Organotypic Cell Culture Model Research Articles

Transferring Microclusters of P. aeruginosa Biofilms to the Air-Liquid Interface of Bronchial Epithelial Cells for Repeated Deposition of Aerosolized Tobramycin.

As an alternative to technically demanding and ethically debatable animal models, the use of organotypic and disease-relevant human cell culture models may improve the throughput, speed, and success rate for the translation of novel anti-infectives into the clinic. Besides bacterial killing, host cell viability and barrier function appear as relevant but seldomly measured readouts. Moreover, bacterial virulence factors and signaling molecules are typically not addressed in current cell culture models. Here, we describe a reproducible protocol for cultivating barrier-forming human bronchial epithelial cell monolayers on Transwell inserts and infecting them with microclusters of pre-grown mature Pseudomonas aeruginosa PAO1 biofilms under the air-liquid interface conditions. Bacterial growth and quorum sensing molecules were determined upon tobramycin treatment. The host cell response was simultaneously assessed through cell viability, epithelial barrier function, and cytokine release. By repeated deposition of aerosolized tobramycin after 1, 24, and 48 h, bacterial growth was controlled (reduction from 10 to 4 log10 CFU/mL), which leads to epithelial cell survival for up to 72 h. E-cadherin's cell-cell adhesion protein expression was preserved with the consecutive treatment, and quorum sensing molecules were reduced. However, the bacteria could not be eradicated and epithelial barrier function was impaired, similar to the currently observed situation in the clinic in lack of more efficient anti-infective therapies. Such a human-based in vitro approach has the potential for the preclinical development of novel anti-infectives and nanoscale delivery systems for oral inhalation.

House Dust Mite Exposure Causes Increased Susceptibility of Nasal Epithelial Cells to Adenovirus Infection

Adenovirus (AdV) infections in the respiratory tract may cause asthma exacerbation and allergic predisposition, and the house dust mite (HDM) may aggravate virus-induced asthma exacerbations. However, the underlying mechanisms of whether and how AdV affects asthmatic patients remains unclear. To address this question, we investigated nasal epithelial cells (NAEPCs) derived from a pediatric exacerbation study cohort for experimental analyses. We analyzed twenty-one different green-fluorescent protein- and luciferase-tagged AdV types in submerged 2D and organotypic 3D cell culture models. Transduction experiments revealed robust transduction of AdV type 5 (AdV5) in NAEPCs, which was associated with an increased uptake of AdV5 in the presence of HDM. In healthy and asthmatic NAEPCs exposed to HDM before infection, we observed a time- and dose-dependent increase of AdV5 uptake associated with upregulation of entry receptors for AdV5. Furthermore, electron microscopic and histologic analyses of 3D cell cultures revealed an impairment of the respiratory cilia after HDM exposition. This ex vivo pilot study shows the impact of AdV infection and HDM exposition in a primary cell culture model for asthma.

‘In vitro clinical trials’ platform for drug testing in patient-derived ex vivo 3D cultured human tumor tissues

Introduction: Despite the widespread utilization of animal models in preclinical drug research these do not fully represent the complexity of human cancer, which leads to significant failure rates for anticancer drugs in clinical development. In recent years, 3D organotypic cell culture models, such as organoids, have emerged to help bridge the gap between conventional in vitro models and patients. However, organoids are clonal and lack tumor associated cells, such as fibroblasts, T cells (TIL) and other immune cells which both modulate the responses to drugs and are themselves important drug targets.

In vitro virucidal activity of Echinaforce\xae, an Echinacea purpurea preparation, against coronaviruses, including common cold coronavirus 229E and SARS-CoV-2

BackgroundCoronaviruses (CoVs) were long thought to only cause mild respiratory and gastrointestinal symptoms in humans but outbreaks of Middle East Respiratory Syndrome (MERS)-CoV, Severe Acute Respiratory Syndrome (SARS)-CoV-1, and the recently identified SARS-CoV-2 have cemented their zoonotic potential and their capacity to cause serious morbidity and mortality, with case fatality rates ranging from 4 to 35%. Currently, no specific prophylaxis or treatment is available for CoV infections. Therefore we investigated the virucidal and antiviral potential of Echinacea purpurea (Echinaforce®) against human coronavirus (HCoV) 229E, highly pathogenic MERS- and SARS-CoVs, as well as the newly identified SARS-CoV-2, in vitro.MethodsTo evaluate the antiviral potential of the extract, we pre-treated virus particles and cells and evaluated remaining infectivity by limited dilution. Furthermore, we exposed cells to the extract after infection to further evaluate its potential as a prophylaxis and treatment against coronaviruses. We also determined the protective effect of Echinaforce® in re-constituted nasal epithelium.ResultsIn the current study, we found that HCoV-229E was irreversibly inactivated when exposed to Echinaforce® at 3.2 μg/ml IC50. Pre-treatment of cell lines, however, did not inhibit infection with HCoV-229E and post-infection treatment had only a marginal effect on virus propagation at 50 μg/ml. However, we did observe a protective effect in an organotypic respiratory cell culture system by exposing pre-treated respiratory epithelium to droplets of HCoV-229E, imitating a natural infection. The observed virucidal activity of Echinaforce® was not restricted to common cold coronaviruses, as both SARS-CoV-1 and MERS-CoVs were inactivated at comparable concentrations. Finally, the causative agent of COVID-19, SARS-CoV-2 was also inactivated upon treatment with 50μg/ml Echinaforce®.ConclusionsThese results show that Echinaforce® is virucidal against HCoV-229E, upon direct contact and in an organotypic cell culture model. Furthermore, MERS-CoV and both SARS-CoV-1 and SARS-CoV-2 were inactivated at similar concentrations of the extract. Therefore we hypothesize that Echinacea purpurea preparations, such as Echinaforce®, could be effective as prophylactic treatment for all CoVs due to their structural similarities.

Targeting IL-11 in the treatment of BK virus-associated haemorrhagic cystitis-A promising new approach.

The BK polyomavirus (BKPyV) has pathogenic relevance especially in immunocompromised patients. No causal therapy has been established yet. Therefore, new therapeutic targets need to be identified in experimental studies. A 3D organotypic cell culture model with primary urothelial cells and fibroblasts was used as infection model. The detection of virus replication was performed with quantitative polymerase chain reaction (qPCR), and immunohistochemistry (IHC) was also used for analysis. Interleukin levels were measured by enzyme‐linked immunosorbent assay (ELISA). Interestingly, the signal transducer and activator of transcription 3 (STAT3) pathway seems to be activated during infection with BKPyV, for example phosphorylated STAT3 is significantly (P < 0.0001) elevated on day 6 following infection. Therefore, we performed ELISAs for involved interleukins in STAT3 pathway. Interleukin 11 (IL‐11) was significantly (P = 0.026) elevated at day 9. Subsequently, 3D cultures were treated with IL‐11 neutralizing antibody. At day 9 following infection, the median virus replication rate is 4.4 × 106 copies/ml. The difference to replication rate without treatment was significantly lower at day 6 (P < 0.0001) and at day 9 (P < 0.0001), respectively. STAT3 pathways seem to be involved during BKPyV infection and need further investigation in experimental studies. A very promising target for treatment might be IL‐11.

Novel 3D organotypic urothelial cell culture model for identification of new therapeutic approaches in urological infections

Purpose3D organotypic cell cultures offer the possibility to study cell growth in a more in vivo like situation. To our knowledge no 3D culture of primary urothelial cells has been established yet. BK Polyomavirus (BKPyV), replicating in urothelial cells, may cause haemorrhagic cystitis in immunocompromised patients. Primary endpoints of this studyEstablishment of a 3D organotypic cell culture of primary urothelial cells and fibroblasts; use of this model as infection model for archetype BKPyV; description of first parts of viral life cycle with identification of therapeutic targets. MethodsThis is an experimental study. Primary urothelial cells were purchased from CellnTec, Bern, Switzerland; fibroblasts were isolated from the ureter of patients with no urothelial malignancy in their medical history. As main methods we used quantitative real-time PCR and immunohistochemistry. Outcomes were analysed using SPSS 23.0. ResultsWe were able to develop a 3D organotypic culture for primary urothelium. An infection with archetype BKPyV was established in this model with virus replication rates up to 6.41 × 108 copies/ml on day 9 following Infection. Interestingly, proliferation rate of the urothelial cells is significantly (p = 0.049 at day 6 following infection) elevated while cells are losing differentiation under infection. Phosphorylated STAT3 is also significantly elevated (p < 0.0001) during infection. ConclusionsThe established of urothelial 3D cultures is a new method to study several urothelial diseases. The archetype BKPyV infection model is novel and the first method to study archetype viral life cycle. The STAT3 pathway might be an interesting target for the development of a causal therapy.

Bacillus/Trapa japonica Fruit Extract Ferment Filtrate enhances human hair follicle dermal papilla cell proliferation via the Akt/ERK/GSK-3\u03b2 signaling pathway

BackgroundDespite advances in medical treatments, the proportion of the population suffering from alopecia is increasing, creating a need for new treatments to control hair loss and prevent balding. Treatments based on plant-derived compounds could potentially prevent hair loss. Human hair follicle dermal papilla (HDP) cells, a type of specialized fibroblast in the hair bulb, play an essential role in controlling hair growth and in conditions such as androgenic alopecia. We examined the effect of Bacillus/Trapa japonica fruit ferment filtrate extracts (TJFs) on HDP cells to determine whether activation of the Akt/ERK/GSK-3β signaling pathway improved HDP cell proliferation.MethodsWe prepared TJFs using various methods. The extract properties were analyzed using WST-1, Lowry, and cell migration assays as well as immunofluorescence staining. We also determined the cell cycle stage and performed western blotting and an in ovo chick chorioallantoic membrane assay. Last, we constructed an organotypic three-dimensional cell culture model for immunohistochemical use.ResultsOur study confirmed that the TJFs contained numerous peptides and five unknown fractions. The TJFs stimulated HDP cell proliferation and migration via the Akt/ERK/GSK-3β signaling pathway. To verify that the Akt/ERK/GSK-3β pathway affected HDP cell proliferation, we treated HDP cells with LY294002 (an Akt inhibitor), BIO (a GSK-3β inhibitor), and PD98059 (an ERK inhibitor). The TJFs also induced cell cycle progression, inhibited type І 5α-reductase, decreased apoptosis, and enhanced angiogenesis (vascular expansion). In addition to these signaling pathways, proteins including insulin-like growth factor-1 and keratinocyte growth factor, stimulating hair growth, were detected in the three-dimensional cell culture model.ConclusionsOur results confirmed that TJFs enhance HDP cell proliferation via the Akt/ERK/GSK-3β signaling pathway, suggesting a potential treatment for alopecia.

High-throughput identification of factors promoting neuronal differentiation of human neural progenitor cells in microscale 3D cell culture.

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose-response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models.

Abstract 1158: Phenotypic screening using AMIDA identifies different drug responses in breast and prostate cancer cell lines in an organotypic cell culture model

Abstract Organotypic 3D cell culture models combined with screening modalities and automated high-content image analyses provide tools to gain a spectrum of biologically relevant information simultaneously from drug responses in tumor cells. This includes information about cell growth, death, differentiation and tumor cell invasion. In this study, we examined the phenotypic drug responses of prostate cancer cell lines PC-3 and LNCaP, and breast cancer cell lines MDA-MB-231 (SA) and MCF-7 cultured in a miniaturized, imaging-optimized, Matrigel-based organotypic screening platform. We demonstrate the use of the 3D cell culture technology combined with automated morphometric image data analysis software AMIDA, for phenotypic, high-content screening. The emerging tumor organoids were treated with the cytostatic drugs doxorubicin, docetaxel and paclitaxel, a selective inhibitor of matrix metalloproteinase-13 (WAY170523), and with ROCK-inhibitors RKI-1447 and Y-27632. Treatments were conducted at seven different drug concentrations for 4-10 days. At the end point, confocal live cell images were captured and analyzed using AMIDA. Among others, the numerical data representing cell growth (Area) and cell invasion (Appendages) were visualized and used for statistics. EC50 values were calculated based on the Area-parameter derived from AMIDA analysis. All cell lines initially formed multicellular, round organoids. PC-3 cells formed round and well-differentiated structures but spontaneously converted around day 9 of culture into structures showing massive, string-like collective invasion into the surrounding matrix. A different pattern of cell invasion was observed in MDA-MB-231 organoids, which developed strong and multicellular extensions by day 7-8. Interestingly, both ROCK-inhibitors promoted the invasion of PC-3 cells, as detected by phenotypic analysis with AMIDA. In contrast, Y-27632 reduced the invasion of MDA-MB-231 cells, whereas both ROCK-inhibitors induced cell invasion in MCF-7 cells at high concentration. WAY170523 inhibited the invasion of PC-3 cells but not of MDA-MB-231 cells, pointing to a different mechanism of invasiveness. All cell lines were highly sensitive to doxorubicin, docetaxel and paclitaxel at nanomolar range, as detected by the decrease of cell invasion, reduced organoid size, and increased cell death. The breast cancer cells were more sensitive to taxanes than PC-3 cells. Organotypic 3D cultures combined with high-content phenotypic analysis with AMIDA software provide a quantitative view of drug effects and enable assessment of differential drug responses on various cell lines. Citation Format: Mervi Toriseva, Katja Fagerlund, Jesse Mattsson, Tiina E. Kähkönen, Ilmari Ahonen, Malin Åkerfelt, Jenni Bernoulli, Jussi M. Halleen, Matthias Nees, Jenni H. Mäki-Jouppila. Phenotypic screening using AMIDA identifies different drug responses in breast and prostate cancer cell lines in an organotypic cell culture model [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 1158.

Engineering Hydrogel Microenvironments to Recapitulate the Stem Cell Niche.

Stem cells are a powerful resource for many applications including regenerative medicine, patient-specific disease modeling, and toxicology screening. However, eliciting the desired behavior from stem cells, such as expansion in a naïve state or differentiation into a particular mature lineage, remains challenging. Drawing inspiration from the native stem cell niche, hydrogel platforms have been developed to regulate stem cell fate by controlling microenvironmental parameters including matrix mechanics, degradability, cell-adhesive ligand presentation, local microstructure, and cell-cell interactions. We survey techniques for modulating hydrogel properties and review the effects of microenvironmental parameters on maintaining stemness and controlling differentiation for a variety of stem cell types. Looking forward, we envision future hydrogel designs spanning a spectrum of complexity, ranging from simple, fully defined materials for industrial expansion of stem cells to complex, biomimetic systems for organotypic cell culture models.

Glutathione peroxidase 7 suppresses cancer cell growth and is hypermethylated in gastric cancer.

Gastric cancer (GC) is one of the most common cancers in the world, and remains the third leading cause of cancer-related deaths worldwide. Glutathione peroxidase 7 (GPX7) is a member of GPX family which is downregulated in some cancer types. In this study, we investigated the expression, regulation, and molecular function of GPX7 in gastric cancer using 2D and 3D in vitro models and de-identified human tissue samples. Quantitative real-time RT-PCR, immunofluorescence, Western blot, 3D organotypic cultures, and pyrosequencing assays were used. We detected downregulation of GPX7 in all 7 gastric cancer cell lines that we tested and in approximately half (22/45) of human gastric cancer samples, as compared to histologically normal gastric tissues. Quantitative bisulfite pyrosequencing methylation analysis demonstrated DNA hypermethylation (> 10% methylation level) of GPX7 promoter in all 7 gastric cancer cell lines and in 56% (25/45) of gastric cancer samples, as compared to only 13% (6/45) in normal samples (p < 0.0001). Treatment of AGS and SNU1 cells with 5-Aza-2′-deoxycytidine led to a significant demethylation of GPX7 promoter and restored the expression of GPX7. In vitro assays showed that reconstitution of GPX7 significantly suppressed gastric cancer cell growth in both 2D and 3D organotypic cell culture models. This growth suppression was associated with inhibition of cell proliferation and induction of cell death. We detected significant upregulation of p27 and cleaved PARP and downregulation of Cyclin D1 upon reconstitution of GPX7. Taken together, we conclude that epigenetic silencing of GPX7 could play an important role in gastric tumorigenesis and progression.

Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models.

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems.

83 - Establishment of a 3D organotypic urothelial cell culture model as infection model system for BK polyomavirus – viral lifecycle and identification of new therapeutic targets

83 - Establishment of a 3D organotypic urothelial cell culture model as infection model system for BK polyomavirus – viral lifecycle and identification of new therapeutic targets

Lessons from Animal Models of Cytoplasmic Intermediate Filament Proteins.

Cytoplasmic intermediate filaments (IFs) represent a major cytoskeletal network contributing to cell shape, adhesion and migration as well as to tissue resilience and renewal in numerous bilaterians, including mammals. The observation that IFs are dispensable in cultured mammalian cells, but cause tissue-specific, life-threatening disorders, has pushed the need to investigate their function in vivo. In keeping with human disease, the deletion or mutation of murine IF genes resulted in highly specific pathologies. Epidermal keratins, together with desmin, are essential to protect corresponding tissues against mechanical force but also participate in stabilizing cell adhesion and in inflammatory signalling. Surprisingly, other IF proteins contribute to tissue integrity to a much lesser extent than anticipated, pointing towards their role in stress situations. In support, the overexpression of small chaperones or the interference with inflammatory signalling in several settings has been shown to rescue severe tissue pathologies that resulted from the expression of mutant IF proteins. It stills remains an open issue whether the wide range of IF disorders share similar pathomechanisms. Moreover, we lack an understanding how IF proteins participate in signalling processes. Now, with a large number of mouse models in hand, the next challenge will be to develop organotypic cell culture models to dissect pathomechanisms at the molecular level, to employ Crispr/Cas-mediated genome engineering to optimize models and, finally, to combine available animal models with medicinal chemistry for the development of molecular therapies.

Association Between Germline Mutation in VSIG10L and Familial Barrett Neoplasia.

Esophageal adenocarcinoma and its precursor lesion Barrett esophagus have seen a dramatic increase in incidence over the past 4 decades yet marked genetic heterogeneity of this disease has precluded advances in understanding its pathogenesis and improving treatment. To identify novel disease susceptibility variants in a familial syndrome of esophageal adenocarcinoma and Barrett esophagus, termed familial Barrett esophagus, by using high-throughput sequencing in affected individuals from a large, multigenerational family. We performed whole exome sequencing (WES) from peripheral lymphocyte DNA on 4 distant relatives from our multiplex, multigenerational familial Barrett esophagus family to identify candidate disease susceptibility variants. Gene variants were filtered, verified, and segregation analysis performed to identify a single candidate variant. Gene expression analysis was done with both quantitative real-time polymerase chain reaction and in situ RNA hybridization. A 3-dimensional organotypic cell culture model of esophageal maturation was utilized to determine the phenotypic effects of our gene variant. We used electron microscopy on esophageal mucosa from an affected family member carrying the gene variant to assess ultrastructural changes. Identification of a novel, germline disease susceptibility variant in a previously uncharacterized gene. A multiplex, multigenerational family with 14 members affected (3 members with esophageal adenocarcinoma and 11 with Barrett esophagus) was identified, and whole-exome sequencing identified a germline mutation (S631G) at a highly conserved serine residue in the uncharacterized gene VSIG10L that segregated in affected members. Transfection of S631G variant into a 3-dimensional organotypic culture model of normal esophageal squamous cells dramatically inhibited epithelial maturation compared with the wild-type. VSIG10L exhibited high expression in normal squamous esophagus with marked loss of expression in Barrett-associated lesions. Electron microscopy of squamous esophageal mucosa harboring the S631G variant revealed dilated intercellular spaces and reduced desmosomes. This study presents VSIG10L as a candidate familial Barrett esophagus susceptibility gene, with a putative role in maintaining normal esophageal homeostasis. Further research assessing VSIG10L function may reveal pathways important for esophageal maturation and the pathogenesis of Barrett esophagus and esophageal adenocarcinoma.

Validation of Novel Biomarkers for Prostate Cancer Progression by the Combination of Bioinformatics, Clinical and Functional Studies.

The identification and validation of biomarkers for clinical applications remains an important issue for improving diagnostics and therapy in many diseases, including prostate cancer. Gene expression profiles are routinely applied to identify diagnostic and predictive biomarkers or novel targets for cancer. However, only few predictive markers identified in silico have also been validated for clinical, functional or mechanistic relevance in disease progression. In this study, we have used a broad, bioinformatics-based approach to identify such biomarkers across a spectrum of progression stages, including normal and tumor-adjacent, premalignant, primary and late stage lesions. Bioinformatics data mining combined with clinical validation of biomarkers by sensitive, quantitative reverse-transcription PCR (qRT-PCR), followed by functional evaluation of candidate genes in disease-relevant processes, such as cancer cell proliferation, motility and invasion. From 300 initial candidates, eight genes were selected for validation by several layers of data mining and filtering. For clinical validation, differential mRNA expression of selected genes was measured by qRT-PCR in 197 clinical prostate tissue samples including normal prostate, compared against histologically benign and cancerous tissues. Based on the qRT-PCR results, significantly different mRNA expression was confirmed in normal prostate versus malignant PCa samples (for all eight genes), but also in cancer-adjacent tissues, even in the absence of detectable cancer cells, thus pointing to the possibility of pronounced field effects in prostate lesions. For the validation of the functional properties of these genes, and to demonstrate their putative relevance for disease-relevant processes, siRNA knock-down studies were performed in both 2D and 3D organotypic cell culture models. Silencing of three genes (DLX1, PLA2G7 and RHOU) in the prostate cancer cell lines PC3 and VCaP by siRNA resulted in marked growth arrest and cytotoxicity, particularly in 3D organotypic cell culture conditions. In addition, silencing of PLA2G7, RHOU, ACSM1, LAMB1 and CACNA1D also resulted in reduced tumor cell invasion in PC3 organoid cultures. For PLA2G7 and RHOU, the effects of siRNA silencing on proliferation and cell-motility could also be confirmed in 2D monolayer cultures. In conclusion, DLX1 and RHOU showed the strongest potential as useful clinical biomarkers for PCa diagnosis, further validated by their functional roles in PCa progression. These candidates may be useful for more reliable identification of relapses or therapy failures prior to the recurrence local or distant metastases.

A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells.

Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the epithelium lining the gastrointestinal tract early in infection. The lack of suitable in vivo and in vitro models to study CVB infection of the gastrointestinal epithelium has limited our understanding of the events that surround infection of these specialized cells. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of human intestinal epithelial cells that better models the gastrointestinal epithelium in vivo. By applying this 3-D model, which recapitulates many aspects of the gastrointestinal epithelium in vivo, to the study of CVB infection, our work provides a new cell system to model the mechanisms by which CVB infects the intestinal epithelium, which may have a profound impact on CVB pathogenesis. Podcast: A podcast concerning this article is available.

Abstract NG06: The Trojan Horse Strategy: Packaging chemotherapeutics can help alleviate toxicity

Abstract NG06: The Trojan Horse Strategy: Packaging chemotherapeutics can help alleviate toxicity

Fibroblasts Protect Melanoma Cells from the Cytotoxic Effects of Doxorubicin

Melanoma is the most aggressive form of skin cancer and until recently, it was extremely resistant to radio-, immuno-, and chemotherapy. Despite the latest success of BRAF V600E-targeted therapies, responses are typically short lived and relapse is all but certain. Furthermore, a percentage (40%) of melanoma cells is BRAF wild type. Emerging evidence suggests a role for normal host cells in the occurrence of drug resistance. In the current study, we compared a variety of cell culture models with an organotypic incomplete skin culture model (the "dermal equivalent") to investigate the role of the tissue microenvironment in the response of melanoma cells to the chemotherapeutic agent doxorubicin (Dox). In the dermal equivalent model, consisting of fibroblasts embedded in type I collagen matrix, melanoma cells showed a decreased cytotoxic response when compared with less complex culture conditions, such as seeding on plastic cell culture plate (as monolayers cultures) or on collagen gel. We further investigated the role of the microenvironment in p53 induction and caspase 3 and 9 cleavage. Melanoma cell lines cultured on dermal equivalent showed decreased expression of p53 after Dox treatment, and this outcome was accompanied by induction of interleukin IL-6, IL-8, and matrix metalloproteinases 2 and 9. Here, we show that the growth of melanoma cells in the dermal equivalent model inflects drug responses by recapitulating important pro-survival features of the tumor microenvironment. These studies indicate that the presence of stroma enhances the drug resistance of melanoma in vitro, more closely mirroring the in vivo phenotype. Our data, thus, demonstrate the utility of organotypic cell culture models in providing essential context-dependent information critical for the development of new therapeutic strategies for melanoma. We believe that the organotypic model represents an improved screening platform to investigate novel anti-cancer agents, as it provides important insights into tumor-stromal interactions, thus assisting in the elucidation of chemoresistance mechanisms.

Polarity in Mammalian Epithelial Morphogenesis

Cell polarity is fundamental for the architecture and function of epithelial tissues. Epithelial polarization requires the intervention of several fundamental cell processes, whose integration in space and time is only starting to be elucidated. To understand what governs the building of epithelial tissues during development, it is essential to consider the polarization process in the context of the whole tissue. To this end, the development of three-dimensional organotypic cell culture models has brought new insights into the mechanisms underlying the establishment and maintenance of higher-order epithelial tissue architecture, and in the dynamic remodeling of cell polarity that often occurs during development of epithelial organs. Here we discuss some important aspects of mammalian epithelial morphogenesis, from the establishment of cell polarity to epithelial tissue generation.