Three-dimensional Organotypic Culture Research Articles

MicroRNAome profiling of breast cancer unveils hsa-miR-5683 as a tumor suppressor microRNA predicting favorable clinical outcome

BackgroundBreast cancer is a heterogeneous disease with diverse molecular subtypes, underscoring a better understanding of its molecular features and underlying regulatory mechanisms. Therefore, identifying novel prognostic biomarkers and therapeutic targets is crucial for advancing the current standard of care for breast cancer patients.MethodsNinety-six formalin-fixed paraffin-embedded (FFPE) breast cancer samples underwent miRNAome profiling using QIAseq microRNA library kit and sequencing on Illumina platform. Mature miRNA quantification was conducted using CLC Genomics Workbench v21.0.5, while Relapse-free survival (RFS) analysis was conducted using RStudio 2023.09.1. Gain-of-function studies were conducted using miRNA mimics, while the effects of miRNA exogenous expression on cancer hallmark were assessed using 2-dimentional (2D) proliferation assay, three-dimensional (3D) organotypic culture, and live-dead staining. TargetScan database and Ingenuity Pathway Analysis (IPA) were used for miRNA target identification.ResultsHierarchical clustering based on miRNA expression revealed distinct patterns in relation to PAM50 classification and identified miRNAs panels associated with luminal, HER2, and basal subtypes. hsa-miR-5683 emerged as a potential prognostic biomarker, showing a favorable correlation with RFS and suppressing tumorigenicity under 2D and 3D conditions in triple-negative breast cancer (TNBC) models. Findings were further extended to the MCF7 hormone receptor positive (HR+) model. Transcriptomic profiling of hsa-miR-5683 overexpressing TNBC cells revealed its potential role in key oncogenic pathways. Integration of downregulated genes and CRISPR-Cas9 perturbational effects identified ACLY, RACGAP1, AK4, MRPL51, CYB5B, MKRN1, TMEM230, NUP54, ANAPC13, PGAM1, and SOD1 as bona fide gene targets for hsa-miR-5683.ConclusionsOur data provides comprehensive miRNA expression atlas in breast cancer subtypes and underscores the prognostic and therapeutic significance of numerous miRNAs, including hsa-miR-5683 in TNBC. The identified gene targets unravel the intricate regulatory network in TNBC progression, suggesting promising avenues for further research and targeted therapeutic interventions.

Involvement of CacyBP/SIP in differentiation and the immune response of HaCaT keratinocytes

CacyBP/SIP is a multifunctional protein present in various cells and tissues. However, its expression and role in the epidermis has not been explored so far. In this work, using RT-qPCR, Western blot analysis and three-dimensional (3D) organotypic cultures of HaCaT keratinocytes we show that CacyBP/SIP is present in the epidermis. To investigate the possible role of CacyBP/SIP in keratinocytes we obtained CacyBP/SIP knockdown cells and studied the effect of CacyBP/SIP deficiency on their differentiation and response to viral infection. We found that CacyBP/SIP knockdown results in reduced expression of epidermal differentiation markers in both undifferentiated and differentiated HaCaT cells. Since epidermis is engaged in immune defense, the impact of CacyBP/SIP knockdown on this process was also analyzed. By applying RT-qPCR and Western blot it was found that poly(I:C), a synthetic analog of double-stranded RNA that mimics viral infection, stimulated the expression of genes involved in antiviral response, such as IFIT1, IFIT2 and OASL. Interestingly, following poly(I:C) stimulation, the level of expression of these genes was significantly lower in cells with CacyBP/SIP knockdown than control ones. Since the signaling pathway mediating cellular responses to viral infection involves, among others, the STAT1 transcription factor, we measured its activity using luciferase assay and found that it was lower in CacyBP/SIP knockdown HaCaT cells. Altogether, the presented results indicate that CacyBP/SIP promotes epidermal differentiation and might be involved in response of the skin cells to viral infection.

Bepotastine Sensitizes Ovarian Cancer to PARP Inhibitors through Suppressing NF-κB-Triggered SASP in Cancer-Associated Fibroblasts.

Therapy-induced senescence (TIS) is common in tumor cells treated with PARP inhibitors (PARPis) and can serve as a promising target for improving PARPi efficacy. However, whether stromal components within the tumor microenvironment undergo TIS caused by PARPis and contribute to consequential treatment failure remain unclear. We previously revealed that PARPis triggered a senescence-like secretory phenotype in stromal fibroblasts. Here, we further explored PARPi-induced senescence in the stroma, its contribution to PARPi resistance, and opportunities to leverage stromal TIS for improved PARPi sensitivity. In this study, we demonstrated that tumor tissues from patients treated with neoadjuvant PARPis showed a significant senescence-like phenotype in the stroma. Moreover, PARPi-induced senescent cancer-associated fibroblasts (CAFs) displayed a senescence-associated secretory phenotype (SASP) profile that was sufficient to induce tumor resistance to PARPis in both homologous recombination-deficient (HRD) and -proficient ovarian cancer cells. Using the GLAD4U database, we found that bepotastine, an approved H1-antihistamine, inhibited the SASP of PARPi-primed CAFs at clinical serum concentrations. We further demonstrated that bepotastine attenuated fibroblast-facilitated tumor resistance to PARPis in three-dimensional organotypic cultures and HRD-positive patient-derived xenograft models. Mechanistically, bepotastine suppressed PARPi-triggered SASP by inhibiting NF-κB signaling independent of the histamine H1 receptor. Taken together, our results highlight the importance of stromal TIS and SASP in PARPi resistance, and targeting SASP with bepotastine may be a promising therapeutic option for improving PARPi sensitivity in ovarian cancer.

Incorporating NK Cells in a Three-Dimensional Organotypic Culture System for Human Skin Stem Cells: Modeling Skin Diseases and Immune Cell Interplay.

Natural killer (NK) cells are a part of a sophisticated immune system that is necessary for the skin because it is a crucial organ that is continually exposed to environmental influences. Recent studies have shown that NK cell incorporation into three-dimensional (3D) organotypic culture systems for human skin stem cells provides a physiologically relevant environment to study the interactions between immune cells and skin cells, making it a powerful tool for simulating skin diseases and researching these interactions. It has been shown that adding NK cells to 3D organotypic culture systems can improve keratinocyte differentiation and control inflammation in a variety of skin conditions, including psoriasis. In order to increase our knowledge of skin diseases and immune cell interactions, this work intends to propose an optimum approach for adding NK cells to a 3D organotypic culturing system for human skin stem cells. By better comprehending these relationships, researchers hope to develop novel treatments for skin diseases that are more effective and cause fewer side effects than current treatments. To completely understand the mechanisms underlying these interactions and to create new treatments for skin diseases, more research is required. In conclusion, NK cell integration into 3D organotypic culture systems offers a potent tool to investigate immune cell interactions with skin cells in a physiologically appropriate setting, which may result in major improvements in the treatment of skin diseases.

Collective cell migration is spatiotemporally regulated during mammary epithelial bifurcation.

Branched epithelial networks are generated through an iterative process of elongation and bifurcation. We sought to understand bifurcation of the mammary epithelium. To visualize this process, we utilized three-dimensional (3D) organotypic culture and time-lapse confocal microscopy. We tracked cell migration during bifurcation and observed local reductions in cell speed at the nascent bifurcation cleft. This effect was proximity dependent, as individual cells approaching the cleft reduced speed, whereas cells exiting the cleft increased speed. As the cells slow down, they orient both migration and protrusions towards the nascent cleft, while cells in the adjacent branches orient towards the elongating tips. We next tested the hypothesis that TGF-β signaling controls mammary branching by regulating cell migration. We first validated that addition of TGF-β1 (TGFB1) protein increased cleft number, whereas inhibition of TGF-β signaling reduced cleft number. Then, consistent with our hypothesis, we observed that pharmacological inhibition of TGF-β1 signaling acutely decreased epithelial migration speed. Our data suggest a model for mammary epithelial bifurcation in which TGF-β signaling regulates cell migration to determine the local sites of bifurcation and the global pattern of the tubular network.

Distinct Dynamics of Migratory Response to PD-1 and CTLA-4 Blockade Reveals New Mechanistic Insights for Potential T-Cell Reinvigoration following Immune Checkpoint Blockade.

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), two clinically relevant targets for the immunotherapy of cancer, are negative regulators of T-cell activation and migration. Optimizing the therapeutic response to CTLA-4 and PD-1 blockade calls for a more comprehensive insight into the coordinated function of these immune regulators. Mathematical modeling can be used to elucidate nonlinear tumor-immune interactions and highlight the underlying mechanisms to tackle the problem. Here, we investigated and statistically characterized the dynamics of T-cell migration as a measure of the functional response to these pathways. We used a previously developed three-dimensional organotypic culture of patient-derived tumor spheroids treated with anti-CTLA-4 and anti-PD-1 antibodies for this purpose. Experiment-based dynamical modeling revealed the delayed kinetics of PD-1 activation, which originates from the distinct characteristics of PD-1 and CTLA-4 regulation, and followed through with the modification of their contributions to immune modulation. The simulation results show good agreement with the tumor cell reduction and active immune cell count in each experiment. Our findings demonstrate that while PD-1 activation provokes a more exhaustive intracellular cascade within a mature tumor environment, the time-delayed kinetics of PD-1 activation outweighs its preeminence at the individual cell level and consequently confers a functional dominance to the CTLA-4 checkpoint. The proposed model explains the distinct immunostimulatory pattern of PD-1 and CTLA-4 blockade based on mechanisms involved in the regulation of their expression and may be useful for planning effective treatment schemes targeting PD-1 and CTLA-4 functions.

A High-Throughput Platform for Culture and 3D Imaging of Organoids.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

Organotypic Human Skin Cultures Incorporating Primary Melanocytes.

Three-dimensional (3D) human organotypic skin cultures provide a physiologically relevant model that recapitulates in vivo skin features. Most commonly, organotypic skin cultures are created by seeding isolated epidermal keratinocytes onto a collagen/fibroblast plug and lifting to an air liquid interface. These conditions are sufficient to drive stratification and differentiation of the keratinocytes to form an epidermal-like sheet with remarkable similarities to human epidermis. Coupled with genetic or pharmacological treatments, these cultures provide a powerful tool for elucidating keratinocyte biology. Recent focus has been placed on increasing the utility of organotypic skin cultures by incorporating other cell types that are present in the skin, such as melanocytes, immune cells, and other cells. Here we describe a step-by-step protocol for the isolation of neonatal human epidermal keratinocytes and melanocytes from foreskins, and the creation of organotypic skin cultures that include both cell types. We also describe methods that can be used to assess melanocyte behavior in these organotypic cultures, including methods for whole mount staining, measurement of melanocyte dendricity, staining for pigment, and 5-bromo-2'-deoxyuridine (BrdU) labeling for identification of proliferating cells. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of primary cells Alternate Protocol: Isolation of primary cells using differential trypsinization Basic Protocol 2: Organotypic culture protocol Support Protocol 1: Culture and maintenance of NHEKs and melanocytes Support Protocol 2: Lentiviral transduction of melanocytes Support Protocol 3: Retroviral transduction of NHEKs Support Protocol 4: Whole mount immunostaining protocol Support Protocol 5: Measuring melanocyte dendricity Support Protocol 6: Fontana-Masson staining protocol Support Protocol 7: BrdU labeling and staining.

Evaluation of the matrix metalloproteinase 9 (MMP9) inhibitor Andecaliximab as an Anti-invasive therapeutic in Head and neck squamous cell carcinoma

ObjectivesLocoregional and lymphovascular involvement of invasive head and neck squamous cell carcinoma (HNSCC) complicates curative treatment. Matrix metalloproteinase (MMP) 9 is a negative prognostic marker in HNSCC and targets multiple extracellular matrix (ECM) substrates, where it contributes to breaching basement membrane and stromal barriers enabling invasive spread. Andecaliximab (ADX) is a second-generation MMP9 inhibitor well tolerated in clinical trials of gastric and pancreatic adenocarcinoma. The impact of selective MMP9 targeting by ADX in HNSCC has not been evaluated. Materials and MethodsEstablished and patient-derived xenograft (PDX) cell lines were utilized in HNSCC invasion assays to determine the inhibitory ability of MMP9-mediated invasion by ADX. MMP9 expression was confirmed using immunohistochemistry (IHC) and immunoblotting. ECM degradation was evaluated with confocal microscopy. Cell invasion from tumor spheroids was monitored by phase microscopy. Histological evaluation was used to determine ADX efficacy in three-dimensional organotypic cultures containing cancer associated fibroblasts (CAFs). ResultsMMP9 was expressed in all established and PDX-derived cell lines. While the broad spectrum clinical MMP inhibitor marimastat (BB2516) blocked HNSCC invadopodia function and tumor spheroid invasion, ADX treatment failed to inhibit invadopodia-based matrix degradation, tumor cell or fibroblast-driven ECM invasion in collagen I-based matrices. ConclusionADX monotherapy was ineffective at blocking initial MMP-dependent events of HNSCC invasion, likely due to redundant functions of additional non-targeted MMPs produced by tumor cells and microenvironment. Combination of ADX with existing and emerging therapies targeting additional MMP activation pathways may warrant future investigation.

Abstract 3456: Discrepancies in the metastatic potential of high-grade serous ovarian cancer cells representing disease progression when comparing two-dimensional assays versus three-dimensional organotypic culture model systems

Abstract Due to the lack of early symptoms and valid screening methods, most patients with the most prevalent histotype of ovarian cancer, high-grade serous ovarian cancer (HGSOC), are diagnosed at late stage, at which point metastasis has already occurred. The purpose of this study was to characterize the migratory, invasive, and adhesive abilities of HGSOC cells, representing different stages of disease evolution, in both two-dimensional (2D) and three-dimensional (3D) organotypic assays. Five HGSOC cell lines, collected at different stages of disease progression were used throughout this study; PEO1, PEO4, and PEO6 cells were obtained from a first patient, whereas PEO14 and PEO23 cells were isolated from a second patient. Both wound healing (WH) and Boyden chamber (BC) assays were performed to study the migratory capacity of each cell line, while invasion was assessed by adding a layer of extracellular matrix to the BC assays. Finally, 2D adhesion assays were performed on fibronectin coated plates. On the other hand, 3D organotypic models composed of a mixture of collagen I and fibroblasts, topped with a monolayer of mesothelial cells, were constructed to further study the metastatic potential of all five HGSOC cell lines; HGSOC were plated on top of the mesothelial cells, and their adhesive behavior and capacity to displace mesothelial cells was followed by fluorescence microscopy. In 2D studies, the migratory and invasive capacities of the HGSOC cells decreased along disease evolution in both cell line series. Moreover, a distinct migration pattern was observed; cell lines representing early-stage disease had a tendency to migrate as individual cells, while cells representing late stage disease migrated in clusters. Also in 2D conditions, adhesion rates were found to be similar between all cell lines. Noteworthy, these results obtained in 2D conditions were somewhat contradicted by the results obtained in the 3D organotypic models, as both late-state disease PEO6 and PEO23 cells adhered more rapidly to the mesothelial cell monolayer than their early-stage counterparts. Furthermore, cell lines previously found to be tumorigenic in vivo such as PEO4, PEO6, and PEO14, demonstrated to trigger higher rates of mesothelial cell displacement in the 3D organotypic model system. This mesothelial cell displacement was also observed after exposure of mesothelial cells to conditioned media derived from PEO4, PEO6, or PEO14 cultures. In conclusion, 2D in vitro assays demonstrated lower migration and invasion capacities at late- rather than early-stage disease, while the opposite was observed in the organotypic models. Mesothelial cell displacement was found to be associated with tumorigenicity in vivo and to be independent of physical interactions between cancer cells and mesothelial cells. Citation Format: Sabrina J. Ritch, Alicia A. Goyeneche, Abu S. Noman, Carlos M. Telleria. Discrepancies in the metastatic potential of high-grade serous ovarian cancer cells representing disease progression when comparing two-dimensional assays versus three-dimensional organotypic culture model systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3456.

Automated high-speed 3D imaging of organoid cultures with multi-scale phenotypic quantification.

Current imaging approaches limit the ability to perform multi-scale characterization of three-dimensional (3D) organotypic cultures (organoids) in large numbers. Here, we present an automated multi-scale 3D imaging platform synergizing high-density organoid cultures with rapid and live 3D single-objective light-sheet imaging. It is composed of disposable microfabricated organoid culture chips, termed JeWells, with embedded optical components and a laser beam-steering unit coupled to a commercial inverted microscope. It permits streamlining organoid culture and high-content 3D imaging on a single user-friendly instrument with minimal manipulations and a throughput of 300 organoids per hour. We demonstrate that the large number of 3D stacks that can be collected via our platform allows training deep learning-based algorithms to quantify morphogenetic organizations of organoids at multi-scales, ranging from the subcellular scale to the whole organoid level. We validated the versatility and robustness of our approach on intestine, hepatic, neuroectoderm organoids and oncospheres.

Three-Dimensional Organotypic Cultures Reshape the microRNAs Transcriptional Program in Breast Cancer Cells.

Simple SummaryThree-dimensional (3D) cell cultures have several advantages over conventional monolayer two-dimensional (2D) cultures as they can better mimic tumor biology. This study delineates the changes in microRNAs (miRNAs) expression patterns of breast cancer cells cultured in 3D and 2D conditions. 3D organotypic cultures showed morphological changes such as cell–cell and cell–extracellular matrix interactions associated with a loss of polarity and reorganization on bulk structures in both basal Hs578T and luminal T47D breast cancer cells. Data indicate that downregulated miRNAs in Hs578T 3D cultures, relative to the 2D condition, contribute to a positive regulation of biological processes such as response to hypoxia and focal adhesion, whereas overexpressed miRNAs were related to negative regulation of the cell cycle. Remarkably, the reprogramming of miRNAs transcriptional profile was accompanied by changes in the expression of key miRNA/mRNA coregulation networks, such as miR-935/HIF-1A, which correlated with the expression found in clinical breast tumors and predicted poor patient outcomes. These data have implications in our understanding of cancer biology and impact the miRNA/mRNA regulatory axes of cells grown in 3D cultures. Our data represent a guide for novel miRNAs candidates for functional analysis, including the response to therapy and biomarkers discovery in breast cancer.The 3D organotypic cultures, which depend on the growth of cells over the extracellular matrix (ECM) used as a scaffold, can better mimic several characteristics of solid cancers that influence tumor biology and the response to drug therapies. Most of our current knowledge on cancer is derived from studies in 2D cultures, which lack the ECM-mediated microenvironment. Moreover, the role of miRNAs that is critical for fine-tuning of gene expression is poorly understood in 3D cultures. The aim of this study was to compare the miRNA expression profiles of breast cancer cells grown in 2D and 3D conditions. On an on-top 3D cell culture model using a basement membrane matrix enriched with laminin, collagen IV, entactin, and heparin-sulfate proteoglycans, the basal B (Hs578T) and luminal (T47D) breast cancer cells formed 3D spheroid-like stellate and rounded mass structures, respectively. Morphological changes in 3D cultures were observed as cell stretching, cell–cell, and cell–ECM interactions associated with a loss of polarity and reorganization on bulk structures. Interestingly, we found prolongations of the cytoplasmic membrane of Hs578T cells similar to tunneled nanotubes contacting between neighboring cells, suggesting the existence of cellular intercommunication processes and the possibility of fusion between spheroids. Expression profiling data revealed that 354 miRNAs were differentially expressed in 3D relative to 2D cultures in Hs578T cells. Downregulated miRNAs may contribute to a positive regulation of genes involved in hypoxia, catabolic processes, and focal adhesion, whereas overexpressed miRNAs modulate genes involved in negative regulation of the cell cycle. Target genes of the top ten modulated miRNAs were selected to construct miRNA/mRNA coregulation networks. Around 502 interactions were identified for downregulated miRNAs, including miR-935/HIF1A and miR-5189-3p/AKT that could contribute to cell migration and the response to hypoxia. Furthermore, the expression levels of miR-935 and its target HIF1A correlated with the expression found in clinical tumors and predicted poor outcomes. On the other hand, 416 interactions were identified for overexpressed miRNAs, including miR-6780b-5p/ANKRD45 and miR-7641/CDK4 that may result in cell proliferation inhibition and cell cycle arrest in quiescent layers of 3D cultures. In conclusion, 3D cultures could represent a suitable model that better resembles the miRNA transcriptional programs operating in tumors, with implications not only in the understanding of basic cancer biology in 3D microenvironments, but also in the identification of novel biomarkers of disease and potential targets for personalized therapies in cancer.

Studying Cell Polarity Dynamics During Cancer Initiation Using Inducible 3D Organotypic Cultures.

Epithelial tissues are highly organized structures that are structured at both the cellular and tissue levels. Individual cells are characterized by an apical membrane facing a central lumen, and a basolateral membrane that contacts adjacent cells and the basement membrane. The maintenance of apical-basal polarity is crucial for maintaining epithelial homeostasis and is considered a barrier to carcinogenesis. Apical-basal cell polarity is compromised in many epithelial cancers, such as breast, lung, and prostate, and has been associated with disease progression. Three-dimensional (3D) organotypic cultures recapitulate the 3D tissue architecture and mechanical properties found in vivo. This chapter describes methods to establish 3D organoids from human cell lines or mouse primary cells with inducible oncogene expression in polarized epithelial structures to investigate mechanisms of tumor initiation, luminal filling, and growth. The method is versatile, and simple modifications can be made to study diverse cell/tissue types and oncogenes.

A novel organ-chip system emulates three-dimensional architecture of the human epithelia and the mechanical forces acting on it

Successful translation of in vivo experimental data to human patients is an unmet need and a bottleneck in the development of effective therapeutics. Organ-on-Chip technology aims to address this need by leveraging recent significant advancements in microfabrication and biomaterials, which enable modeling of organs and their functionality. These microengineered chips offer researchers the possibility to recreate critical elements of native tissue architecture such as in vivo relevant tissue-tissue interface, air-liquid interface, and mechanical forces, including mechanical stretch and fluidic shear stress, which are crucial to recapitulate tissue level functions. Here, we present the development of a new, comprehensive 3D cell-culture system, where we combined our proprietary Organ-Chip technology with the advantages offered by three-dimensional organotypic culture. Leveraging microfabrication techniques, we engineered a flexible chip that consists of a chamber containing an organotypic epithelium, surrounded by two vacuum channels that can be actuated to stretch the hydrogel throughout its thickness. Furthermore, the ceiling of this chamber is a removable lid with a built-in microchannel that can be perfused with liquid or air and removed as needed for direct access to the tissue. The bottom part of this chamber is made from a porous flexible membrane which allows diffusive mass transport to and from the microfluidic channel positioned below the membrane. This additional microfluidic channel can be coated with endothelial cells to emulate a blood vessel and recapitulate endothelial interactions. Our results show that the Open-Top Chip design successfully addresses common challenges associated with the Organs-on-Chip technology, including the capability to incorporate a tissue-specific extracellular matrix gel seeded with primary stromal cells, to reproduce the architectural complexity of tissues by micropatterning the gel, and to extract the gel for H&E staining. We also provide proof-of-concept data on the feasibility of using the system with primary human skin and alveolar epithelial cells.

Inhibition of dipeptidyl-peptidase 4 induces upregulation of the late cornified envelope cluster in keratinocytes.

Dipeptidyl-peptidase 4 (DPP4) is a multifunctional type II transmembrane glycoprotein that is expressed on various cell surfaces. While DPP4 inhibitors have a therapeutic role in the treatment of diabetes mellitus, they are an independent risk factor in the development of bullous pemphigoid. Contrarily, there are reports of improvement in psoriasis with DPP4 inhibition. We investigated the effect of DPP4 inhibition on primary human keratinocytes to determine whether DPP4 modulates keratinocyte inflammatory signaling and keratinocyte homeostasis. We performed RNA sequencing of primary adult human keratinocytes treated with DPP4 inhibitor, identifying 424 differentially expressed genes. Gene ontology analysis revealed significant enrichment of epidermal differentiation and cornified envelope genes. Using three-dimensional organotypic cultures and a pan-late cornified envelope 2 (LCE2) antibody, we demonstrate a dose dependent relationship between DPP4 inhibition and increased expression of LCE2 during epidermal development. The late cornified envelope gene clusters are expressed at the late stages of epithelial development, responding tostimuli such as calcium and ultraviolet light. While its biologic function is not fully understood, mutations in LCE3B/LCE3C confer a 40% increased risk in the development of plaque psoriasis. While we did not identify significant modulation of keratinocyte inflammatory markers, DPP4 inhibition increased expression of the late cornified envelope may offer a potential alternative therapeutic mechanism in psoriasis.

In vitro three-dimensional organotypic culture models of the oral mucosa.

Three-dimensional, organotypic models of the oral mucosa have been developed to study a wide variety of phenomena occurring in the oral cavity. Although a number of models have been developed in academic research labs, only a few models have been commercialized. Models from academic groups offer a broader range of phenotypes while the commercial models are more focused on the oral and gingival mucosa. The commercialized models are manufactured under highly controlled conditions and meet the requirements of quality standards, which leads to high levels of reproducibility. These in vitro models have been used to evaluate the irritancy of oral care products such as toothpastes, mouthwashes, and mucoadhesives. The effects of cigarette smoke on oral cavity tissues have been studied and compared to those of e-cigarettes. Oral tissue models have facilitated investigation of the mechanisms of oral mucositis and oral candidiasis and have been used to examine transbuccal drug delivery rates and the absorption of nanoparticles. Infection studies have investigated the effects of HIV-1 along with the effects of commensal and pathogenic bacteria. More recently, a differentiated oral tissue model has been shown to express the ACE2 receptor, which is known to be important for the receptor-mediated entry of the SARS-CoV-2 coronavirus into human cells and tissues. Hence, oral mucosal models may find application in determining whether viral infection of the oral mucosa is possible and whether such infection has implications vis-a-vis the current COVID-19 pandemic. As is apparent, these models are used in a broad variety of applications and often offer advantages versus animal models in terms of reproducibility, avoiding species extrapolation, and the ethical concerns related to human and animal experimentation. The goals of this paper are to review commercially available models of the human buccal and gingival mucosa and highlight their use to gain a better understanding of a broad range of phenomena affecting tissues in the oral cavity.

Extracellular Matrix Composition Modulates the Responsiveness of Differentiated and Stem Pancreatic Cancer Cells to Lipophilic Derivate of Gemcitabine.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance. Methods: We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs. Results: PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters’ expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions. Conclusion: We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment.

Sphingosine-1-phosphate Receptor-1 Promotes Vascular Invasion and EMT in Hepatocellular Carcinoma

BackgroundIt remains unknown whether epithelial–mesenchymal transition (EMT)-mediated vascular invasion and cancer stemness are associated with sphingosine-1-phosphate receptor-1 (S1PR1) expression in human hepatocellular carcinoma (HCC). The aim of this study was to investigate the correlation between S1PR1 expression and prognosis of patients with primary HCC and to define the potential of S1PR as a therapeutic target. Materials and methodsWe investigated 108 patients who underwent primary surgical resection for HCC treatment. Expression of S1PR1 and EMT markers was analyzed to predict prognosis of patients with HCC. Furthermore, three-dimensional organotypic culture, anoikis assay, and cell invasion were performed to validate the association of S1PR1 with EMT and cancer stemness. ResultsAmong patients with HCC, the high S1PR1 expression group had significantly shorter overall survival than the low expression group. Moreover, high S1PR1 expression was significantly associated with shorter recurrence-free survival, increased risk of portal and hepatic vein invasion, and intrahepatic metastasis. Multivariate analyses revealed that S1PR1 overexpression was an independent prognostic factor in patients with HCC. S1PR1 overexpression positively correlated with vimentin and MMP-9 expression and negatively correlated with E-cadherin. In addition, S1PR1 overexpression induced EMT and enhanced tumor invasion and cancer stemness. ConclusionsS1PR1 overexpression, via EMT-induced vascular invasion and increased cancer stem cell properties, establishes a metastatic niche, enhances the capacity of hematogenous metastasis, and associates with poor outcomes in patients with HCC. Hence, S1PR1 may serve as a therapeutic target for patients with HCC with vascular invasion.

A Perspective on Organoids for Virology Research.

Animal models and cell lines are invaluable for virology research and host–pathogen interaction studies. However, it is increasingly evident that these models are not sufficient to fully understand human viral diseases. With the advent of three-dimensional organotypic cultures, it is now possible to study viral infections in the human context. This perspective explores the potential of these organotypic cultures, also known as organoids, for virology research, antiviral testing, and shaping the virology landscape.

Stem-cell-derived models: tools for studying role of microbiota in intestinal homeostasis and disease.

In this review, we will summarize the recent progress made in generating stem-cell-based organoid and enteroid models of the gastrointestinal tract and their importance in understanding the role of microbes in intestinal epithelial homeostasis and disease. Intestinal stem-cell-derived culture systems are self-organizing three-dimensional organotypic cultures that recapitulate many cellular, architectural and functional aspects of the human intestine. Progress has been made in the development of methods to incorporate additional cell lineages and physiological cues to better mimic the complexity of the intestine. Current model systems have facilitated both the study of gastrointestinal infections and interactions with normally nonpathogenic microbial residents of the gastrointestinal tract. These studies have illustrated how live microbes, or their metabolites, ligands and virulence factors influence epithelial cell differentiation, maintenance, repair, function and intestine development. Organotypic models are invaluable tools for studying host-microbe interactions that complement in-vivo experimental model systems. These models have evolved in terms of complexity and fidelity. The stem-cell-based models are already at forefront for studying host-microbe interactions and with continued development, the future looks even more promising.