Reconstituted Basement Membrane Research Articles

Oncofetal morphogenesis similar to embryonic gut formation by a subpopulation of DLD-1 human colon cancer cells

Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers.

Soft Polyethylene Glycol Hydrogels Support Human PSC Pluripotency and Morphogenesis.

Lumenogenesis within the epiblast represents a critical step in early human development, priming the embryo for future specification and patterning events. However, little is known about the specific mechanisms that drive this process due to the inability to study the early embryo in vivo. While human pluripotent stem cell (hPSC)-based models recapitulate many aspects of the human epiblast, most approaches for generating these 3D structures rely on ill-defined, reconstituted basement membrane matrices. Here, we designed synthetic, nonadhesive polyethylene glycol (PEG) hydrogel matrices to better understand the role of matrix mechanical cues in iPSC morphogenesis, specifically elastic modulus. First, we identified a narrow range of hydrogel moduli that were conducive to the hPSC viability, pluripotency, and differentiation. We then used this platform to investigate the effects of the hydrogel modulus on lumenogenesis, finding that matrices of intermediate stiffness yielded the most epiblast-like aggregates. Conversely, stiffer matrices impeded lumen formation and apico-basal polarization, while the softest matrices yielded polarized but aberrant structures. Our approach offers a simple, modular platform for modeling the human epiblast and investigating the role of matrix cues in its morphogenesis.

Breast organoid suspension cultures maintain long-term estrogen receptor expression and responsiveness.

Organoid cultures offer a powerful technology to investigate many different aspects of development, physiology, and pathology of diverse tissues. Unlike standard tissue culture of primary breast epithelial cells, breast organoids preserve the epithelial lineages and architecture of the normal tissue. However, existing organoid culture methods are tedious, difficult to scale, and do not robustly retain estrogen receptor (ER) expression and responsiveness in long-term culture. Here, we describe a modified culture method to generate and maintain organoids as suspension cultures in reconstituted basement membrane (™Matrigel). This method improves organoid growth and uniformity compared to the conventional Matrigel dome embedding method, while maintaining the fidelity of the three major epithelial lineages. Using this adopted method, we are able to culture and passage purified hormone sensing (HS) cells that retain ER responsiveness upon estrogen stimulation in long-term culture. This culture system presents a valuable platform to study the events involved in initiation and evolution of ER-positive breast cancer.

Abstract PO1-24-04: Gedatolisib, a pan-PI3K/mTOR inhibitor, shows superior potency and efficacy relative to other PI3K/AKT/mTOR pathway inhibitors in breast cancer models

Abstract Background: The PI3K, AKT, and mTOR (PAM) pathway is frequently activated in breast cancer (BC). Current standard-of-care therapy options for patients with advanced BC (ABC) include PAM inhibitors (PAMi), such as everolimus and alpelisib, and hormonal therapy, such as letrozole and fulvestrant. Most PAMi selectively inhibit one or only a few PAM pathway components, which can lead to drug resistance. To minimize drug resistance, a more comprehensive inhibition of the multiple PI3K isoforms and downstream mTOR complexes may be required. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, as well as mTORC1 and mTORC2, can be more effective in BC cells than a PAMi targeting single PAM pathway components. Methods: A panel of BC cell lines with mutated or non-mutated PAM pathway genes (PIK3CA, PTEN, AKT) were evaluated for their sensitivity to gedatolisib and other PAMi (PI3K-α: alpelisib; AKT: capivasertib; mTORC1: everolimus; pan-PI3K/mTOR: gedatolisib). Cell viability, growth rate inhibition, and cell death were evaluated by luciferase- and fluorescence-based assays, both in monolayer cultures and three-dimensional (3D) cultures on reconstituted basement membrane. Flow cytometry analytical assays were conducted to assess DNA synthesis (EdU incorporation), protein synthesis (OPP incorporation), and PAM pathway activity (RPS6 and 4EBP1 phosphorylation). BC xenograft studies evaluating gedatolisib in vivo were also performed. Results: Gedatolisib strongly inhibited PAM pathway activity and reduced cell viability and growth rate in the cell lines tested. Compared to the other PAMi, gedatolisib exhibited more potent and efficacious anti-proliferative and cytotoxic effects, regardless of the cell lines’ PI3K pathway mutational status. Gedatolisib effects were confirmed in 3D culture on reconstituted basement membrane, where gedatolisib inhibited tumor cell spheroid growth and induced regression. Mechanistically, gedatolisib decreased both DNA and protein synthesis more effectively than the other PAMi tested. Gedatolisib also exerted superior anti-proliferative and cytotoxic effects relative to the other PAMi tested in estrogen receptor-positive BC cell lines concomitantly treated with fulvestrant. BC xenografts confirmed growth inhibitory effects of gedatolisib in vivo. Conclusions: The pan-PI3K/mTOR inhibitor, gedatolisib, may more effectively address potential drug resistance mechanisms associated with narrowly targeted PAM inhibitors. Gedatolisib has previously demonstrated promising preliminary clinical efficacy and safety data in ABC in combination with hormonal therapy. A Phase 3 study (VIKTORIA-1) evaluating gedatolisib plus fulvestrant with and without palbociclib is underway in patients with ABC. Citation Format: Stefano Rossetti, Aaron Broege, Adrish Sen, Salmaan Khan, Ian MacNeil, Jhomary Molden, Igor Gorbatchevsky, Brian F. Sullivan, Lance Laing. Gedatolisib, a pan-PI3K/mTOR inhibitor, shows superior potency and efficacy relative to other PI3K/AKT/mTOR pathway inhibitors in breast cancer models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-04.

Interferon-Alpha Impairs Neoplastic Vasculogenesis in a 3D iPSC-Based Model of JAK2V617F+ Myeloproliferative Neoplasms

Overt Myelofibrosis (MF) is an end-stage subtype of BCR-ABL1 negative myeloproliferative neoplasms (MPN), which is characterized by progressive bone marrow (BM) fibrosis resulting in loss of normal hematopoietic support as well as inflammatory and vascular complications. While aberrant neoangiogenesis is known to participate in the pathogenesis of MF, Erba et al. (Am J Pathol, 2017) suggested the occurrence of endothelial-to-mesenchymal transition (EndMT) in BM and spleen of MF patients (pts). Given the relevance of endothelial cells (EC) for MF development and the occurrence of JAK2V617F-mutated EC in MPN pts, targeting of the vascular niche has emerged as a promising therapeutic strategy. To untangle the impact of the JAK2V617F mutation on human vasculogenesis, we leveraged high-purity and clonal patient-specific induced pluripotent stem cell (iPSC)-derived EC (iEC) harboring either the JAK2V617F mutation in hetero- (JAK2V617F HET) or homozygosis (JAK2V617F HOM). JAK2 WT and CRISPR-repaired isogenic JAK2 WT (both referred to as JAK2 WT) were used as controls. In a first set of in vitro experiments, JAK2V617F mutated iEC showed a significantly increased proliferation rate in comparison to JAK2 WT iEC. We next evaluated the effect of selected drugs on iEC viability to assess their vascular toxicity. While treatment with 1 µM each of fedratinib, anagrelide or venetoclax led to a decrease in iEC viability (all **p ≤ .01), no significant alterations were observed with 1 µM ruxolitinib (RUX) or 1000 U/ml interferon-alpha (IFNa). Overall, no differences between the 3 genotypes were observed, pointing towards a genotype-independent mode of action. Given the favorable vascular toxicity profile of IFNa, as shown above, its anti-angiogenic potential was then assessed via endothelial tube formation assays. Pre-incubation with 1000 U/ml IFNa prior to seeding of the cells onto a reconstituted basement membrane matrix led to a significant inhibition of capillary-like structures formation in JAK2 WT and JAK2V617F HET or HOM iEC. One of the limitations of 2D culture assays is the lack of 3D homotypic cellular organization, compromising its translational value. Therefore, we established an adaptive 3D fibrin-based model to recapitulate, in vitro, the critical EC-pericyte crosstalk required for formation and maturation of blood vessels. Recent studies highlighted the capacity of BM mesenchymal stem cells (MSC) to differentiate into pericytes. Therefore, we seeded JAK2 WT and JAK2V617F HETor HOM iEC with or without MSC in fibrin hydrogels. MSC and iEC were visualized using different PKH dyes as fluorescent cell linkers to enable live tracking of vascular tree formation. Although self-assembly of JAK2 WT and JAK2V617F HET or HOM iEC into vessel-like structures was observed in the generated monocultures, the presence of MSC in the cocultures led to a significant increase of the formed network area (Fig. 1), hence recapitulating the critical support of pericytes. While acute IFNa exposure did not alter vascular tree formation (Fig. 1), a separate pre-treatment of iEC and MSC prior to cocultivation, significantly impaired neoplastic vasculogenesis (Fig. 2). Interestingly, interactive microscopy image analysis revealed an increased MSC/iEC interaction in JAK2V617F HOM pre-treated cocultures (*p ≤ .05). Bulk RNA-sequencing of JAK2 WT and JAK2V617F HET or HOM iEC treated for 24h with 1000 U/ml showed a strong response to IFNa. Future analysis may unravel a JAK2V617F specific transcription signature. Moreover, we induced EndMT in our 3D iEC-MSC coculture model by treatment with pro-inflammatory cytokines, reflected by the loss of EC markers (e.g. Claudin 5) and upregulation of mesenchymal markers (e.g. aSMA) by immunofluorescence. We are currently evaluating the capacity of RUX vs IFNa to inhibit EndMT. In summary, we show that IFNa impairs neoplastic vasculogenesis in a robust in vitro 3D coculture system that enables precise control of cellularity and genotype. Our model is a valuable tool to decipher the poorly investigated impact of IFNa on the BM microenvironment. In addition, its translational and scalable properties (e.g. by integrating primary or iPSC-derived hematopoietic cells) will allow us to study the impact of additional drugs on MPN-associated fibrotic transformation of the BM.

Fibrin Stiffness Regulates Phenotypic Plasticity of Metastatic Breast Cancer Cells.

The extracellular matrix (ECM)-regulated phenotypic plasticity is crucial for metastatic progression of triple negative breast cancer (TNBC). While ECM faithful cell-based models are available for in situ and invasive tumors, such as cell aggregate cultures in reconstituted basement membrane and in collagenous gels, there are no ECM faithful models for metastatic circulating tumor cells (CTCs). Such models are essential to represent the stage of metastasis where clinical relevance and therapeutic opportunities are significant. Here, CTC-like DU4475 TNBC cells are cultured in mechanically tunable 3D fibrin hydrogels. This is motivated, as in circulation fibrin aids CTC survival by forming a protective coating reducing shear stress and immune cell-mediated cytotoxicity and promotes several stages of late metastatic processes at the interface between circulation and tissue. This work shows that fibrin hydrogels support DU4475 cell growth, resulting in spheroid formation. Furthermore, increasing fibrin stiffness from 57 to 175Pa leads to highly motile, actin and tubulin containing cellular protrusions, which are associated with specific cell morphology and gene expression patterns that markedly differ from basement membrane or suspension cultures. Thus, mechanically tunable fibrin gels reveal specific matrix-based regulation of TNBC cell phenotype and offer scaffolds for CTC-like cells with better mechano-biological properties than liquid.

Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation.

The complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885. The interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers. Zilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies. Our findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.

Abstract 332: Simultaneous inhibition of EZH2 and activation of dopamine D1 in an isotropic triple-negative breast cancer microgel model

Abstract Triple negative breast cancer (TNBC) cells overexpress enhancer of zeste homolog 2 (EZH2) the catalytic subunit of polycomb repressive complex 2 (PRC2). EZH2 is a potential driver of TNBC metastasis, and its high expression strongly associates with the TNBC phenotype as compared with other molecular subtypes of breast cancer. Furthermore, several studies have also shown the involvement of the neurotransmitter, dopamine, in proliferation, apoptosis, tumor angiogenesis, and drug resistance among different cancers, including the breast. Dopamine D1 receptor (D1R) activation in TNBC cell line induces apoptosis, autophagy, inhibit the invasion, and regress mammary tumors. The oncogenic nature of EZH2 in driving aggressiveness in TNBC cells led us to simultaneously target D1R and EZH2 to completely ablate tumor growth and metastasis. Conventional in vitro models utilize monolayers to investigate the effect of drugs on cancer cells but do not accurately recapitulate the in vivo microenvironment as the cells are subjected to homogeneous growth conditions. Microgels are miniature compartmentalized hydrogels that yield isotropic cell culture conditions unbiased by gravitational or hydrodynamic forces. As such, microgels lend suitable microenvironments conducive to the growth of tumor spheroids, which impose radial-dependent growth conditions as observed in vivo. While non-transformed epithelial cells with reconstituted basement membrane form hollow, growth-arrested, polarized three-dimensional (3D) structures, tumorigenic cells form large, solid, proliferating and invasive structures characteristic of in vivo tumor, which are recapitulated by tumor spheroids. As such, tumor spheroids become an ideal model to study the sensitivity of drugs combination, which led us to investigate the efficacy of EZH2 inhibitor and D1R agonist on TNBC tumor spheroids. Tumor spheroids were formed in monodisperse calcium-alginate microgels generated via droplet-based microfluidics, which mimic the viscoelastic properties of decellularized ECM and induce aggregation by removing integrin-binding sites. Tumor spheroids formed after 2 days were subjected to single and combination therapies of GSK126, an EZH2 inhibitor, and A77636, a dopamine agonist over the span of 4 days. Single doses demonstrated a dose-dependent decrease in tumor spheroid area. Furthermore, the combination therapy demonstrated a regression in growth, synergistic decrease in area (CI < 0.6), and the induction of necrosis. Furthermore, knockout of EZH2 in the TNBC cells resulted in inability for tumor spheroid formation and a sensitivity to A77636. Our findings imply that EZH2 is critical for spheroid formation and growth in TNBC and suggest that targeting the EZH2 alongside D1R presents a novel strategy for enhancing EZH2 inhibition (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: Xilal Y. Rima, Gautham Sarathy, Lara Rizzotto, Anagh Kulkarni, Dario Palmieri, Meisam Naeimi Kararoudi, Eduardo Reátegui, Bhuvaneswari Ramaswamy, Eswar Shankar. Simultaneous inhibition of EZH2 and activation of dopamine D1 in an isotropic triple-negative breast cancer microgel model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 332.

Novel cryo-tomography workflow reveals nanometer-scale responses of epithelial cells to matrix stiffness and dimensionality.

Matrix stiffness and dimensionality have been shown to be major determinants of cell behavior. However, a workflow for examining nanometer-scale responses of the associated molecular machinery is not available. Here, we describe a comprehensive, quantitative workflow that permits the analysis of cells responding to mechanical and dimensionality cues in their native state at nanometer scale by cryogenic electron tomography. Using this approach, we quantified distinct cytoskeletal nanoarchitectures and vesicle phenotypes induced in human mammary epithelial cells in response to stiffness and dimensionality of reconstituted basement membrane. Our workflow closely recapitulates the microenvironment associated with acinar morphogenesis and identified distinct differences in situ at nanometer scale. Using drug treatment, we showed that molecular events and nanometer-scale rearrangements triggered by engagement of apical cell receptors with reconstituted basement membrane correspond to changes induced by reduction of cortical tension. Our approach is fully adaptable to any kind of stiffness regime, extracellular matrix composition, and drug treatment.

ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells.

Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.

Toward Measuring the Mechanical Stresses Exerted by Branching Embryonic Airway Epithelial Explants in 3D Matrices of Matrigel.

Numerous organs in the bodies of animals, including the lung, kidney, and mammary gland, contain ramified networks of epithelial tubes. These structures arise during development via a process known as branching morphogenesis. Previous studies have shown that mechanical forces directly impact this process, but the patterns of mechanical stress exerted by branching embryonic epithelia are not well understood. This is, in part, owing to a lack of experimental tools. Traditional traction force microscopy assays rely on the use of compliant hydrogels with well-defined mechanical properties. Isolated embryonic epithelial explants, however, have only been shown to branch in three-dimensional matrices of reconstituted basement membrane protein, or Matrigel, a biomaterial with poorly characterized mechanical behavior, especially in the regime of large deformations. Here, to compute the traction stresses generated by branching epithelial explants, we quantified the finite-deformation constitutive behavior of gels of reconstituted basement membrane protein subjected to multi-axial mechanical loads. We then modified the mesenchyme-free assay for the ex vivo culture of isolated embryonic airway epithelial explants by suspending fluorescent microspheres within the surrounding gel and tracking their motion during culture. Surprisingly, the tracked bead motion was non-zero in regions of the gel far away from the explants, suggestive of passive swelling deformations within the matrix. To compute accurate traction stresses, these swelling deformations must be decomposed from those generated by the branching explants. We thus tracked the motion of beads suspended within cell-free matrices and quantified spatiotemporal patterns of gel swelling. Taken together, these passive swelling data can be combined with the measured mechanical properties of the gel to compute the traction forces exerted by intact embryonic epithelial explants.

Generation of Alveolar Epithelium Using Reconstituted Basement Membrane and hiPSC‐Derived Organoids (Adv. Healthcare Mater. 6/2022)

Reconstituted Basement Membranes In article number 2101972 by Juan Peng, Yong Chen, and co-workers, an artificial basement membrane is obtained by protein self-assembling in the pore areas of a monolayer of crosslinked nanofibers. Alveolar epithelium is achieved by replating dissociated single cells of alveolar organoids.

Generation of Alveolar Epithelium Using Reconstituted Basement Membrane and hiPSC-Derived Organoids.

In vitro modeling of alveolar epithelium needs to recapitulate features of both cellular and noncellular components of the lung tissues. Herein, a method is presented to generate alveolar epithelium by using human induced pluripotent stem cells (hiPSCs) and reconstituted or artificial basement membrane (ABM). The ABM is obtained by self-assembling type IV collagen and laminin with a monolayer of crosslinked gelatin nanofibers as backbone and a patterned honeycomb microframe for handling. Alveolar organoids are obtained from hiPSCs and then dissociated into single cells. After replating the alveolar cells on the ABM and a short-period incubation under submerged and air-liquid interface culture conditions, an alveolar epithelium is achieved, showing high-level expressions of both alveolar cell-specific proteins and characteristic tight junctions. Besides, endothelial cells derived from the same hiPSCs are cocultured on the backside of the epithelium, forming an air-blood barrier. The method is generic and can potentially be applied to other types of artificial epithelium and endothelium.

Application of 3D Culture Assays to Study Breast Morphogenesis, Epithelial Plasticity, and Cellular Interactions in an Epithelial Progenitor Cell Line.

Capturing breast morphogenesis and cancer progression in 3D culture using cell lines with stem cell properties can greatly increase understanding of the underlying mechanisms involved in these processes, highlighting the importance of the culture method. D492 is a breast epithelial progenitor cell line that provides a model for branching morphogenesis when cultured in 3D reconstituted basement membrane matrix (rBM). Along with its derivate cell lines D492M and D492HER2, D492 also serves as a robust model for epithelial to mesenchymal transition (EMT) and tumorigenicity, respectively. Here, we describe the routine maintenance and application of the D492 cell lines in 3D culture for the study of branching morphogenesis, EMT and epithelial-endothelial interaction.

A Unified Protocol to Streamline Molecular and Cellular Analysis for Three-Dimensional Cell Cultures.

Three-dimensional (3D) cell cultures based on reconstituted basement membrane materials recapitulate features of extracellular matrix (ECM) and tissue stiffness in vivo and provide a physiologically relevant platform to study complex cellular processes, such as stem cell differentiation and tissue morphogenesis, that are otherwise difficult in animal models. The form and composition of 3D matrices in culture can interfere with and pose challenges for different experimental setups and assays, which necessitate alterations to facilitate analysis. Here, we provide a unified protocol for 3D cell cultures with modular workflows that streamline procedures for compatibility with common molecular and cellular assays such as live-cell imaging, immunofluorescence , qPCR, RNAseq, western blotting, and quantitative mass spectrometry.

Peroxidasin Enhances Basal Phenotype and Inhibits Branching Morphogenesis in Breast Epithelial Progenitor Cell Line D492

The human breast is composed of terminal duct lobular units (TDLUs) that are surrounded by stroma. In the TDLUs, basement membrane separates the stroma from the epithelial compartment, which is divided into an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Stem cells and progenitor cells also reside within the epithelium and drive a continuous cycle of gland remodelling that occurs throughout the reproductive period. D492 is an epithelial cell line originally isolated from the stem cell population of the breast and generates both luminal and myoepithelial cells in culture. When D492 cells are embedded into 3D reconstituted basement membrane matrix (3D-rBM) they form branching colonies mimicking the TDLUs of the breast, thereby providing a well-suited in vitro model for studies on branching morphogenesis and breast development. Peroxidasin (PXDN) is a heme-containing peroxidase that crosslinks collagen IV with the formation of sulfilimine bonds. Previous studies indicate that PXDN plays an integral role in basement membrane stabilisation by crosslinking collagen IV and as such contributes to epithelial integrity. Although PXDN has been linked to fibrosis and cancer in some organs there is limited information on its role in development, including in the breast. In this study, we demonstrate expression of PXDN in breast epithelium and stroma and apply the D492 cell line to investigate the role of PXDN in cell differentiation and branching morphogenesis in the human breast. Overexpression of PXDN induced basal phenotype in D492 cells, loss of plasticity and inhibition of epithelial-to-mesenchymal transition as is displayed by complete inhibition of branching morphogenesis in 3D culture. This is supported by results from RNA-sequencing which show significant enrichment in genes involved in epithelial differentiation along with significant negative enrichment of EMT factors. Taken together, we provide evidence for a novel role of PXDN in breast epithelial differentiation and mammary gland development.

Generation of Interconnected Neural Clusters in Multiscale Scaffolds from Human-Induced Pluripotent Stem Cells.

The development of in vitro neural networks depends to a large extent on the scaffold properties, including the scaffold stiffness, porosity, and dimensionality. Herein, we developed a method to generate interconnected neural clusters in a multiscale scaffold consisting of a honeycomb microframe covered on both sides with a monolayer of cross-linked gelatin nanofibers. Cortical neural precursor cells were first produced from human-induced pluripotent stem cells and then loaded into the scaffold for a long period of differentiation toward cortical neural cells. As a result, neurons and astrocytes self-organized in the scaffold to form clusters in each of the honeycomb compartments with remarkable inter-cluster connections. These cells highly expressed neuron- and astrocyte-specific proteins, including NF200, tau, synapsin I, and glial fibrillary acidic protein, and showed spatially correlated neural activities. Two types of neural clusters, that is, spheroid-like and hourglass-like clusters, were found, indicating the complexity of neural-scaffold interaction and the variability of three-dimensional neural organization. Furthermore, we incorporated a reconstituted basement membrane into the scaffold and performed co-culture of the neural network with brain microvascular endothelial cells. As a proof of concept, an improved neurovascular unit model was tested, showing large astrocytic end-feet on the back side of the endothelium.

Assessing a Novel 3D Assay System for Drug Screening against OS Metastasis.

Osteosarcoma (OS) is an aggressive mesenchymal cell tumor that carries a poor long-term prognosis. Despite definitive surgery for the primary tumor and adjuvant chemotherapy, pulmonary metastasis is common and is the primary cause of morbidity. To improve outcomes for patients, we have developed and optimized a phenotypic screen for drugs that may target OS disseminated tumor cells (DTCs) and inhibit their metastatic outbreak rather than merely screening for cytotoxic activity against proliferating cells, as is commonly conducted in conventional drug discovery approaches. We report on the validation of a previously described 3D reconstituted basement membrane extract (3D BME) model system for tumor dormancy and metastatic outgrowth adapted to clonal pairs of high and low metastatic OS cells. A post-hoc validation of the assay was possible by comparing the activity of a drug in our assay with early evidence of activity in human OS clinical trials (regorafenib and saracatinib). In this validation, we found concordance between our assay and human clinical trial experience We then explored an approved veterinary small molecule inhibitor of Janus kinase-1 (oclacitinib) as a potential drug candidate to take advantage of the high prevalence of OS in pet dogs and its translational value to humans. Despite the biological rationale, we found no evidence to support the use of oclacitinib as an antimetastatic agent in OS. The findings support our 3D BME assay as a highly efficient method to examine drugs for activity in targeting OS DTCs.

Viscoelasticity and Adhesion Signaling in Biomaterials Control Human Pluripotent Stem Cell Morphogenesis in 3D Culture.

Organoids are lumen-containing multicellular structures that recapitulate key features of the organs, and are increasingly used in models of disease, drug testing, and regenerative medicine. Recent work has used 3D culture models to form organoids from human induced pluripotent stem cells (hiPSCs) in reconstituted basement membrane (rBM) matrices. However, rBM matrices offer little control over the microenvironment. More generally, the role of matrix viscoelasticity in directing lumen formation remains unknown. Here, viscoelastic alginate hydrogels with independently tunable stress relaxation (viscoelasticity), stiffness, and arginine-glycine-aspartate (RGD) ligand density are used to study hiPSC morphogenesis in 3Dculture. A phase diagram that shows how these properties control hiPSC morphogenesis is reported. Higher RGD density and fast stress relaxation promote hiPSC viability, proliferation, apicobasal polarization, and lumen formation, while slow stress relaxation at low RGD densities triggers hiPSC apoptosis. Notably, hiPSCs maintain pluripotency in alginate hydrogels for much longer times than is reported in rBM matrices. Lumen formation is regulated by actomyosin contractility and is accompanied by translocation of Yes-associated protein (YAP) from the nucleus to the cytoplasm. The results reveal matrix viscoelasticity as a potent factor regulating stem cell morphogenesis and provide new insights into how engineered biomaterials may be leveraged to build organoids.

Steroid hormones and human choriogonadotropin influence the distribution of alpha6-integrin and desmoplakin 1 in gland-like endometrial epithelial spheroids

In human glandular endometrial epithelial cells, desmosomal and adherens junction proteins have been shown to extend from a subapically restricted lateral position to the entire lateral membrane during the implantation window of the menstrual cycle. Similarly, a menstrual cycle stage-dependent redistribution of the extracellular matrix adhesion protein α6-integrin has been reported. These changes are believed to be important for endometrial receptiveness and successful embryo implantation. To prove the hypothesis that steroid hormones and human choriogonadotropin can induce the redistribution of these adhesion molecules, we used the human endometrial cell line Ishikawa in a 3D culture system. Gland-like spheroids were grown in reconstituted basement membrane (Matrigel™). The lumen-bearing spheroids were treated for 2 or 4 days with ovarian steroids or human choriogonadotropin and then assessed by immunofluorescence microscopy. In addition, human endometrial biopsies were obtained from patients, who were in therapy for assisted reproductive technology, and were examined in parallel. Lateral redistribution of the desmosomal plaque protein desmoplakin 1 was observed in the spheroids treated either with progesterone, medroxyprogesterone acetate or human choriogonadotropin. Furthermore, the extracellular matrix adhesion protein α6-integrin showed an increased lateral membrane localization upon gestagen stimulation in the 3D culture system. The results of this study demonstrate that the 3D endometrial Ishikawa cell culture might be suited as an experimental model system to prove the effect of hormonal changes like those occurring during the window of implantation.