, particularly the structure of corpus luteum. The granulosa cells secreted stromal cell-derived factor 1 (SDF-1, also known as CXCL12). SDF-1 attracts TICs that express CXCR4 (the receptor of SDF-1) to ovulatory wounds. Upon ovulation the ovulatory wounds expose the stroma of ovary, which provides a collagen enriched ECM for TICs to attach. Conclusion: The unique ovulatory wound microenvironment attracts the malignant cells to migrate towards the ovary and creates access for the malignant cells to implant in the collagen-enriched ECM in the stroma of ovary. The new mechanisms revealed by our data may explain why increased ovulation is associated with epithelial ovarian cancer. Our findings support the “extra-ovarian origin” theory and will help us better understand the initiation and progression of HGSOC. Citation Format: Yang Yang-Hartwich, Marta Gurrea, Natalia Sumi, Jennie Holmberg, Vinicius Craveiro, Ayesha Alvero, Gil Mor. Ovulatory wound: the site of origin for ovarian serous carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1974. doi:10.1158/1538-7445.AM2014-1974

Hypertensive Rat Lungs Retain Hallmarks of Vascular Disease upon Decellularization but Support the Growth of Mesenchymal Stem Cells

There are an insufficient number of donor organs available to meet the demand for lung transplantation. This issue could be addressed by regenerating functional tissue from diseased or damaged lungs that would otherwise be deemed unsuitable for transplant. Detergent-mediated whole-lung decellularization produces a three-dimensional natural scaffold that can be repopulated with various cell types. In this study, we investigated the decellularization and initial recellularization of diseased lungs using a rat model of monocrotaline-induced pulmonary hypertension (MCT-PHT). Decellularization of control and MCT-PHT Sprague-Dawley rat lungs was accomplished by treating the lungs with a combination of Triton X-100, sodium deoxycholate, NaCl, and DNase. The resulting acellular matrices were characterized by DNA quantification, Western blotting, immunohistochemistry, and proteomic analyses revealing that decellularization was able to remove cells while leaving the extracellular matrix (ECM) components and lung ultrastructure intact. Decellularization significantly reduced DNA content (∼30-fold in MCT-PHT lungs and ∼50-fold in the control lungs) and enriched ECM components (>60-fold in both the control and MCT-PHT lungs) while depleting cellular proteins. MicroCT visualization of MCT-PHT rat lungs indicated that the vasculature was narrowed as a result of MCT treatment, and this characteristic was unchanged by decellularization. Mean arterial vessel diameter of representative decellularized MCT-PHT and control scaffolds was estimated to be 0.152±0.134 mm and 0.247±0.160 mm, respectively. Decellularized MCT-PHT lung scaffolds supported attachment and survival of rat adipose-derived stem cells (rASCs), seeded into the airspace or the vasculature, for at least 2 weeks. The cells seeded in MCT-PHT lung scaffolds proliferated and underwent apoptosis similar to control scaffolds; however, the initial percentage of apoptotic cells was slightly higher in MCT-PHT lungs (2.79±2.03% vs. 1.05±1.02% of airway-seeded rASCs, and 4.47±1.21% vs. 2.66±0.10% of vascular seeded rASCs). The ECM of cell-seeded scaffolds showed no signs of degradation by the cells after 14 days in culture. These data suggest that diseased hypertensive lungs can be efficiently decellularized similar to control lungs and have the potential to be recellularized with mesenchymal stem cells with the ultimate goal of generating healthy, functional pulmonary tissue.

Global Analysis Reveals the Complexity of the Human Glomerular Extracellular Matrix

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456.

Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage

Cancer stem-like cell subpopulations, referred to as "side-population" (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, alpha-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into alpha-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.

Metabolic Dysregulation and Adipose Tissue Fibrosis: Role of Collagen VI

Adipocytes are embedded in a unique extracellular matrix whose main function is to provide mechanical support, in addition to participating in a variety of signaling events. During adipose tissue expansion, the extracellular matrix requires remodeling to accommodate adipocyte growth. Here, we demonstrate a general upregulation of several extracellular matrix components in adipose tissue in the diabetic state, therefore implicating "adipose tissue fibrosis" as a hallmark of metabolically challenged adipocytes. Collagen VI is a highly enriched extracellular matrix component of adipose tissue. The absence of collagen VI results in the uninhibited expansion of individual adipocytes and is paradoxically associated with substantial improvements in whole-body energy homeostasis, both with high-fat diet exposure and in the ob/ob background. Collectively, our data suggest that weakening the extracellular scaffold of adipocytes enables their stress-free expansion during states of positive energy balance, which is consequently associated with an improved inflammatory profile. Therefore, the disproportionate accumulation of extracellular matrix components in adipose tissue may not be merely an epiphenomenon of metabolically challenging conditions but may also directly contribute to a failure to expand adipose tissue mass during states of excess caloric intake.