Basal Epithelial Cells Research Articles

Non-invasive in vivo imaging of human corneal microstructures with optical coherence microscopy.

Non-invasive imaging systems with cellular-level resolution offer the opportunity to identify biomarkers of the early stage of corneal diseases, enabling early intervention, monitoring of disease progression, and evaluating treatment efficacy. In this study, a non-contact polarization-dependent optical coherence microscope (POCM) was developed to enable non-invasive in vivo imaging of human corneal microstructures. The system integrated quarter-wave plates into the sample and reference arms of the interferometer to enable deeper penetration of light in tissues as well as mitigate the strong specular reflection from the corneal surface. A common-path approach was adopted to enable control over the polarization in a free space configuration, thus alleviating the need for a broadband polarization-maintained fiber. The POCM achieved volumetric imaging of corneal microstructures, including endothelial cells over a field of view 0.5 × 0.5 mm2 with an almost isotropic resolution of ∼2.2 µm and a volume (500 × 500 × 2048 voxels) rate of 1 Hz. A self-interference approach between the corneal surface and underlying layers was also developed to lessen the corneal curvature and axial motion artifacts, thus enabling high-resolution imaging of microstructures in the anterior cornea, including squamous epithelial cells, wing epithelial cells, basal epithelial cells, sub-basal nerve plexus, and stromal keratocytes.

Bioconversion of chicken feather wastes into value added bioactive peptide by Geobacillus thermodenitrificans PS41 strain

A need for an economically attractive method for feather waste utilization, lead us to research for a new effective technology assuring an outcome of valuable product. In this study, keratinase producing Geobacillus thermodenitrificans a bacterial isolate PS41 was used to convert feather waste into bioactive peptides. Feather peptide under 15 kDa had shown a maximum antioxidant activity and it was identified as radical oxygenase of Propionibacterium acne. The purified feather peptide from PS41 showed antibacterial activity, anticancer activity against human alveolar basal epithelial cells, and larvicidal activity against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus larvae in comparison to non-targeted organisms like zebra fish embryo and brine shrimp. The wound healing rate was significantly higher in peptide treated groups in comparison to standard and control groups respectively. Peptide treated wound had exhibited promisingly complete healing after 5 days of treatment in cat fish model and after 12 days of treatment in wistar albino rat. Histological analysis on experimental animal model showed that the tissue regenerating efficacy of peptide was more than the standard.

Dysfunctional Epithelial Barrier Is Characterized by Reduced E-Cadherin in Idiopathic Subglottic Stenosis.

To aim of the study was to characterize the molecular profile and functional phenotype of idiopathic subglottic stenosis (iSGS)-scar epithelium. Human tracheal biopsies from iSGS scar (n = 6) and matched non-scar (n = 6) regions were analyzed using single-cell RNA sequencing (scRNA-seq). Separate specimens were used for epithelial cell expansion in vitro to assess average growth rate and functional capabilities using transepithelial-electrical resistance (TEER), fluorescein isothiocyanate-dextran flux permeability assay, ciliary coverage, and cilia beating frequency (CBF). Finally, epithelial tight junction protein expression of cultured cells was quantified using immunoblot assay (n = 4) and immunofluorescence (n = 6). scRNA-seq analysis revealed a decrease in goblet, ciliated, and basal epithelial cells in the scar iSGS cohort. Furthermore, mRNA expression of proteins E-cadherin, claudin-3, claudin-10, occludin, TJP1, and TJP2 was also reduced (p < 0.001) in scar epithelium. Functional assays demonstrated a decrease in TEER (paired 95% confidence interval [CI], 195.68-890.83 Ω × cm2 , p < 0.05), an increase in permeability (paired 95% CI, -6116.00 to -1401.99 RFU, p < 0.05), and reduced epithelial coverage (paired 95% CI, 0.1814-1.766, fold change p < 0.05) in iSGS-scar epithelium relative to normal controls. No difference in growth rate (p < 0.05) or CBF was found (paired 95% CI, -2.118 to 3.820 Hz, p > 0.05). Immunoblot assay (paired 95% CI, 0.0367-0.605, p < 0.05) and immunofluorescence (paired 95% CI, 13.748-59.191 mean grey value, p < 0.05) revealed E-cadherin reduction in iSGS-scar epithelium. iSGS-scar epithelium has a dysfunctional barrier and reduced structural protein expression. These results are consistent with dysfunctional epithelium seen in other airway pathology. Further studies are warranted to delineate the causality of epithelial dysfunction on the downstream fibroinflammatory cascade in iSGS. NA Laryngoscope, 134:374-381, 2024.

Estrogen receptor alpha regulates uterine epithelial lineage specification and homeostasis

Postnatal development of the uterus involves specification of undifferentiated epithelium into uterine-type epithelium. That specification is regulated by stromal-epithelial interactions as well as intrinsic cell-specific transcription factors and gene regulatory networks. This study utilized mouse genetic models of Esr1 deletion, endometrial epithelial organoids (EEO), and organoid-stromal co-cultures to decipher the role of Esr1 in uterine epithelial development. Organoids derived from wild-type (WT) mice developed a normal single layer of columnar epithelium. In contrast, EEO from Esr1 null mice developed a multilayered stratified squamous type of epithelium with basal cells. Co-culturing Esr1 null epithelium with WT uterine stromal fibroblasts inhibited basal cell development. Of note, estrogen treatment of EEO-stromal co-cultures and Esr1 conditional knockout mice increased basal epithelial cell markers. Collectively, these findings suggest that Esr1 regulates uterine epithelium lineage plasticity and homeostasis and loss of ESR1 promotes altered luminal-to-basal differentiation driven by ESR1-mediated paracrine factors from the stroma.

Single-cell transcriptional profiling reveals immunomodulatory properties of stromal and epithelial cells in periodontal immune milieu with diabetes in rats

Periodontitis is the sixth major complication of diabetes. Gingiva, as an important component of periodontal tissues, serves as the first defense barrier against infectious stimuli. However, relatively little is known about cellular heterogeneity and cell-specific changes in gingiva in response to diabetes-associated periodontitis. To characterize molecular changes linking diabetes with periodontitis, we profiled single-cell transcriptome analyses of a total of 45,259 cells from rat gingiva with periodontitis under normoglycemic and diabetic condition. The single-cell profiling revealed that stromal and epithelial cells of gingiva contained inflammation-related subclusters enriched in functions of immune cell recruitment. Compared to normoglycemic condition, diabetes led to a reduction in epithelial basal cells, fibroblasts and smooth muscle cells in gingiva with periodontitis. Analysis of differentially expressed genes indicated that stromal and epithelial populations were reprogrammed towards pro-inflammatory phenotypes promoting immune cell recruitment in diabetes-related periodontitis. In aspect of immune cells, diabetes prominently enhanced neutrophil and M1 macrophage infiltration in periodontitis lesions. Cell-cell communications revealed enhanced crosstalk between stromal/epithelial cells and immune cells mediating by chemokine/chemokine receptor interplay in diabetes-associated periodontitis. Our findings deconvolved cellular heterogeneity of rat gingiva associated with periodontitis and diabetes, uncovered altered immune milieu caused by the disease, and revealed immunomodulatory functions of stromal and epithelial cells in gingival immune niche. The present study improves the understanding of the link between the diabetes and periodontitis and helps in formulating precise therapeutic strategies for diabetes-enhanced periodontitis.

Antiinflammatory effect of the ethanolic extract of Korean native herb Potentilla rugulosa Nakai in Bisphenol-a-stimulated A549 cells

ABSTRACT Potentilla rugulosa Nakai (P. rugulosa) is a perennial herb in the Rosaceae family and found in the Korean mountains. Previously, our findings demonstrated that P. rugulosa contains numerous polyphenols and flavonoids exhibiting important antioxidant and anti-obesity bioactivities. Bisphenol A (BPA) is a xenoestrogen that was shown to produce pulmonary inflammation in humans. However, the mechanisms underlying BPA-induced inflammation remain to be determined. The aim of this study was to examine whether ethanolic extract of P. rugulosa exerted an inhibitory effect on BPA-induced inflammation utilizing an adenocarcinoma human alveolar basal epithelial cell line A549. The P. rugulosa extract inhibited BPA-mediated cytotoxicity by reducing levels of reactive oxygen species (ROS). Further, P. rugulosa extract suppressed the upregulation of various pro-inflammatory mediators induced by activation of the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, inhibition of the NF-κB and MAPK signaling pathways by P. rugulosa extract was found to occur via decrease in the transcriptional activity of NF-κB. Further, blockade of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) was noted. Thus, our findings suggest that the ethanolic extract of P. rugulosa may act as a natural anti-inflammatory therapeutic agent.

A Comparative Study of the Effects of Electronic Cigarette and Traditional Cigarette on the Pulmonary Functions of C57BL/6 Male Mice.

Electronic cigarettes (E-cigs) are in a controversial state. Although E-cig aerosol generally contains fewer harmful substances than smoke from burned traditional cigarettes, aerosol along with other compounds of the E-cigs may also affect lung functions and promote the development of lung-related diseases. We investigated the effects of E-cig on the pulmonary functions of male C57BL/6 mice and reveal the potential underlying mechanisms. A total of 60 male C57BL/6 mice were randomly divided into four groups. They were exposed to fresh-air, traditional cigarette smoke, E-cig vapor with 12 mg/mL of nicotine, and E-cig with no nicotine for 8 weeks. Lung functions were evaluated by using quantitative analysis of the whole body plethysmograph, FlexiVent system, lung tissue histological and morphometric analysis, and RT-PCR analysis of mRNA expression of inflammation-related genes. In addition, the effects of nicotine and acrolein on the survival rate and DNA damage were investigated using cultured human alveolar basal epithelial cells. Exposure to E-cig vapor led to significant changes in lung functions and structures including the rupture of the alveolar cavity and enlarged alveolar space. The pathological changes were also accompanied by increased expression of interleukin-6 and tumor necrosis factor-α. The findings of the present study indicate that the safety of E-cig should be further evaluated. Some people currently believe that using nicotine-free E-cigs is a safe way to smoke. However, our research shows that E-cigs can cause lung damage regardless of whether they contain nicotine.

QbD driven targeted pulmonary delivery of dexamethasone-loaded chitosan microspheres: Biodistribution and pharmacokinetic study

Inhaling drugs, on the other hand, is limited mainly by the natural mechanisms of the respiratory system, which push drug particles out of the lungs or make them inefficient once they are there. Because of this, many ways have been found to work around the problems with drug transport through the lungs. Researchers have made polymeric microparticles (MP) and nanoparticles as a possible way to get drugs into the lungs. They showed that the drug could be trapped in large amounts and retained in the lungs for a long time, with as little contact as possible with the bloodstream. MP were formulated in this study to get dexamethasone (DMC) into the pulmonary area. The Box-Behnken design optimized microspheres preparation to meet the pulmonary delivery prerequisites. Optimized formulation was figured out based on the desirability approach. The mass median aerodynamic diameter (MMAD) of the optimized formula (O-DMC-MP) was 8.46 ± 1.45 µm, and the fine particle fraction (FPF) was 77.69 ± 1.26%. This showed that it made suitable drug delivery system, which could make it possible for MP to settle deeply in the lung space after being breathed in. With the first burst of drug release, it was seen that drug release could last up to 16 h. Also, there was no clear sign that the optimized formulation was toxic to the alveoli basal epithelial cells in the lungs, as supported by cytotoxic studies in HUVEC, A549, and H1299 cell lines. Most importantly, loading DMC inside MP cuts the amount of drug into the bloodstream compared to plain DMC, as evident from biodistribution studies. Stability tests have shown that the product can stay the same over time at both the storage conditions. Using chitosan DMC-MP can be a better therapeutic formulation to treat acute respiratory distress syndrome (ARDS).

Morphological Characteristics of Various Cells in Esophageal Squamous Dysplasia: Extremely Wide Morphological Spectrum.

The WHO classification of esophageal tumors divides esophageal squamous intraepithelial dysplasia into high and low grades, but does not specify its morphological spectrum. Here, the morphological characteristics of various cells were investigated in esophageal squamous (high-grade) dysplasia, and a morphological spectrum and terminology for this lesion were proposed to avoid misdiagnosis. The clinicopathological data of 540 patients with esophageal squamous dysplasia were analyzed retrospectively. According to the unique cytomorphological characteristics of the lesions and the predominant cell type, the esophageal squamous dysplasia was divided into the following morphological groups: classic type (34.6%, 187/540), basaloid subtype (10.7%, 58/540), spindle-cell subtype (4.6%, 25/540), differentiated subtype (48.9%, 264/540), and verrucous subtype (1.1%, 6/540). Gender, age, and lesions location did not differ among the subtypes (P > 0.05), while Paris classification and lesions diameter significantly differed among the subtypes (P < 0.01). Classic-type cells showed severe atypia. In the basaloid subtype, the cells were small, and resembled basal cells; most of these lesions were of the 0-IIb type with small lesion diameter. In the spindle-cell subtype, the cells and nuclei were spindle-shaped or long and spindle-shaped and arranged in parallel. Differentiated-subtype showed well-to-moderately differentiated cells, and epithelial basal cells were mature. Verrucous-subtype showed well-differentiated cells, and were characterized by verrucous or papillary structures. Esophageal squamous dysplasia has extremely wide morphological spectrum. Awareness of the spectrum of morphological presentations of this lesion, specifically the basaloid subtype, spindle-cell subtype, differentiated subtype, and verrucous subtype, is important for accurate diagnosis.

Chemical composition and in vitro cytotoxicity of Cyclotrichium niveum essential oil against human breast adenocarcinoma (MCF-7) and human lung adenocarcinoma (A549) cells

The current study is aimed to investigate the essential oil yields, chemical compositions and in vitro cytotoxic activities of Cyclotrichium niveum (Boiss.) Manden et Scheng. C. niveum is an endemic species of Turkiye, generally used as a spice in local cuisine and traditional medicine. The plant material was collected from Yeşilyurt province of Malatya in August. Hydrodistillation yielded 1.41% (g/g) essential oil. The majority of the essential oil was dominated by oxygenated monoterpenes mainly pulegone (56.83%) and isomenthone (29.06%). Cytotoxicity tests of C. niveum essential oil on MCF-7 (human breast adenocarcinoma cells) and A549 (human lung adenocarcinoma alveolar basal epithelial cells) cell lines represented promising results. IC50 values on MCF-7 and A549 were found to be 31.68 μg/mL and 66.70 μg/mL respectively. In the current work, we have shown for the first time that C. niveum essential oil has cytotoxic properties in the cancer cell lines tested.

Melatonin Ameliorates Apoptosis of A549 Cells Exposed to Chicken House PM2.5: A Novel Insight in Poultry Production.

The particulate matter 2.5 (PM2.5) from the chicken production system can cause lung injury and reduce productivity through prolonged breath as it attaches large amounts of harmful substances and microbes. Melatonin has acted to regulate physiological and metabolic disorders and improve growth performance during poultry production. This research would investigate the apoptosis caused by chicken house PM2.5 on lung pulmonary epithelial cells and the protective action of melatonin. Here, the basal epithelial cells of human lung adenocarcinoma (A549 cells) were subjected to PM2.5 from the broiler breeding house to investigate the apoptosis induced by PM2.5 as well as the alleviation of melatonin. The apoptosis was aggravated by PM2.5 (12.5 and 25 μg/mL) substantially, and the expression of Bcl-2, Bad, Bax, PERK, and CHOP increased dramatically after PM2.5 treatment. Additionally, the up-regulation of cleaved caspase-9 and cleaved caspase-3 as well as endoplasmic reticulum stress (ERS)-related proteins, including ATF6 and CHOP, was observed due to PM2.5 exposure. It is worth noting that melatonin could support A549 cells' survival, in which reduced expression of Bax, Bad, cleaved caspase-3, and cleaved caspase-9 appeared. Concurrently, the level of malondialdehyde (MDA) was down-regulated and enhanced the intracellular content of total superoxide dismutase (T-SOD) and catalase (CAT) after treatment by PM2.5 together with melatonin. Collectively, our study underlined that melatonin exerted an anti-apoptotic action on A549 cells by strengthening their antioxidant capacity.

Oxidative Stress and Cellular Protein Accumulation Are Present in Keratoconus, Macular Corneal Dystrophy, and Fuchs Endothelial Corneal Dystrophy

The aim of the study was to investigate oxidative stress as well as cellular protein accumulation in corneal diseases including keratoconus (KC), macular corneal dystrophy (MCD), and Fuchs endothelial corneal dystrophy (FECD) at their primary affecting sites. Corneal buttons from KC, MCD, and FECD patients, as well as healthy controls, were analyzed immunohistochemically to evaluate the presence of oxidative stress and the function of the proteostasis network. 4-Fydroxynonenal (4-HNE) was used as a marker of oxidative stress, whereas the levels of catalase and heat-shock protein 70 (HSP70) were analyzed to evaluate the response of the antioxidant defense system and molecular chaperones, respectively. Sequestosome 1 (SQSTM1) levels were determined to assess protein aggregation and the functionality of autophagic degradation. Basal epithelial cells of the KC samples showed increased levels of oxidative stress marker 4-HNE and antioxidant enzyme catalase together with elevated levels of HSP70 and accumulation of SQSTM1. Corneal stromal cells and endothelial cells from MCD and FECD samples, respectively, showed similarly increased levels of these markers. All corneal diseases showed the presence of oxidative stress and activation of the molecular chaperone response to sustain protein homeostasis. However, the accumulation of protein aggregates suggests insufficient function of the protective mechanisms to limit the oxidative damage and removal of protein aggregates via autophagy. These results suggest that oxidative stress has a role in KC, MCD, and FECD at the cellular level as a secondary outcome. Thus, antioxidant- and autophagy-targeted therapies could be included as supporting care when treating KC or corneal dystrophies.

Investigation of ocular involvement in patients with Fabry disease

Purpose To investigate ocular abnormalities in Fabry disease (FD). Methods Forty-five patients with FD diagnosed by genetic analysis were enrolled in a single medical centre. The following ocular examinations were performed: slit-lamp examination, ophthalmic fundus imaging, in vivo confocal microscopy (IVCM) and optical coherence tomography (OCT). The prevalences of typical abnormalities in the cornea, conjunctiva and retina were recorded; their differences between hemizygote and heterozygote were compared. Results In this study, the prevalence of corneal verticillata was 97.8% (44/45). Corneal examination with IVCM demonstrated hyper-reflective intracellular inclusions located within basal epithelial cells. Conjunctival vessel malformations were observed in 64.4% (29/45) of patients, and retinal vessel tortuosity was observed in 62.2% (28/45) of patients. OCT revealed many strong hyper-reflective foci (HRF) in the inner retinal layer (in 66.7% [30/45] of patients); these foci may represent retinal vascular plexi. The prevalences of conjunctival vessel malformation, retinal vessel tortuosity and HRF were higher in hemizygote than in heterozygote. Conclusions Corneal verticillata, HRF on OCT, conjunctival vessel malformation and retinal vessel tortuosity exhibit high prevalence in patients with FD. These ocular manifestations are characteristic and easily accessible; thus, they should be considered diagnostic criteria for FD.

Highly-specific aptamer targeting SARS-CoV-2 S1 protein screened on an automatic integrated microfluidic system for COVID-19 diagnosis

Variants of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) have evolved such that it may be challenging for diagnosis and clinical treatment of the pandemic coronavirus disease-19 (COVID-19). Compared with developed SARS-CoV-2 diagnostic tools recently, aptamers may exhibit some advantages, including high specificity/affinity, longer shelf life (vs. antibodies), and could be easily prepared. Herein an integrated microfluidic system was developed to automatically carry out one novel screening process based on the systematic evolution of ligands by exponential enrichment (SELEX) for screening aptamers specific with SARS-CoV-2. The new screening process started with five rounds of positive selection (with the S1 protein of SARS-CoV-2). In addition, including non-target viruses (influenza A and B), human respiratory tract-related cancer cells (adenocarcinoma human alveolar basal epithelial cells and dysplastic oral keratinocytes), and upper respiratory tract-related infectious bacteria (including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), and human saliva were involved to increase the specificity of the screened aptamer during the negative selection. Totally, all 10 rounds could be completed within 20 h. The dissociation constant of the selected aptamer was determined to be 63.0 nM with S1 protein. Limits of detection for Wuhan and Omicron clinical strains were found to be satisfactory for clinical applications (i.e. 4.80 × 101 and 1.95 × 102 copies/mL, respectively). Moreover, the developed aptamer was verified to be capable of capturing inactivated SARS-CoV-2 viruses, eight SARS-CoV-2 pseudo-viruses, and clinical isolates of SARS-CoV-2 viruses. For high-variable emerging viruses, this developed integrated microfluidic system can be used to rapidly select highly-specific aptamers based on the novel SELEX methods to deal with infectious diseases in the future.

Engineering artesunate-loaded micelles using spray drying for pulmonary drug delivery

Novel d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) micelles containing the anti-cancer drug artesunate (ART) were developed in a dry powder form for inhalation for the treatment of lung cancer. Targeted delivery to the lungs via inhalation presents a challenge, and the current study involved the development, characterization, and cytotoxicity determination of the dry powder formulations. TPGS micelles encapsulating ART were successfully prepared using thin-film hydration method, followed by the preparation of a dry powder formulation through spray-drying, which included lactose and leucine as carrier molecules. Characterization of the dry powder formulation using Sympatec laser diffraction particle size analyzer and a next generation impactor. The analysis showed a unimodal particle size distribution with a mean particle size of 3.27 μm and a fine particle fraction of 31%, respectively. Artesunate-loaded micelle dry powder formulation demonstrated higher cytotoxicity against the A549 adenocarcinomic human alveolar basal epithelial cell line when compared to currently used cancer therapies docetaxel and doxorubicin. Overall, this study demonstrates that spray-dried micelles containing artesunate have the potential to be used as a dry powder formulation for inhalation in the treatment of lung cancer.

The diagnosis and treatment of contact lens-induced limbal stem cell deficiency

Objective: To analyze the clinical features and treatment outcomes of eyes with contact lens-induced limbal stem cell deficiency (CL-iLSCD). Methods: This cross-sectional study involved patients diagnosed with CL-iLSCD at the Eye, Ear, Nose & Throat Hospital of Fudan University between October 2018 and September 2022. A total of 17 patients (25 eyes) with a mean age of (36.4±6.9) years were enrolled. Among them, 14 were females (82.4%). Corneal and limbal abnormalities, especially the range of epitheliopathy, were observed under a slit lamp biomicroscope with fluorescein staining. Anterior segment optical coherence tomography and in vivo laser scanning confocal microscopy were performed to obtain the central corneal epithelial thickness, density of basal epithelial cells and corneal nerve fiber length. The clinical features of CL-iLSCD, along with their treatment outcomes and related risk factors, were analyzed. Results: All patients wore soft contact lenses, with an average daily wearing time of (10.5±2.5) hours and a median wearing duration of 10 (4 to 30) years. Ocular symptoms, including decreased vision, ocular discomfort or pain, redness, and photophobia, were present in 22 eyes (88.0%). The most characteristic clinical sign of CL-iLSCD was comb-or whorl-pattern late fluorescein staining under cobalt blue light, which was most commonly seen at the superior limbus (25/25, 100.0%). Additionally, reductions in central corneal epithelial thickness, basal cell density, and corneal nerve fiber length were observed. A comprehensive score was assigned to each eye based on clinical findings and in vivo imaging biomarkers. LSCD was mild, moderate, and severe in 5, 11, and 8 eyes, respectively. A history of misdiagnosis was found in 20 eyes (80.0%). After discontinuing the use of contact lenses and receiving medical treatment, significant improvement was observed in all eyes, with 13 eyes fully recovered. Conclusions: The symptoms and clinical signs of CL-iLSCD can be subtle at the early stage. Discontinuing contact lens wear and medication are effective to treat CL-iLSCD.

Abstract B005: The oncogenic transcription factor ERG has distinct roles and signaling regulation in different prostate cancer cell types

Abstract Background: ERG is the most common oncogene in prostate cancer, as it is aberrantly expressed in about 50% of cases due to the TMPRSS2/ERG gene rearrangement. ERG is not normally expressed in the adult prostate. The normal function of ERG is confined to hematopoietic stem cells and blood vessels. Aberrant expression of ERG in prostate epithelial cells is oncogenic when combined with mutations that activate the PI3K/AKT signaling pathway. The RAS/MAPK pathway can also regulate ERG function through direct phosphorylation of ERG by ERK. The function of these regulatory events and how they can be exploited to develop therapy for ERG-positive prostate cancer remains unclear. Results: ERG expression in prostate basal epithelial cells drives epithelial to mesenchymal transition and stemness properties. This function requires ERG phosphorylation via a positive feedback loop with the TLR4 signaling pathway, and can be repressed by a TLR4 inhibitor. Activation of the PI3K/AKT pathway rearranges the ERG cistrome and alters ERG function, where ERG now promotes luminal epithelial differentiation. In the presence of activated AKT, new ERG targets include genes involved in vascular development and ERG phosphorylation is regulated through a positive feedback loop with the VEGF signaling pathway. Conclusions: ERG function in basal and luminal prostate epithelial cells appear to mirror ERG functions in hematopoietic stem cells and blood vessels, respectively. These findings suggest strategies of combinatorial inhibition of ERG function in both cell types. Citation Format: Peter C. Hollenhorst, Saranya Rajendran, Travis J. Jerde. The oncogenic transcription factor ERG has distinct roles and signaling regulation in different prostate cancer cell types [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B005.

Abstract A073: Human prostate-on-chip models to define stromal and epithelial interactions in normal and cancerous prostate

Abstract Background: The secretion of morphogenic factors from the stroma, whose production is dependent on stromal androgen receptor (AR) action, is crucial for maintaining glandular structure and homeostatic function of the prostate gland. However, the precise nature of the factors involved, their regulation, and how they impact homeostasis in the human prostate is not clear. We hypothesize that loss of stromal AR function as seen in prostate cancer progression and during androgen-deprivation therapy likely contribute to tumor progression through disruption of this homeostasis. It is important to understand these interactions in the human context to better aid in improving patient outcomes. Approach: Microfluidic-based technology was used to develop the first human Prostate-on-Chip (PoC) where human prostate fibroblasts are co-cultured with human prostate basal epithelial cells under flow. Addition of androgen to the stromal cells is sufficient to induce luminal cell differentiation on top of the neighboring basal cells – recapitulating the cellular architecture of the human gland. To study prostate cancer in this context, we replaced the normal epithelial cells with tumor cells to interrogate the tumor-stromal interactions; thus, generating a human Prostate Cancer-on-Chip model (PCoC). Cytokine arrays and RT-qPCR was used to identify androgen-induced stromal morphogens and tumor cytokines involved in tumor/stromal interactions. Molecular biology approaches were used to identify the mechanisms involved. Results: Within the PCoC model, tumor cells invaded into the stroma and conversely stromal cells invaded into the tumors. This was accompanied by localized CAF conversion of the stroma. Invasion of tumor cells into the stroma could be blocked with an integrin-α6 binding peptide. Strikingly, the level of AR expression in the stroma was also reduced, recapitulating the loss of stromal AR expression seen in patients. A cytokine array identified TNFα and TGFβ as tumor secreted factors responsible for suppressing stromal AR protein and mRNA expression. TNFα suppressed stromal AR expression in the absence of CAF conversion via NF-κB binding to the AR promoter. On the other hand, p38-MAPK signaling suppressed AR expression independent of TNFα. FGF10 and Wnt16, but not KGF, were identified as androgen-induced stromal secreted morphogens. Loss of stromal AR expression, led to suppression of FGF10 and Wnt16, but not KGF expression. Thus, stromal AR inhibition by tumor secreted TNFα or p38-MAPK signaling suppresses the stromal morphogens required to maintain normal prostate homeostasis. Conclusions: We have developed the first human Prostate-on-Chip and Prostate Cancer-on-Chip models which recapitulate the biology of both the normal human prostate as well as prostate cancer stromal interactions. These models can be used to address a range of clinical problems in cancer development, drug response, and biomarker identification in the context of the tumor microenvironment. This model can easily be adapted for personalized medicine approaches. Citation Format: Shekha K. Tahsin, Linan Jiang, Neha Sane, Kailie Szewczyk, Hunain Khawaja, Yitshak Zohar, Cindy K. Miranti. Human prostate-on-chip models to define stromal and epithelial interactions in normal and cancerous prostate [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A073.

The expression of PKM1 and PKM2 in benign and cancerous prostatic tissues.

e17058 Background: Prostate cancer (PCa) is the most diagnosed cancers in American men. After androgen deprivation therapy (ADT) fails, there is an increase in the aggressive neuroendocrine (NE) phenotype. Currently, there is no effective treatment for NE prostate cancer (NEPC). The metabolic reprogramming, one of the cancer hallmarks, regulates PCa progression and therapy resistance. However, the energy metabolism in NEPC has not been well studied yet. Pyruvate kinase (PK), catalyzing the final step of glycolysis, has PKM1, PKM2, PKL and PKR four isoforms. PKL and PKR are expressed in the liver and erythrocytes, respectively. Alternative splicing of PKM results in two isoforms, PKM1 and PKM2, which are expressed in most tissues. In prostate adenocarcinoma, loss of PKM1 promotes PCa progression whereas loss of PKM2 suppresses tumor growth. However, the expression pattern of PKM1 and PKM2 in NEPCa remains largely unknown. Methods: Immunofluorescence (IF), immunohistochemistry (IHC), Western blot and RT-qPCR were conducted to examine the expression of PKM1 and PKM2 in both murine and human prostatic tissues. The bioinformatics analysis was done using the publicly available RNA-Seq data obtained from the cBioportal, the Cancer Genome Atlas (TCGA), and the Cancer Cell Line Encyclopedia websites (CCLE). Results: TRAMP is a widely used PCa mouse model. TRAMP mice develop prostatic intraepithelial neoplasia (PIN) and the tumors progress into NEPC following castration. We found that PKM1 expression was detected in normal prostate but not in the PIN lesions. In the TRAMP NEPC tumors, PKM1 expression was detected in the NE areas but not in the adjacent PIN lesions. Compared with the adjacent PIN, NEPC cells displayed lower PKM2 expression. Further, we examined the expression of PKM1 and PKM2 in human prostatic tissues including benign prostatic hyperplasia (BPH), low-grade adenocarcinomas (AdPCa), high-grade AdPCa, and NEPC. We found that in BPH, basal epithelial cells express both PKM1 and PKM2. In PCa, PKM1 was robustly expressed in the stromal cells but its expression was absent in the cancer cells in majority of the specimens examined except a small number of samples where low level of PKM1 was detected in the cancer cells. However, PKM1 expression was detected in 9 out of 12 NEPC samples and colocalized with NE marker chromogranin A. This co-expression was also detected in the NE cells scattered on the prostate adenocarcinoma tissues. As for PKM2, its expression was detected in all the samples examined. However, different from previous report, PKM2 expression levels did not correlate with cancer grade in this cohort. Conclusions: PKM1 is expressed in the basal epithelial cells of benign prostates, a small subset of prostate adenocarcinomas, and 75% NEPC tumors. PKM2 is expressed throughout prostate development, in both benign and cancerous prostate. However, PKM2 expression does not correlate with tumor grade.

Modulation of the canonical Wnt activity by androgen signaling in prostate epithelial basal stem cells.

Both the canonical Wnt and androgen receptor (AR) signaling pathways are important for prostate organogenesis and homeostasis. How they crosstalk to regulate prostate stem cell behaviors remains unclear. Here, we show in lineage-tracing mouse models that although Wnt is essential for basal stem cell multipotency, ectopic Wnt activity promotes basal cell over-proliferation and squamous phenotypes, which are counteracted by elevated levels of androgen. In prostate basal cell organoids, dihydrotestosterone (DHT) antagonizes R-spondin-stimulated growth in a concentration-dependent manner. DHT down-regulates the expressions of a Wnt reporter and target genes, and RNA sequencing (RNA-seq) analyses identify Wnt signaling as a key altered pathway. Mechanistically, DHT enhances AR and β-catenin protein binding, and CUT&RUN analyses reveal that ectopic AR sequesters β-catenin away from its Wnt-related cistrome. Our results suggest that an intermediate level of Wnt activity in prostate basal stem cells, achieved via AR-β-catenin interaction, is essential for normal prostate homeostasis.