Presence Of Extracellular Matrix Research Articles

Microscopic study of cultural and diffusion kinetics of the effects of a prototype extracellular matrix on the photodynamic therapy of periopathogenic bacteria - a pilot study.

In dental medicine, photodynamic therapy is a promising treatment for bacterial infections. Oral biofilms, on the other hand, can produce an extracellular matrix that provides protection and stability against external forces.

Characterization and scanning electron microscopy of Malassezia species isolated from central line tips of neonates on total parenteral nutrition

Background: Aim of this study was to demonstrate colonization by Malassezia species in the central lines of very low birth weight newborns on total parental nutrition (TPN). We also aimed to perform scanning electron microscopy (SEM) of central line tip cut sections to document the quality of the biofilm, cell structure, micro-colony characteristics, and the presence of extracellular matrix. Methods: We collected central line tips of very low birth weight newborns (<1.5 kg) over a one-year period. We included a total of 63 samples, which were cultured on slants along with controls purchased from CBS Netherlands. We incubated the cultures at 32°C and observed them every two days for three weeks. Once growth occurred, we phenotypically identified the cultures and observed the central line tip cut sections with SEM. Results: Among the 63 central line tips, two (3.1%) were colonized by Malassezia. We observed a visible biofilm on the tips. We confirmed the phenotypic identification of the isolates as M. furfur and M. restricta by gene sequencing. Conclusions: Our study revealed Malassezia colonization in the central line used for total parenteral nutrition. Hence, it is important to have a high index of suspicion towards Malassezia catheter-related blood stream infection in newborns on TPN with a clinical picture of fever (in spite of antibiotic therapy), leukocytosis, thrombocytopenia with cardiac disease, and pulmonary infiltrates. The outbreak potential of Malassezia warrants preventive steps, and early identification and treatment.

Polycaprolactone/Testicular Extracellular Matrix/Graphene Oxide-Based Electrospun Tubular Scaffolds for Reproductive Medicine: Biomimetic Architecture of Seminiferous Tubules.

Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively replicating the spatial characteristics of native tissue, poses a challenge in maintaining the requisite cellular arrangement essential for spermatogenesis. In order to mimic the structural properties of seminiferous tubules, the objective is to fabricate a biocompatible tubular scaffold. Following the decellularization process of the testicular tissue, validation of cellular remnants' elimination from the specimens is conducted using 4',6-diamidino-2-phenylindole staining, hematoxylin and eosin staining, and DNA content analysis. The presence of extracellular matrix (ECM) components is confirmed through Alcian blue, Orcein, and Masson's trichrome staining techniques. The electrospinning technique is employed to synthesize the scaffolds using polycaprolactone (PCL), extracted ECM, and varying concentrations of graphene oxide (GO) (0.5%, 1%, and 2%). Subsequently, comprehensive evaluations are performed to assess the properties of the synthetic scaffolds. These evaluations encompass Fourier-transform infrared spectroscopy, scanning electron microscopy imaging, scaffold degradation testing, mechanical behavior analysis, methylthiazolyldiphenyl-tetrazolium bromide assay, and in vivo biocompatibility assessment. The PCL/decellularized extracellular matrix with 0.5% GO formulation exhibits superior fiber morphology and enhanced mechanical properties, and outperforms other groups in terms of in vitro biocompatibility. Consequently, these scaffolds present a viable option for implementation in "in vitro spermatogenesis" procedures, holding promise for future sperm production from spermatogonial cells.

Cell-matrix interactions control biliary organoid polarity, architecture, and differentiation.

Cholangiopathies are an important cause of morbidity and mortality. Their pathogenesis and treatment remain unclear in part because of the lack of disease models relevant to humans. Three-dimensional biliary organoids hold great promise; however, the inaccessibility of their apical pole and the presence of extracellular matrix (ECM) limits their application. We hypothesized that signals coming from the extracellular matrix regulate organoids' 3-dimensional architecture and could be manipulated to generate novel organotypic culture systems. Biliary organoids were generated from human livers and grown embedded into Culturex Basement Membrane Extract as spheroids around an internal lumen (EMB). When removed from the EMC, biliary organoids revert their polarity and expose the apical membrane on the outside (AOOs). Functional, immunohistochemical, and transmission electron microscope studies, along with bulk and single-cell transcriptomic, demonstrate that AOOs are less heterogeneous and show increased biliary differentiation and decreased expression of stem cell features. AOOs transport bile acids and have competent tight junctions. When cocultured with liver pathogenic bacteria (Enterococcus spp.), AOOs secrete a range of proinflammatory chemokines (ie, MCP1, IL8, CCL20, and IP-10). Transcriptomic analysis and treatment with a beta-1-integrin blocking antibody identified beta-1-integrin signaling as a sensor of the cell-extracellular matrix interaction and a determinant of organoid polarity. This novel organoid model can be used to study bile transport, interactions with pathobionts, epithelial permeability, cross talk with other liver and immune cell types, and the effect of matrix changes on the biliary epithelium and obtain key insights into the pathobiology of cholangiopathies.

Pericyte heterogeneity identified by 3D ultrastructural analysis of the microvessel wall.

Confident identification of pericytes (PCs) remains an obstacle in the field, as a single molecular marker for these unique perivascular cells remains elusive. Adding to this challenge is the recent appreciation that PC populations may be heterogeneous, displaying a range of morphologies within capillary networks. We found additional support on the ultrastructural level for the classification of these PC subtypes-"thin-strand" (TSP), mesh (MP), and ensheathing (EP)-based on distinct morphological characteristics. Interestingly, we also found several examples of another cell type, likely a vascular smooth muscle cell, in a medial layer between endothelial cells (ECs) and pericytes (PCs) harboring characteristics of the ensheathing type. A conserved feature across the different PC subtypes was the presence of extracellular matrix (ECM) surrounding the vascular unit and distributed in between neighboring cells. The thickness of this vascular basement membrane was remarkably consistent depending on its location, but never strayed beyond a range of 150-300nm unless thinned to facilitate closer proximity of neighboring cells (suggesting direct contact). The density of PC-EC contact points ("peg-and-socket" structures) was another distinguishing feature across the different PC subtypes, as were the apparent contact locations between vascular cells and brain parenchymal cells. In addition to this thinning, the extracellular matrix (ECM) surrounding EPs displayed another unique configuration in the form of extensions that emitted out radially into the surrounding parenchyma. Knowledge of the origin and function of these structures is still emerging, but their appearance suggests the potential for being mechanical elements and/or perhaps signaling nodes via embedded molecular cues. Overall, this unique ultrastructural perspective provides new insights into PC heterogeneity and the presence of medial cells within the microvessel wall, the consideration of extracellular matrix (ECM) coverage as another PC identification criteria, and unique extracellular matrix (ECM) configurations (i.e., radial extensions) that may reveal additional aspects of PC heterogeneity.

Lack of extracellular matrix switches TGF-β induced apoptosis of endometrial cells to epithelial to mesenchymal transition

The extracellular matrix and the correct establishment of epithelial cell polarity plays a critical role in epithelial cell homeostasis and cell polarity. In addition, loss of tissue structure is a hallmark of carcinogenesis. In this study, we have addressed the role of extracellular matrix in the cellular responses to TGF-β. It is well known that TGF-β is a double-edged sword: it acts as a tumor suppressor in normal epithelial cells, but conversely has tumor-promoting effects in tumoral cells. However, the factors that determine cellular outcome in response to TGF-β remain controversial. Here, we have demonstrated that the lack of extracellular matrix and consequent loss of cell polarity inhibits TGF-β-induced apoptosis, observed when endometrial epithelial cells are polarized in presence of extracellular matrix. Rather, in absence of extracellular matrix, TGF-β-treated endometrial epithelial cells display features of epithelial-to-mesenchymal transition. We have also investigated the molecular mechanism of such a switch in cellular response. On the one hand, we found that the lack of Matrigel results in increased AKT signaling which is sufficient to inhibit TGF-β-induced apoptosis. On the other hand, we demonstrate that TGF-β-induced epithelial-to-mesenchymal transition requires ERK and SMAD2/3 activation. In summary, we demonstrate that loss of cell polarity changes the pro-apoptotic function of TGF-β to tumor-associated phenotype such as epithelial-to-mesenchymal transition. These results may be important for understanding the dual role of TGF-β in normal versus tumoral cells.

Histological classification and collagen distribution in atherosclerotic plaques in aorta of hypercholesterolemic rabbits

Objective: To analyze the histopathological aspect of atheroma plaques in the aorta of rabbits submitted to diet modification and different evolution times. Methods: Aortic fragments of rabbits submitted to the following protocols were analyzed: all animals received a diet plus 0.5% cholesterol for 3months and/or followed by: group A (n=8) - sacrificed at 3months; group B (n=10) - followed by 3months of standard diet; group C (n=20) followed by 3months of 0.1% cholesterol diet; group D (n=12) - followed by 9months of 0.1% cholesterol diet. Histological sections of aortic arch fragment were analyzed in hematoxylin-eosin and picro-sirius red staining. The atherosclerotic plaques were analyzed, classifying them according to the predominance of cellularity (type 2) or presence of extracellular matrix and fibromuscular cap (type 3) and associated with a large amount of extracellular cholesterol crystals (type 4). Results: We did not observe a statistically significant difference in the intima/mediarelationship, although there was a tendency to larger plaques in groups with lipid diet for prolonged time (p<0.06). We found after 3months of hypercholesterolemic diet (group A), high serum cholesterol levels (1972±127 mg/dL) and plaques with higher cellularity (type 2), destructuring of the media layer, especially along the internal elastic lamina. The modification of the diet for 3months (groups B and C) reduced serum cholesterol levels, modifying the characteristics of the plaque, verifying more developed plaques (types 3 and 4) in animals with lipid diet. After 12months (group D) plaques with matrix predominance in relation to cellularity were observed, with a large amount of crystal of cholesterol and collagen fibers. Conclusion: The normalization of the diet or reduction of the lipid content modifies the histology of the plaque, evolving with higher extracellular matrix content.

Transcription Factors of CAT1, EFG1, and BCR1 Are Effective in Persister Cells of Candida albicans-Associated HIV-Positive and Chemotherapy Patients.

BackgroundBiofilm is an accumulation of cells, which are formed on mucosal surfaces of the host as well as on medical devices. The inherent resistance of Candida strains producing biofilms to antimicrobial agents is an important and key feature for biofilm growth, which can lead to treatment failure. This resistance is due to the regulatory increase of the output pumps, the presence of extracellular matrix, and the existence of persister cells. Persister cells are phenotypic variants that have MICs similar to antibiotic-sensitive populations and are able to tolerate high doses of antibiotics. The current study investigated the possible role of EFG1, BCR1, and CAT1 in the establishment or maintenance of persister cells in Candida albicans strains that produce biofilms.MethodsAfter identifying Candida isolates by molecular methods, C. albicans isolates were confirmed by sequencing. Isolation of persister cells and determination of their MIC were performed by microdilution method. Then, RNA extraction and cDNA synthesis were performed from 60 C. albicans isolates under promoting and inducing conditions. Afterward, the mean expression of BCR1, EFG1, and CAT1 genes in both persister and non-persister groups was calculated using real-time qPCR. Phylogeny tree of persister and non-persister group isolates was drawn using ITS fragment.ResultsA total of 77 persister isolates were taken from the oral cavity of HIV patients as well as from patients undergoing chemotherapy. Biofilm intensity in persister isolates separated from HIV-infected patients was different from the non-persister group. The mean fold change of BCR1 (10.73), CAT1 (15.34), and EFG1 (2.41) genes in persister isolates was significantly higher than these genes in isolates without persister.ConclusionIt can be concluded that the most important factor in the production of persister cells is biofilm binding and production, not biofilm development or mature biofilm production, which was found in the expression of BCR1 gene without change in the expression of EFG1 gene in the persister group. Also, catalase plays an essential role in the production of persister in C. albicans biofilm producers with ROS detoxification.

Approaches for Mitigating Microbial Biofilm-Related Drug Resistance: A Focus on Micro- and Nanotechnologies.

Biofilms play an essential role in chronic and healthcare-associated infections and are more resistant to antimicrobials compared to their planktonic counterparts due to their (1) physiological state, (2) cell density, (3) quorum sensing abilities, (4) presence of extracellular matrix, (5) upregulation of drug efflux pumps, (6) point mutation and overexpression of resistance genes, and (7) presence of persister cells. The genes involved and their implications in antimicrobial resistance are well defined for bacterial biofilms but are understudied in fungal biofilms. Potential therapeutics for biofilm mitigation that have been reported include (1) antimicrobial photodynamic therapy, (2) antimicrobial lock therapy, (3) antimicrobial peptides, (4) electrical methods, and (5) antimicrobial coatings. These approaches exhibit promising characteristics for addressing the impending crisis of antimicrobial resistance (AMR). Recently, advances in the micro- and nanotechnology field have propelled the development of novel biomaterials and approaches to combat biofilms either independently, in combination or as antimicrobial delivery systems. In this review, we will summarize the general principles of clinically important microbial biofilm formation with a focus on fungal biofilms. We will delve into the details of some novel micro- and nanotechnology approaches that have been developed to combat biofilms and the possibility of utilizing them in a clinical setting.

Selection of Effective Therapies Using Three-Dimensional in vitro Modeling of Chondrosarcoma.

Purpose: Chondrosarcomas are a group of cartilaginous malignant neoplasms characterized by the deposition of chondrogenic extracellular matrix. Surgical resection is currently the only curative treatment option, due to their high resistance to conventional chemotherapy and radiotherapy. Novel therapeutic treatment options may improve outcome. Predominantly used cell line monolayer in vitro models lack in vivo complexity, such as the presence of extracellular matrix, and differing oxygen access. Hence, we aimed to improve pre-clinical chondrosarcoma research by developing an alginate-based 3D cell culture model.Method: An alginate scaffold was applied to generate spheroids of three chondrosarcoma cell lines (CH2879, JJ012, SW1353). Morphological, histological and immunohistochemical assessment of the spheroids were used to characterize the chondrosarcoma model. Presto blue assay, morphological and immunohistochemical assessment were applied to assess spheroid response to a panel of chemotherapeutics and targeted therapies, which was compared to conventional 2D monolayer models. Synergistic effect of doxorubicin and ABT-737 (Bcl-2 inhibitor) was compared between monolayer and spheroid models using excess over Bliss. A 3D colony formation assay was developed for assessment of radiotherapy response.Results: Chondrosarcoma spheroids produced chondrogenic matrix and remained proliferative after 2 weeks of culture. When treated with chemotherapeutics, the spheroids were more resistant than their monolayer counterparts, in line with animal models and clinical data. Moreover, for sapanisertib (mTOR inhibitor) treatment, a recovery in chondrosarcoma growth, previously observed in mice models, was also observed using long-term treatment. Morphological assessment was useful in the case of YM-155 (survivin inhibitor) treatment where a fraction of the spheroids underwent cell death, however a large fraction remained proliferative and unaffected. Synergy was less pronounced in 3D compared to 2D. A 3D clonogenic assay confirmed increased resistance to radiotherapy in 3D chondrosarcoma spheroids.Conclusion: We demonstrate that the chondrosarcoma alginate spheroid model is more representative of chondrosarcoma in vivo and should be used instead of the monolayer model for therapy testing. Improved selection at in vitro stage of therapeutic testing will increase the amount of information available for experimental design of in vivo animal testing and later, clinical stages. This can potentially lead to increased likelihood of approval and success at clinical trials.

Spheroid-Like Cultures for Expanding Angiopoietin Receptor-1 (aka. Tie2) Positive Cells from the Human Intervertebral Disc.

Lower back pain is a leading cause of disability worldwide. The recovery of nucleus pulposus (NP) progenitor cells (NPPCs) from the intervertebral disc (IVD) holds high promise for future cell therapy. NPPCs are positive for the angiopoietin-1 receptor (Tie2) and possess stemness capacity. However, the limited Tie2+ NPC yield has been a challenge for their use in cell-based therapy for regenerative medicine. In this study, we attempted to expand NPPCs from the whole NP cell population by spheroid-formation assay. Flow cytometry was used to quantify the percentage of NPPCs with Tie2-antibody in human primary NP cells (NPCs). Cell proliferation was assessed using the population doublings level (PDL) measurement. Synthesis and presence of extracellular matrix (ECM) from NPC spheroids were confirmed by quantitative Polymerase Chain Reaction (qPCR), immunostaining, and microscopy. Compared with monolayer, the spheroid-formation assay enriched the percentage of Tie2+ in NPCs’ population from ~10% to ~36%. Moreover, the spheroid-formation assay also inhibited the proliferation of the Tie2- NPCs with nearly no PDL. After one additional passage (P) using the spheroid-formation assay, NPC spheroids presented a Tie2+ percentage even further by ~10% in the NPC population. Our study concludes that the use of a spheroid culture system could be successfully applied to the culture and expansion of tissue-specific progenitors.

Generation of Scalable Hepatic Micro-Tissues as a Platform for Toxicological Studies.

Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects of therapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to human hepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and the presence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this study was to generate the scalable and functional hepatic micro-tissues (HMTs). The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cells and human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, using an air-driven droplet generator. The generated HMTs were functional throughout a culture period of 28days, as assessed by monitoring glycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretion levels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involved in hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional (2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity to hepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450 enzymes confirmed that the HMTs can be used for in vitro drug screening. Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7, with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.

Short-term organoid culture for drug sensitivity testing of high-grade serous carcinoma

ObjectiveCancer patient-derived organoids (PDOs) grow as three dimensional (3D) structures in the presence of extracellular matrix and have been found to represent the original tumor's genetic complexity. In addition, PDOs can be grown and subjected to drug sensitivity testing in a shorter time course and with lesser expense than patient-derived xenograft models. Many patients with recurrent ovarian cancer develop malignant effusions that become refractory to chemotherapy. Since these same patients often present for palliative aspiration of ascites or pleural effusions, there is a potential opportunity to obtain tumor specimens in the form of multicellular spheroids (MCS) present in malignant effusion fluids. Our objective was to develop a short duration culture of MCS from ovarian cancer malignant effusions in conditions selected to support organoid growth and use them as a platform for empirical drug sensitivity testing. MethodsIn this study, malignant effusion specimens were collected from patients with high-grade serous ovarian carcinoma (HGSOC). MCS were recovered and subjected to culture conditions designed to support organoid growth. In a subset of specimens, RNA-sequencing was performed at two time points during the short-term culture to determine changes in transcriptome in response to culture conditions. Organoid induction was also characterized in these specimens using Ki67 staining and histologic analysis. Drug sensitivity testing was performed on all specimens. ResultsOur model describes organoids formed within days of primary culture, which can recapitulate the histological features of malignant ascites fluid and can be expanded for at least 6 days. RNA-seq analysis of four patient specimens showed that within 6 days of culture, there was significant up-regulation of genes related to cellular proliferation, epithelial-mesenchymal transition, and KRAS signaling pathways. Drug sensitivity testing identified several agents with therapeutic potential. ConclusionsShort duration organoid culture of MCS from HGSOC malignant effusions can be used as a platform for empiric drug sensitivity testing. These ex vivo models may be helpful in screening new or existing therapeutic agents prior to individualized treatment options.

In vitro morphogenetic responses from obligatory apomictic Taraxacum belorussicum Val. N. Tikhom seedlings explants

Taraxacum belorussicum Val. N. Tikhom, a poorly known and obligatory apomictic species, is an attractive plant material for studying the embryological, genetic and molecular mechanisms of apomixis. This work aims to obtain an efficient protocol for Taraxacum belorussicum regeneration. Four types of explants (cotyledons, hypocotyls, meristems and roots) that were taken from 2-weeks-old seedlings were used for in vitro cultures, and a fast and efficient protocol of T. belorussicum regeneration was obtained. Various ½ MS-based media containing IAA (5.71 µM), TDZ (4.54 µM) and PSK (100 nM) were chosen to assess the morphogenetic abilities of selected T. belorussicum explants. Studies on the role of PSK were done in three independent experiments, where the most significant factors were always light and darkness. All explants produced callus by the third day of culture and adventitious shoots after 7 days, although in an asynchronous indirect manner, and with different intensities for all explant types. The most preferred medium culture for hypocotyl, cotyledon and meristem explants was ½ MS + TDZ, and ½ MS + IAA + TDZ + PSK for roots which were the only explant sensitive to PSK. A short darkness pretreatment (8 days) in PSK medium was found suitable to enhance organogenesis. Secondary organogenesis was observed for regenerated plants on meristem explants from the ½ MS + IAA + TDZ + PSK medium. A weak somatic embryogenesis was observed for hypocotyl and cotyledon explants from ½ MS + IAA + TDZ and ½ MS + IAA + TDZ + PSK media. Histological and scanning electron microscope images (SEM) of T. belorussicum confirmed indirect organogenesis and somatic embryogenesis. Plant material treated with aniline blue solution revealed the presence of callose in the cell walls of cotyledon and hypocotyl explants. The presence of extracellular matrix (ECM) and heterogenic structure of callus was also verified by scanning electron microscopy and light microscopy, confirming the high morphogenetic ability of T. belorussicum.

Functional MRI for evaluation of hyaline cartilage extracelullar matrix, a physiopathological-based approach.

MRI of articular cartilage (AC) integrity has potential to become a biomarker for osteoarthritis progression. Traditional MRI sequences evaluate AC morphology, allowing for the measurement of thickness and its change over time. In the last two decades, more advanced, dedicated MRI cartilage sequences have been developed aiming to assess AC matrix composition non-invasively and detect early changes in cartilage not captured on morphological sequences. T2-mapping and T1ρ sequences can be used to estimate the relaxation times of water inside the AC. These sequences have been introduced into clinical protocols and show promising results for cartilage assessment. Extracelullar matrix can also be assessed using diffusion-weighted imaging and diffusion tensor imaging as the movement of water is limited by the presence of extracellular matrix in AC. Specific techniques for glycosaminoglycans (GAG) evaluation, such as delayed gadolinium enhanced MRI of cartilage or Chemical Exchange Saturation Transfer imaging of GAG, as well as sodium imaging have also shown utility in the detection of AC damage. This manuscript provides an educational update on the physical principles behind advanced AC MRI techniques as well as a comprehensive review of the strengths and weaknesses of each approach. Current clinical applications and potential future applications of these techniques are also discussed.

Influence of Matrigel on Single- and Multiple-Spheroid Cultures in Breast Cancer Research

This study aimed to develop and compare single and multiple 3D models such as multicellular tumor spheroids and to investigate the influence of Matrigel on their morphological and functional behavior. MDA-MB-231 3D models were generated in the presence and absence of Matrigel and their key biological properties within 6 days of culture were monitored. Our results revealed the formation of well-defined 3D models in the presence of Matrigel, with a uniform morphology, increased diameter, good circularity, and increased expression of a proliferation marker (PCNA). In comparison, 3D models generated without Matrigel were characterized by an irregular border, reduced dimensions and circularity, and a decrease of PCNA expression. Similarities between the single and multiple 3D cultures were found in their viability, Nrf2 expression, and glutathione (GSH) content. The influence of Matrigel on MDA-MB-231 spheroids metabolism under hypoxic conditions was highlighted by released lactate dehydrogenase and nitric oxide, GSH levels and expression of Nrf2 and Hsp70 proteins. Based on the increased expression of PCNA and the development of the hypoxia process in the presence of extracellular matrix, our study showed that the addition of Matrigel improves the growing environment of tumor spheroids, making it closer to that of in vivo tumor conditions.

Characterization and Differentiation of Circulating Blood Mesenchymal Stem Cells and the Role of Phosphatidylinositol 3-Kinase in Modulating the Adhesion.

Bone marrow mesenchymal stem cells (BM MSCs) can differentiate into multi-lineage tissues. However, obtaining BM MSCs by aspiration is difficult and can be painful; therefore peripheral blood (PB) MSCs might provide an easier alternative for clinical applications. Here, we show that circulating PB MSCs proliferate as efficiently as BM MSCs in the presence of extracellular matrix (ECM) and that differentiation potential into osteoblast in vitro and in vivo. Both BM MSCs and PB MSCs developed into new bone when subcutaneously transplanted into immune-compromised mice using hydroxyapatite/tricalcium phosphate as a carrier. Furthermore, LY294002 and Wortmannin blocked mesenchymal stem cell attachment in a dose-dependent manner, suggesting a role of phosphatidylinositol 3-kinase in MSC attachment. Our data showed that the growth of PB MSCs could be regulated by interaction with the ECM and that these cells could differentiate into osteoblasts, suggesting their potential for clinical applications.

The cytoplasmic domain of RAGE (Receptor for Advanced Glycation Endproduct) is selfsufficient to contribute cell adhesion in the presence of extracellular matrix

PurposeThe Receptor for Advanced Glycation Endproducts (RAGE) is a pattern‐recognition, cell‐surface receptor of the immunoglobulins‐like receptor superfamily. RAGE activation by binding of its soluble ligands has been documented to promote pro‐inflammatory signaling, cytokine release, and phenotypic changes. While the pathophysiological significance of RAGE as a signaling receptor is undisputed, it is not clear whether RAGE may also have critical roles in cell‐cell and cell‐matrix adhesion. A more defined understanding of the biochemical and biophysical properties of RAGE will be of tremendous value to rationalize its biological function in disease development and progression. Identifying the biological functions of individual protein domains of the RAGE receptor molecule will accelerate the rational discovery of drugs targeting RAGE.MethodsFor this study, a protein engineering approach is used to express full length RAGE (F‐RAGE) and a panel of domain deletion constructs (ΔV, ΔC1, ΔC2, DN‐RAGE, TmCyto RAGE) of the receptor. The necessary expression constructs were assembled in the pcDNA 3 vector, and the RAGE variants were expressed in Hek 293 cells. The contribution of individual domains of RAGE towards cell adherence and spreading were evaluated by cell adhesion and cell spreading assays for all domain deletion constructs.ResultsConfocal fluorescence microscopy and flow cytometry was used to determine the cellular localization of the RAGE constructs. All constructs localized to the plasma membrane, except for the TmCyto and ΔV‐RAGE constructs, which had an intercellular or nuclear localization. Cell adhesion measurements to collagen IV using a Xcelligence RTCA DP system revealed that deletion of individual domains gradually reduced cell adhesion compared to full‐length RAGE. Surprisingly, the DN‐RAGE construct, which differs from F‐RAGE only in the deletion of the intracellular tail, showed complete loss of RAGE‐mediated cell adhesion. This suggests that the cytoplasmic tail is required to modulate cell adhesion via RAGE. Interestingly, expression of the membrane‐anchored cytoplasmic tail (TmCyto), restored cell adhesion to a level exceeding those observed for F‐RAGE expressing cells. Cell spreading data showed a similar pattern as the cell adhesion results in which F‐RAGE and TmCyto transfected cells had a dense, almost neuronal‐like, spread pattern compared to mock and DN‐RAGE which were clearly rounded in shape.ConclusionRAGE does modulate cell adhesion and cell spreading properties through direct interaction of the extracellular domains of RAGE with matrix proteins, as well as through RAGE‐signaling, which does require the intracellular tail of RAGE. Drugs targeting the intracellular cytoplasmic domain of RAGE might become useful to modulate tumor cell adhesion, spreading, and migration in cancers with high levels of RAGE expression.Support or Funding InformationThis research was supported by the NDSU College of Health Professions, the NDSU Department of Pharmaceutical Sciences, NIH Grant GM103332 and XCELLigence Research grant. Cartoon representation of the domain organization of full‐length RAGE (F‐RAGE) and the domain deletion variants used in this study.imageCartoon representation of the domain organization of full‐length RAGE (F‐RAGE) and the domain deletion variants used in this study.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3738: Three-dimensional treatment-resistant breast cancer spheroids as a predictive model of in vivo response to endocrine therapy

Abstract 1 out of every 8 U.S. women will develop invasive breast cancer during her lifetime, making it the second most common form of cancer affecting women. Breast cancer is also a leading cause of cancer related deaths for women in the U.S., second only to lung cancer. Activation of estrogen receptor alpha (ERα) is the primary proliferative mechanism of breast cancer cells, making it a logical target for therapy. ER ligands with antiestrogenic activity, such as the selective estrogen receptor modulator (SERM), tamoxifen, and selective estrogen receptor degrader (SERD), fulvestrant, have proven clinically successful as treatments for breast cancer; however, resistance in up to 50% of patients provides a therapeutic challenge. Once resistant, breast cancer cells become endocrine-independent, because of this, there is an urgent need for both novel therapy and improved models of resistant breast cancer. Our lab has created a panel of various endocrine-independent cell lines to mimic SERM and SERD resistance. Along with traditional 2D cell culturing, 3D spheroids have also been utilized to gain a better understanding of resistance. Importantly, the response to therapeutic agents, of cell lines in 2D versus 3D cultures is not identical. We observe that 3D cultures better replicate observations in mouse xenograft models, demonstrating that elements of the spheroid microenvironment, such as cell-cell interactions and the presence of extracellular matrix (EM), mimic aspects of the tumor microenvironment in vivo. Cells cultured as spheroids are therefore a suitable in vitro model for drug discovery, predictive of response in preclinical animal models, in contrast to 2D monolayer cell cultures. Citation Format: Carlo I. Rosales, Jiong Zhao, Lauren M. Gutgesell, Rui Xiong, Debra A. Tonetti, Gregory R. Thatcher. Three-dimensional treatment-resistant breast cancer spheroids as a predictive model of in vivo response to endocrine therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3738.

Collagen overlays can inhibit leptin and adiponectin secretion but not lipid accumulation in adipocytes.

BackgroundWhite adipose tissue (WAT) is essential for energy storage as well as being an active endocrine organ. The secretion of adipokines by adipocytes can affect whole body metabolism, appetite, and contribute to overall health. WAT is comprised of lipid-laden mature adipocytes, as well as immune cells, endothelial cells, pre-adipocytes, and adipose-derived stem cells. In addition, the presence of extracellular matrix (ECM) proteins in WAT can actively influence adipocyte differentiation, growth, and function. Type I collagen is an abundant fibrous ECM protein in WAT that is secreted by developing adipocytes. However, the extent and overall effect of Type I collagen on adipokine secretion in mature adipocytes when added exogenously has not been established.MethodsWe characterized the effects of Type I collagen overlays prepared using two different buffers on adipocyte physiology and function when added at different times during differentiation. In addition, we compared the effect of collagen overlays when adipocytes were cultured on two different tissue culture plastics that have different adherent capabilities. Triglyceride accumulation was analyzed to measure adipocyte physiology, and leptin and adiponectin secretion was determined to analyze effects on adipokine secretion.ResultsWe found that collagen overlays, particularly when added during the early differentiation stage, impaired adipokine secretion from mature adipocytes. Collagen prepared using PBS had a greater suppression of leptin than adiponectin while collagen prepared using HANKS buffer suppressed the secretion of both adipokines. The use of CellBind plates further suppressed leptin secretion. Triglyceride accumulation was not substantially impacted with any of the collagen overlays.DiscussionAdipokine secretion can be selectively altered by collagen overlays. Thus, it is feasible to selectively manipulate the secretion of adipokines by adipocytes in vitro by altering the composition or timing of collagen overlays. The use of this technique could be applied to studies of adipokine function and secretion in vitro as well as having potential therapeutic implications to specifically alter adipocyte functionality in vivo.