Extracellular Matrix Matrigel Research Articles

An Extracellular Matrix Overlay Model for Bioluminescence Microscopy to Measure Single-Cell Heterogeneous Responses to Antiandrogens in Prostate Cancer Cells.

Prostate cancer (PCa) displays diverse intra-tumoral traits, impacting its progression and treatment outcomes. This study aimed to refine PCa cell culture conditions for dynamic monitoring of androgen receptor (AR) activity at the single-cell level. We introduced an extracellular matrix-Matrigel (ECM-M) culture model, enhancing cellular tracking during bioluminescence single-cell imaging while improving cell viability. ECM-M notably tripled the traceability of poorly adherent PCa cells, facilitating robust single-cell tracking, without impeding substrate permeability or AR response. This model effectively monitored AR modulation by antiandrogens across various PCa cell lines. Single-cell imaging unveiled heterogeneous antiandrogen responses within populations, correlating non-responsive cell proportions with drug IC50 values. Integrating ECM-M culture with the PSEBC-TSTA biosensor enabled precise characterization of ARi responsiveness within diverse cell populations. Our ECM-M model stands as a promising tool to assess heterogeneous single-cell treatment responses in cancer, offering insights to link drug responses to intracellular signaling dynamics. This approach enhances our comprehension of the nuanced and dynamic nature of PCa treatment responses.

Abstract 2088: Targeting transcriptional factor IWS1 as a potential treatment of advanced dedifferentiated liposarcoma

Abstract Introduction. Surgery remains the only curative treatment for patients with liposarcoma (LPS). The translation of novel therapies has been hampered, and there has been no significant improvement in prognosis for 30 years. Our recent findings demonstrate that IWS1, a transcription elongation and alternative splicing factor, is elevated in dedifferentiated LPS (ddLPS) patient tumor samples and is associated with worse survival and a shorter time to relapse in ddLPS. In this study, we present preliminary results on a novel treatment for advanced ddLPS: an IWS1 inhibitor, serving as a way to discover a spliceosome as a novel therapeutic target. Methods. Utilizing computer-aided, structure-based virtual screening approaches, we identified a class of compounds that exhibited inhibitory potential. A subset of FDA-approved drugs was evaluated by molecular docking calculations, and compounds with the highest calculated theoretical potency were selected for further analysis. To assess the ability of specific compounds to disrupt IWS1/Spt6 binding, Co-immunoprecipitation (Co-IP) experiments on ddLPS cell line Lipo863 were conducted. Transwell invasion and migration assays were performed to evaluate the effect of the drug candidate on the metastatic potential of cells. Additionally, to evaluate the impact of IWS1 inhibition on spheroid formation, a matrigel extracellular matrix scaffold was employed. Results. Through virtual screening, we identified a class of compounds characterized by a specific chemical pattern featuring a tricyclic fused-ring structure. This class of compounds includes the well-known FDA-approved drug Ketotifen, an antihistamine and mast cell stabilizer. Co-immunoprecipitation of IWS1/Spt6 complex after 48-hour treatment with Ketotifen showed a decrease of over 50% of the isolated complex. Migration and invasion of the ddLPS cell line, Lipo863, were reduced in a dose-dependent manner by 18-44% and 17-52%, respectively, when treated with Ketotifen, without affecting cell viability. In a 3D model, a 14-day treatment of Ketotifen significantly reduced spheroid formation, confirming its impact on tumor growth and cell proliferation. Conclusion. Ketotifen effectively disrupted IWS1/Spt6 complex in vitro. Importantly, it did not affect cell viability while reducing the migratory and invasive potential of ddLPS. The dose-dependent effect of Ketotifen suggests that there may be an optimal in vivo concentration to prevent cells from invading nearby structures and/or metastasizing. We are currently testing the ability of these compounds in preclinical mouse models of ddLPS. Citation Format: Marina Goryunova, Yiran He, Nipin Sp, Elizaveta K. Titerina, Sayumi Tahara, Carson Karakis, Alessandro La Ferlita, Michael Sorkin, Alex Kim, Hua Zhu, Christopher M. Hadad, Raphael E. Pollock, Joal D. Beane. Targeting transcriptional factor IWS1 as a potential treatment of advanced dedifferentiated liposarcoma [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 2088.

Phenotypic Switch and Growth of Melanoma Spheroids in the Presence of Mast Cells: Potential Impact of Nutrient-starvation Effects.

Mast cells are abundant in melanoma tumors, and studies suggest that they can be either detrimental or protective for melanoma growth. However, the underlying mechanisms are not fully understood. Here, we adopted an established hanging-drop spheroid system to investigate how mast cells influence melanoma growth and phenotype in a 3-D context. To address the underlying mechanism, we conducted transcriptomic and pathway analyses. In the presence of mast cells or mast cell-conditioned medium, growth of melanoma spheroids was profoundly reduced. Transcriptomic analysis revealed that mast cell-conditioned medium had extensive effects on the gene-expression patterns of melanoma. Pathway analyses revealed profound effects on the expression of genes related to amino acid and protein metabolism. The conditioned medium also induced up-regulation of cancer-related genes, including adhesion molecules implicated in metastatic spreading. In line with this, after transfer to a Matrigel extracellular matrix milieu, spheroids that had been developed in the presence of mast cell-conditioned medium displayed enhanced growth and adhesive properties. However, when assessing the possible impact of nutrient starvation, i.e., reduced nutrient content in mast cell-conditioned medium, we found that the observed effects on growth of melanoma spheroids could potentially be explained by such a scenario. Our findings suggest that the phenotypic alterations of melanoma spheroids grown in the presence of mast cells or mast cell-conditioned media are, at least partly, due to nutrient starvation rather than to the action of factors secreted by mast cells. Our findings may provide insight into the effects on gene-expression events that occur in melanoma tumors under nutrient stress.

Three dimensional models of dedifferentiated liposarcoma cell lines: scaffold-based and scaffold-free approaches.

Sarcomas are rare malignancies, the number of reports is limited, and this rarity makes further research difficult even though liposarcoma is one of major sarcomas. 2D cell culture remains an important role in establishing basic tumor biology research, but its various shortcomings and limitations are still of concern, and it is now well-accepted that the behavior of 3D-cultured cells is more reflective of in vivo cellular responses compared to 2D models. This study aimed to establish 3D cell culture of liposarcomas using two different methods: scaffold-based (Matrigel extracellular matrix [ECM] scaffold method) and scaffold-free (Ultra-low attachment [ULA] plate). Lipo246, Lipo224 and Lipo863 cell lines were cultured, and distinctive differences in structures were observed in Matrigel 3D model: Lipo224 and Lipo863 formed spheroids, whereas Lipo246 grew radially without forming spheres. In ULA plate approaches, all cell lines formed spheroids, but Lipo224 and Lipo863 spheroids showed bigger size and looser aggregation than Lipo246. Formalin fixed, paraffin embedded (FFPE) blocks were obtained from all 3D models, confirming the spheroid structures. The expression of MDM2, Ki-67 positivity and MDM2 amplification were confirmed by IHC and DNAscope™, respectively. Protein and DNA were extracted from all samples and MDM2 upregulation was confirmed by western blot and qPCR analysis. After treatment with MDM2 inhibitor SAR405838, DDLPS spheroids demonstrated different sensitivity patterns from 2D models. Taken together, we believed that 3D models would have a possibility to provide us a new predictability of efficacy and toxicity, and considered as one important process in in vitro pre-clinical phase prior to moving forward to clinical trials.

Effect of LOXL2 on metastasis through remodeling of the cell surface matrix in non-small cell lung cancer cells

Lung cancer is the prominent cause of cancer-associated death primarily because of distant metastatic disease. The metastatic potential of non-small cell lung cancer (NSCLC) is associated with tumor cell aggregation. However, the systemic mechanotransduction mechanism by which tumor cells dynamically aggregate and disseminate is poorly understood, especially in NSCLC. In this study, we examine whether the cell surface matrix plays an important role in metastasis. We used poly-2-hydroxyethyl methacrylate-based 3D spheroid formation methods to mimic in vivo metastatic lesions. Supra-structural analysis of human NSCLC A549 cells stained with ruthenium red for transmission electron microscopy (TEM) showed that glycocalyx surrounding the cell surface in 2D culture decreases in 3D culture. Comprehensive gene expression analysis revealed that the genes associated with cell adhesion were distinctly enriched in A549 cell spheroids. Of these, downregulation of the tumor metastatic microenvironment facilitator LOXL2, a copper-dependent enzyme catalyzing posttranslational oxidative deamination of peptidyl lysine, was of special interest. Knockdown of LOXL2 thickened the cell surface matrix in 2D culture and impaired compact aggregate formation in 3D culture. Moreover, A549 cell spheroids with endogenous overexpression of LOXL2 increased their dissemination on basement extracellular matrix Matrigel. Overall, these data imply that cell detachment-downregulated LOXL2 contributes to cell surface matrix remodeling, leading to collective dissemination of free-floating aggregates.

Ortho-coumaric acid derivatives with therapeutic potential in a three-dimensional culture of the immortalised U-138 MG glioblastoma multiforme cell line

IntroductionThe treatment of such primary brain tumours such as glioblastoma multiforme (GBM) presents numerous difficulties, considerably impacting patient survival rates. The search for new compounds with therapeutic potential focuses on the identification of molecules that can be produced through chemical and enzymatic processes and that present anticancer effects in different types of tumours. MethodsGiven the need for new treatments for GBM, we conducted a study to synthesise ortho-coumaric acid alkyl esters (OCAAE) and to evaluate them in an appropriate study model, a three-dimensional (3D) culture using the Matrigel extracellular matrix. We tested cytotoxicity with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) technique and by determining phosphatidylserine externalisation with annexin V and active caspase-3 expression as markers of apoptosis, and the subsequent degradation of DNA as a marker of cell death. ResultsMedium-chain OCAAEs, such as butyl-o-coumarate (BOC) and its isomer, showed the greatest effect on most of the parameters evaluated in 3D cultures of GBM, followed by methyl-, propyl-, and ethyl-o-coumarate (MOC, POC and EOC); these compounds showed effects both in reducing cell viability and in increasing the expression of active caspase-3 and annexin V and the degradation of DNA, in comparison with the first-line treatment for GBM, temozolomide. ConclusionThese results, together with those of previous researchers, show that the different OCAAEs have a potential therapeutic effect on GBM, and that 3D culture of GBM cells is an appropriate model for the study of new antitumour molecules.

Three-Dimensional Culture of Ameloblast-Originated HAT-7 Cells for Functional Modeling of Defective Tooth Enamel Formation.

Background: Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca2+, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization. Aims: Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation. Methods: To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca2+ and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF. Results: A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis. Conclusion: In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca2+ signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure.

Electrokinetic Perfusion Through Three-Dimensional Culture Reduces Cell Mortality

Cell proliferation and survival are dependent on mass transfer. In vivo, fluid flow promotes mass transfer through the vasculature and interstitial space, providing a continuous supply of nutrients and removal of cellular waste products. In the absence of sufficient flow, mass transfer is limited by diffusion and poses significant challenges to cell survival during tissue engineering, tissue transplantation, and treatment of degenerative diseases. Artificial perfusion may overcome these challenges. In this work, we compare the efficacy of pressure driven perfusion (PDP) with electrokinetic perfusion (EKP) toward reducing cell mortality in three-dimensional cultures of Matrigel extracellular matrix. We characterize electro-osmotic flow through Matrigel to identify conditions that generate similar interstitial flow rates to those induced by pressure. We also compare changes in cell mortality induced by continuous or pulsed EKP. We report that continuous EKP significantly reduced mortality throughout the perfusion channels more consistently than PDP at similar flow rates, and pulsed EKP decreased mortality just as effectively as continuous EKP. We conclude that EKP has significant advantages over PDP for promoting tissue survival before neovascularization and angiogenesis. Impact statement Interstitial flow helps promote mass transfer and cell survival in tissues and organs. This study generated interstitial flow using pressure driven perfusion (PDP) or electrokinetic perfusion (EKP) to promote cell viability in three-dimensional cultures. EKP through charged extracellular matrices possesses significant advantages over PDP and may promote cell survival during tissue engineering, transplantations, and treatment of degenerative diseases.

Reconstituting neurovascular unit with primary neural stem cells and brain microvascular endothelial cells in three‐dimensional matrix

Neurovascular dysfunction is a primary or secondary cause in the pathogenesis of several cerebrovascular and neurodegenerative disorders, including stroke. Therefore, the overall protection of the neurovascular unit (NVU) is a promising therapeutic strategy for various neurovascular diseases. However, the complexity of the NVU limits the study of the pathological mechanisms of neurovascular dysfunction. Reconstituting the in vitro NVU is important for the pathological study and drug screening of neurovascular diseases. In this study, we generated a spontaneously assembled three‐dimensional NVU (3D NVU) by employing the primary neural stem cells and brain microvascular endothelial cells in a Matrigel extracellular matrix platform. This novel model exhibits the fundamental structures and features of the NVU, including neurons, astrocytes, oligodendrocytes, vascular‐like structures, and blood–brain barrier‐like characteristics. Additionally, under oxygen‐glucose deprivation, the 3D NVU exhibits the neurovascular‐ or oxidative stress‐related pathological characteristics of cerebral ischemia and the injuries can be mitigated, respectively, by supplementing with the vascular endothelial growth factor or edaravone, which demonstrated that the availability of 3D NVU in ischemic stroke modeling. Finally, the 3D NVU promoted the angiogenesis and neurogenesis in the brain of cerebral ischemia rats. We expect that the proposed in vitro 3D NVU model will be widely used to investigate the relationships between angiogenesis and neurogenesis and to study the pathology and pharmacology of neurovascular diseases.

Abstract PO-092: Development of an organoid assay for studying racial disparity in pancreatic acinar ductal metaplasia

Abstract Blacks have higher rates of incidence and mortality of pancreatic cancer compared to Whites and non-White Hispanics. In an effort to identify a biological basis for this disparity, we propose examining the early events in the development of pancreatic cancer. The process by which pancreatic acini transdifferentiate into ductal epithelial cells (i.e. acinar ductal metaplasia or ADM) is one of the earliest events in the development of pancreatic ductal adenocarcinoma. We adapted the classic in vitro ADM assay, by which primary mouse pancreatic acini undergo ADM once plated onto the extracellular matrix matrigel, to an assay to culture and transdifferentiate human pancreatic organoids. We have received normal, human pancreatic acini from pancreatic islet transplantation centers from Black (n=8), White (n=19) and Hispanic (n=16) donors. The gender distribution was 29 males and 14 females. The acinar cells were cultured on matrigel and allowed to undergo ADM over a 5 to 6 day period. The number of ductal epithelial cells were microscopically counted and the rate of transdifferentiation was determined by kinetic modeling of the data. We have successfully cultured and transdifferentiated 40 of 43 human pancreatic acinar cells. Data from our study suggests that females undergo ADM at a faster rate than males and among the different races, the rate of acinar transdifferentiation decreases in the following order Blacks > Whites > non-White Hispanics. In summary, we demonstrate that women and Blacks have the highest rate of pancreatic transdifferentiation. Future studies include sequencing of the RNA isolated from the tissues to identify molecular drivers of acinar transdifferentiation and their impact on health disparities of pancreatic cancer. Citation Format: Alyssa Gosling, Jinmai Jiang, Corey M. Perkins, Yingwei Yao, Thomas D. Schmittgen. Development of an organoid assay for studying racial disparity in pancreatic acinar ductal metaplasia [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-092.

Microvesicles Produced by Natural Killer Cells Regulate the Formation of Blood Vessels.

We studied the effect of microvesicles derived from cells of the NK-92 cell line on the formation of tube-like structures by endothelial cells of the ЕА.Hy926 cell line. Microvesicles were isolated by differential centrifugation and their size was controlled by granulometric analysis using dynamic light scattering method. The effect of microvesicles produced by NK cells on angiogenesis was evaluated by cultural methods. In the course of the research, a model of co-culturing of microvesicles and endothelial cells on extracellular matrix Matrigel was developed. It was found that microvesicles derived from NK-92 cells promoted elongation of tube-like structures formed by endothelial ЕА.Hy926 cells. Microvesicles produced by NK cells can modulate functional activity of endothelial cells by affecting their ability to form blood vessels.

Abstract D112: Development of an organoid assay for studying racial disparity in pancreatic acinar ductal metaplasia

Abstract Blacks have a higher rate of incidence and mortality of pancreatic cancer compared to whites. In an effort to identify a biological basis for this disparity, we propose examining the early events in the development of pancreatic cancer. The process by which pancreatic acini transdifferentiate into ductal epithelial cells (i.e. acinar ductal metaplasia or ADM) is one of the earliest events in the development of pancreatic ductal adenocarcinoma. We adapted the classic in vitro ADM assay, by which primary mouse pancreatic acini undergo ADM once plated onto the extracellular matrix matrigel, to an assay to culture and transdifferentiate human pancreatic organoids. Human pancreatic acini was received from black (n=5), white (n=3) and Hispanic (n=6) donors from pancreatic islet transplantation centers. The gender distribution was 8 males and 6 females. The acinar cells were cultured on matrigel and allowed to undergo ADM over a 5 to 6 day period. The number of ductal epithelial cells were microscopically counted and the rate of transdifferentiation was determined. Using this technique, we successfully cultured and transdifferentiated 14 of 14 human pancreatic acinar cells. Preliminary data from our study suggests that females undergo ADM at a greater rate than males. Data on race was inconclusive due to the low number of samples. In summary, we have developed an assay to culture and study pancreatic ADM using human specimens. Future studies using increased sample size will investigate if racial disparity exists for pancreatic ADM. Citation Format: Julie Bray, Sihem Ait-Oudhia, Martha Campbell-Thompson, Thomas D Schmittgen. Development of an organoid assay for studying racial disparity in pancreatic acinar ductal metaplasia [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D112.

Sample preparation strategies for high-throughput mass spectrometry imaging of primary tumor organoids.

Patient-derived 3D organoids show great promise for understanding patient heterogeneity and chemotherapy response in human-derived tissue. The combination of organoid culture techniques with mass spectrometry imaging provides a label-free methodology for characterizing drug penetration, patient-specific response, and drug biotransformation. However, current methods used to grow tumor organoids employ extracellular matrices that can produce small molecule background signal during mass spectrometry imaging analysis. Here, we develop a method to isolate 3D human tumor organoids out of a Matrigel extracellular matrix into gelatin mass spectrometry compatible microarrays for high-throughput mass spectrometry imaging analysis. The alignment of multiple organoids in the same z-axis is essential for sectioning organoids together and for maintaining reproducible sample preparation on a single glass slide for up to hundreds of organoids. This method successfully removes organoids from extracellular matrix interference and provides an organized array for high-throughput imaging analysis to easily identify organoids by eye for area selection and further analysis. With this method, mass spectrometry imaging can be readily applied to organoid systems for preclinical drug development and personalized medicine research initiatives.

705 Cyclin-dependent kinase 9 (CDK9) as a therapeutic target in uveal and triple-wild type melanoma

Despite extraordinary gains in molecular therapies for melanoma, about half of all melanomas lack a “druggable” oncogene. One proposed approach for treating these therapeutically-orphaned tumors is by targeting transcriptional dependencies (“oncogene starvation”), whereby survival factors are depleted through inhibition of various transcriptional regulators. Considering the published success of MITF suppression through inhibition of CDK7, we sought to assess the utility of targeting CDK9, which regulates transcriptional elongation and which is being studied in various clinical trials. We undertook 2 strategies to inhibit CDK9- a PROTAC linked to thalidomide (THAL-SNS-032, TS-032) to rapidly degrade CDK9 and a selective CDK9 kinase inhibitor (NVP-2). We performed 2D cell viability studies using CellTiter-Glo luminescence in 2 uveal melanoma (UM), 1 triple-wild type melanoma (TWT), 6 BRAF and NRAS-driven melanoma, and 3 non-malignant fibroblast and melanocyte cell lines. At 500nM, both TS-032 and NVP-2 demonstrated greater suppression of UM and TWT melanomas with mean fractional survivals of 0.16 and 0.10, respectively, compared to 0.87 and 0.60 in BRAF/NRAS mutated melanomas (p = 0.0045 and 0.0005) and 0.64 and 0.77 in non-malignant controls (p = 0.0035 and 0.0009). The range of GI50 for TS-032 and NVP2 in UM and TWT was 23-107nM and 10-26nM compared to 300-1584nM and 96-130nM in BRAF/NRASmelanoma and 379-970nM and 742-2196nM in controls. These results were confirmed in a 3D culture system whereby cells were arranged in spheroids using Matrigel extracellular matrix and viability was visualized using operetta and LIVE/DEAD staining assay. Intracellular depletion of CDK9 was rapid (within 24 hrs). Our results suggest that transcriptional blockade through selective targeting of CDK9 is an effective method of suppressing melanomas which ostensibly lack single oncogenic drivers, such as UM and TWT tumors.

Chemically defined and xenogeneic-free differentiation of human pluripotent stem cells into definitive endoderm in 3D culture

In vitro differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) represents a key step towards somatic cells of lung, liver and pancreas. For future clinical applications, mass production of differentiated cells at chemically defined conditions and free of xenogeneic substances is envisioned. In this study we adapted our previously published two-dimensional (2D) DE induction protocol to three-dimensional (3D) static suspension culture in the absence of the xenogeneic extracellular matrix Matrigel. Next, fetal calf serum and bovine serum albumin present in the standard medium were replaced by a custom-made and xeno-free B-27. This yielded in a chemically defined and xenogeneic-free 3D culture protocol for differentiation of hPSCs into DE at efficiencies similar to standard 2D conditions. This novel protocol successfully worked with different hPSC lines including hESCs and hiPSCs maintained in two different stem cell media prior to differentiation. DE cells obtained by our novel BSA-free 3D protocol could be further differentiated into PDX1- or NKX6.1-expressing pancreatic progenitor cells. Notably, upon DE differentiation, we also identified a CXCR4+/NCAM+/EpCAMlow cell population with reduced DE marker gene expression. These CXCR4+/NCAM+/EpCAMlow cells emerge as a result of Wnt/beta-catenin hyperactivation via elevated CHIR-99021 concentrations and likely represent misspecified DE.

Inhibiting lncRNA ROR suppresses growth, migration and angiogenesis in microvascular endothelial cells by up-regulating miR-26.

Lower extremity arteriosclerosis is one of leading causes of death worldwide. Arteriosclerosis is closely related to microvascular endothelial cells. This study was aimed to explore the role of long non-coding RNA ROR in regulations of growth, migration, and angiogenesis of microvascular endothelial cells. Angiogenesis was determined by the number of tube-like cells on a matrigel extracellular matrix. Cell viability, apoptosis, and migration were determined by CCK-8 assay, PI/FITC-Annexin V staining method, and transwell assay, respectively. Relative RNA expression of ROR, miR-26, and angiogenesis-associated genes were analyzed by qRT-PCR. The protein expression of apoptosis- and angiogenesis-associated genes, as well as main factors in NF-κB and JAK1/STAT3 pathways, were analyzed by Western blot. LncRNA ROR silence inhibited viability, migration, and angiogenesis of HMEC-1 cells but promoted apoptosis of them. miR-26 expression was promoted after knocking down ROR expression. miR-26 overexpression enhanced the inhibitory effects of ROR silence on growth, migration, and angiogenesis in HMEC-1 cells, whereas, miR-26 silence impaired the effects of ROR silence. Finally, we found that NF-κB and JAK1/STAT3 signaling pathways were inhibited by ROR down-regulation. Similarly, miR-26 overexpression enhanced the inhibitory effect of ROR down-regulation on the pathways and miR-26 inhibition abrogated it. Down-regulating lncRNA ROR inhibited growth, migration and angiogenesis of microvascular endothelial cells possibly through up-regulation of miR-26. During this process, the activations of NF-κB and JAK1/STAT3 pathways were inhibited after interaction of ROR and miR-26.

Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF’s blood vessel forming potential prior to clinical implantation.

Abstract 2114: Targeting invasive and drug-resistant PDAC with photodynamic therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is associated with an extensive desmoplastic reaction and a characteristic extracellular matrix (ECM)-rich stromal microenvironment which is a major determinant of disease progression and therapeutic response. Using 3D cell culture models of PDAC, we recently showed that an invasion-promoting ECM composition leads to differential response to chemotherapy agents and photodynamic therapy (PDT), in which cytotoxic response is triggered by light activation of a photosensitizing agent. In particular, ECM-infiltrating populations with characteristic mesenchymal phenotype display a marked increase in sensitivity to PDT (using the photosensitizer verteporfin), while the same cells exhibit increased chemoresistance. Here, motivated by these findings we systematically disentangle influences associated with the physical and biochemical properties of ECM as regulators of PDAC growth behavior and treatment response. In 3D tumor spheroids transplanted into invasion-promoting type 1 collagen gels, we use riboflavin-mediated photocrosslinking as a tool to regulate ECM stiffness (independent of its concentration), combined with high-content imaging to co-register invasive progression and differential response to chemotherapy and PDT. Increased extent of photocrosslinking correlates with increased collagen stiffness as confirmed by bulk oscillatory rheology, and is associated with a decrease in the invasive velocity (migration speed directed away from the primary transplanted spheroid) of ECM-infiltrating PDAC cells. Preliminary comparisons of PDT and oxaliplatin chemotherapy in low and high collagen crosslinking conditions show that PDT is significantly more effective than oxaliplatin in ECM-invading populations regardless of collagen stiffness. We also compare oxaliplatin chemotherapy and PDT before and after PDAC invasion into Matrigel ECM, and find that PDT efficacy is only enhanced after extensive invasion into the surrounding matrix. These results support the efficacy of PDT in targeting locally invasive PDAC while stressing the importance of elucidating how both biochemical and biophysical ECM interactions regulate therapeutic susceptibilities. Citation Format: Gwendolyn Cramer, Seyedehrojin Jafari, Hamid El-Hamidi, Dustin Jones, Jonathan Celli. Targeting invasive and drug-resistant PDAC with photodynamic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2114. doi:10.1158/1538-7445.AM2017-2114

Scaffold-Free Coculture Spheroids of Human Colonic Adenocarcinoma Cells and Normal Colonic Fibroblasts Promote Tumorigenicity in Nude Mice

The aim of this study was to form a scaffold-free coculture spheroid model of colonic adenocarcinoma cells (CACs) and normal colonic fibroblasts (NCFs) and to use the spheroids to investigate the role of NCFs in the tumorigenicity of CACs in nude mice. We analysed three-dimensional (3D) scaffold-free coculture spheroids of CACs and NCFs. CAC Matrigel invasion assays and tumorigenicity assays in nude mice were performed to examine the effect of NCFs on CAC invasive behaviour and tumorigenicity in 3D spheroids. We investigated the expression pattern of fibroblast activation protein-α (FAP-α) by immunohistochemical staining. CAC monocultures did not form densely-packed 3D spheroids, whereas cocultured CACs and NCFs formed 3D spheroids. The 3D coculture spheroids seeded on a Matrigel extracellular matrix showed higher CAC invasiveness compared to CACs alone or CACs and NCFs in suspension. 3D spheroids injected into nude mice generated more and faster-growing tumors compared to CACs alone or mixed suspensions consisting of CACs and NCFs. FAP-α was expressed in NCFs-CACs cocultures and xenograft tumors, whereas monocultures of NCFs or CACs were negative for FAP-α expression. Our findings provide evidence that the interaction between CACs and NCFs is essential for the tumorigenicity of cancer cells as well as for tumor propagation.

Células madre limbocorneales: actualidades y aplicaciones terapéuticas

Stem cells are those poorly differentiated bodily cells capable of self-renewing and differentiating into specialized cells in the human body. They can be found in all self-renewing tissues like epithelia. It is well known that in corneal epithelium stem cells are housed in the limbal region, more concentrated in the upper and lower corneal limbus. Some of the markers that have been used to identify them are: p63, ABCG2, C/EBPd, Bmi1, Notch1, K19, vimentin, among others. Currently, research has been made on the use of limbal stem cells to treat conditions that involve their deficiency, such as: chemical or thermal damage to the ocular surface, Stevens-Johnson syndrome, and aniridia. To culture stem cells it is necessary to take a 1-2 mm2 biopsy from the sclerocorneal limbus which can be directly seeded or previously treated with trypsin and dispase to create a cell suspension. Feeder layers have been used as scaffold based on 3T3 fibroblasts, amniotic membrane, matrigel extracellular matrix, collagen, thermosensible gelation polymers, and anterior lens capsule. Culture media also vary, being the following the most used: Dulbecco's modified Eagle medium, Ham F12, MCDB151, EpiLife medium, PCT, etc. Various substrates have been studied to transport stem cells to the ocular surface. Amniotic membrane has been the most used to date, but others such as contact lenses, fibrin gels, and thermosensible biopolymers have also been reported