Establishment and characterization of prostate organoids from treatment-naïve patients with prostate cancer.

Three-dimensional (3D) organoid culture systems are emerging as potential reliable tools to investigate basic developmental processes of human disease, especially cancer. The present study used established and modified culture conditions to report successful generation and characterization of patient-derived organoids from fresh primary tissue specimens of patients with treatment-naïve prostate cancer (PCa). Fresh tissue specimens were collected, digested enzymatically and the resulting cell suspensions were plated in a 3D environment using Matrigel as an extracellular matrix. Previously established 12-factor medium for organoid culturing was modified to create a minimal 5-factor medium. Organoids and corresponding tissue specimens were characterized using transcriptomic analysis, immunofluorescent analysis, and immunohistochemistry. Furthermore, patient-derived organoids were used to assess the drug response. Treatment-naïve patient-derived PCa organoids were obtained from fresh radical prostatectomy specimens. These PCa organoids mimicked the heterogeneity of corresponding parental tumor tissue. Histopathological analysis demonstrated similar tissue architecture and cellular morphology, as well as consistent immunohistochemical marker expression. Also, the results confirmed the potential of organoids as an in vitro model to assess potential personalized treatment responses as there was a differential drug response between different patient samples. In conclusion, the present study investigated patient-derived organoids from a cohort of treatment-naïve patients. Derived organoids mimicked the histological features and prostate lineage profiles of their corresponding parental tissue and may present a potential model to predict patient-specific treatment response in a pre-clinical setting.

Epidermal Growth Factor Is Essential for the Maintenance of Novel Prostate Epithelial Cells Isolated From Patient-Derived Organoids.

Prostate cancer (PCa) is the second leading cause of cancer-related mortality and morbidity among males worldwide. Deciphering the biological mechanisms and molecular pathways involved in PCa pathogenesis and progression has been hindered by numerous technical limitations mainly attributed to the limited number of cell lines available, which do not recapitulate the diverse phenotypes of clinical disease. Indeed, PCa has proven problematic to establish as cell lines in culture due to its heterogeneity which remains a challenge, despite the various in vitro and in vivo model systems available. Growth factors have been shown to play a central role in the complex regulation of cell proliferation among hormone sensitive tumors, such as PCa. Here, we report the isolation and characterization of novel patient-derived prostate epithelial (which we named as AUB-PrC) cells from organoids culture system. We also assessed the role of epidermal growth factor (EGF) in culturing those cells. We profiled the AUB-PrC cells isolated from unaffected and tumor patient samples via depicting their molecular and epithelial lineage features through immunofluorescence staining and quantitative real-time PCR (qRT-PCR), as well as through functional assays and transcriptomic profiling through RNA sequencing. In addition, by optimizing a previously established prostate organoids culture system, we were able to grow human prostate epithelial cells using growth medium and EGF only. With these data collected, we were able to gain insight at the molecular architecture of novel human AUB-PrC cells, which might pave the way for deciphering the mechanisms that lead to PCa development and progression, and ultimately improving prognostic abilities and treatments.

PulseNet Lebanon: An Overview of Its Activities, Outbreak Investigations, and Challenges.

Background: Foodborne diseases are still a major health issue in Lebanon, although some steps have been taken forward in food safety. To this purpose, PulseNet Lebanon, a foodborne diseases tracking network, was established in 2009, through the collaboration between the Ministry of Public Health (MoPH) and the American University of Beirut (AUB). Materials and Methods: Three papers published regarding the PulseNet project were summarized. Initially, clinical and food samples, collected within the surveillance network scope, were identified by using the respective API for Salmonella and Listeria spp. Salmonella spp. were further serotyped by using the Kauffman and White method. Campylobacter spp. were determined by the 16 S rRNA sequencing method. Antimicrobial susceptibility to a number of antibiotics was determined by using the disk diffusion method for Samonella and Campylobacter spp. Genomic diversity was determined by using pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD). Results: Results indicated that 290 clinical and 49 food isolates were identified as Salmonella. Serotyping revealed the prevalence of ten and seven serotypes in the clinical and food samples, respectively. Fifty-one isolates from chicken ceca and carcass were identified to be Campylobacter spp. Fifty-nine samples were identified to be Listeria monocytogenes. Antimicrobial susceptibility testing revealed a wide range of resistance among the different samples. PFGE showed a variation in pulsotypes among the Salmonella serotypes. PFGE also linked certain outbreaks to their food sources. This method also demonstrated 13 subtypes with 100% similarity among the L. monocytogenes isolates. Finally, the Camplyobcater spp. were grouped into nine clusters with a minimum similarity of 43.5% using RAPD. Conclusion: This summary of results shows the importance of implementing a "farm-to-fork" approach in the surveillance of foodborne disease outbreaks in Lebanon, allowing the detection of pathogens causing foodborne disease outbreaks in a timely fashion.

Assessing Radiosensitivity of Bladder Cancer in vitro: A 2D vs. 3D Approach.

Background: Bladder cancer is the fourth most commonly diagnosed cancer among males worldwide. Current treatment strategies established for bladder cancer mainly consist of cystectomy yet advances in radiation therapy have pointed to the value of organ-preserving strategies in preserving patients' quality of life. Aim: To study and compare the radiosensitivity in two-dimension (2D) and physiologically-relevant three-dimension (3D) in vitro culture of three human bladder cancer cell lines, RT4, T24, and UM-UC-3. Materials and Methods: Clonogenic assay was performed to assess cells' radiosensitivity in 2D. Employing the 3D Matrigel™-based cultures to enrich for cancer stem cells (CSCs) allowed us to assess the survival of this subpopulation of cells via evaluating the number, i.e., sphere forming unit (SFU), and the sizes of cultured spheres, formed from cells exposed to different radiation doses compared to non-irradiated cells. Results: Irradiating cells with increasing radiation doses revealed highest survival rates with RT4 cells in 2D, followed by T24 and UM-UC-3. In 3D, however, UM-UC-3 cells were shown to be the most radio-resistant as evidenced by the number of spheres formed, yet they displayed the least efficient volume reduction/regression (VR), whilst the volume decreased significantly for both RT4 and T24 cells. Sphere VR and sphere ratio (SR) values were then plotted against each other demonstrating a linear correlation between volume and number with RT4 and UM-UC-3 cell lines, but not T24. Lastly, multiple regression model was employed to evaluate the possibility of obtaining a function combining both 3D parameters, SR and VR, with the surviving fraction (SF) in 2D, and showed a linear regression for T24 cells only, with a correlation coefficient of 0.97 for the combined parameters. Conclusion: We were able to radiobiologically characterize 3 human bladder cancer cell lines showing differential effects of radiation between 2D and 3D culture systems, paving the way for achieving better assessment of radiosensitivity of bladder cancer in vitro.

EMT Markers in Locally-Advanced Prostate Cancer: Predicting Recurrence?

Background: Prostate cancer (PCa) is the second most frequent cause of cancer-related death in men worldwide. It is a heterogeneous disease at molecular and clinical levels which makes its prognosis and treatment outcome hard to predict. The epithelial-to-mesenchymal transition (EMT) marks a key step in the invasion and malignant progression of PCa. We sought to assess the co-expression of epithelial cytokeratin 8 (CK8) and mesenchymal vimentin (Vim) in locally-advanced PCa as indicators of EMT and consequently predictors of the progression status of the disease. Methods: Co-expression of CK8 and Vim was evaluated by immunofluorescence (IF) on paraffin-embedded tissue sections of 122 patients with PCa who underwent radical prostatectomies between 1998 and 2016 at the American University of Beirut Medical Center (AUBMC). EMT score was calculated accordingly and then correlated with the patients' clinicopathological parameters and PSA failure. Results: The co-expression of CK8/Vim (EMT score), was associated with increasing Gleason group. A highly significant linear association was detected wherein higher Gleason group was associated with higher mean EMT score. In addition, the median estimated biochemical recurrence-free survival for patients with < 25% EMT score was almost double that of patients with more than 25%. The validity of this score for prediction of prognosis was further demonstrated using cox regression model. Our data also confirmed that the EMT score can predict PSA failure irrespective of Gleason group, pathological stage, or surgical margins. Conclusion: This study suggests that assessment of molecular markers of EMT, particularly CK8 and Vim, in radical prostatectomy specimens, in addition to conventional clinicopathological prognostic parameters, can aid in the development of a novel system for predicting the prognosis of locally-advanced PCa.

The synthetic retinoid ST1926 attenuates prostate cancer growth and potentially targets prostate cancer stem-like cells.

Retinoids are vitamin A derivatives that regulate crucial biological processes such as cellular proliferation, apoptosis, and differentiation. The use of natural retinoids in cancer therapy is limited due to their toxicity and the acquired resistance by cancer cells. Therefore, synthetic retinoids were developed, such as the atypical adamantyl retinoid ST1926 that provides enhanced bioavailability and reduced toxicity. We have assessed the in vitro and in vivo antitumor properties and mechanism of action of ST1926 in targeting cancer stem-like cells population of human prostate cancer (PCa) cell lines, DU145 and PC3, and mouse PCa cell lines, PLum-AD and PLum-AI. We demonstrated that ST1926 substantially reduced proliferation of PCa cells and induced cell cycle arrest, p53-independent apoptosis, and early DNA damage. It also decreased migration and invasion of PCa cells and significantly reduced prostate spheres formation ability in vitro denoting sufficient eradication of the self-renewal ability of the highly androgen-resistant cancer stem cells. Importantly, ST1926 potently inhibited PCa tumor growth and progression in vivo. Our results highlight the potential of ST1926 in PCa therapy and warrant its clinical development.

Sphere-Formation Assay: Three-Dimensional in vitro Culturing of Prostate Cancer Stem/Progenitor Sphere-Forming Cells.

Cancer Stem Cells (CSCs) are a sub-population of cells, identified in most tumors, responsible for the initiation, recurrence, metastatic potential, and resistance of different malignancies. In prostate cancer (PCa), CSCs were identified and thought to be responsible for the generation of the lethal subtype, commonly known as Castration-Resistant Prostate Cancer (CRPC). In vitro models to investigate the properties of CSCs in PCa are highly required. Sphere-formation assay is an in vitro method commonly used to identify CSCs and study their properties. Here, we report the detailed methodology on how to generate and propagate spheres from PCa cell lines and from murine prostate tissue. This model is based on the ability of stem cells to grow in non-adherent serum-free gel matrix. We also describe how to use these spheres in histological and immuno-fluorescent staining assays to assess the differentiation potential of the CSCs. Our results show the sphere-formation Assay (SFA) as a reliable in vitro assay to assess the presence and self-renewal ability of CSCs in different PCa models. This platform presents a useful tool to evaluate the effect of conventional or novel agents on the initiation and self-renewing properties of different tumors. The effects can be directly evaluated through assessment of the sphere-forming efficiency (SFE) over five generations or other downstream assays such as immuno-histochemical analysis of the generated spheres.

Modeling Human Neurological and Neurodegenerative Diseases: From Induced Pluripotent Stem Cells to Neuronal Differentiation and Its Applications in Neurotrauma.

With the help of several inducing factors, somatic cells can be reprogrammed to become induced pluripotent stem cell (iPSCs) lines. The success is in obtaining iPSCs almost identical to embryonic stem cells (ESCs), therefore various approaches have been tested and ultimately several ones have succeeded. The importance of these cells is in how they serve as models to unveil the molecular pathways and mechanisms underlying several human diseases, and also in its potential roles in the development of regenerative medicine. They further aid in the development of regenerative medicine, autologous cell therapy and drug or toxicity screening. Here, we provide a comprehensive overview of the recent development in the field of iPSCs research, specifically for modeling human neurological and neurodegenerative diseases, and its applications in neurotrauma. These are mainly characterized by progressive functional or structural neuronal loss rendering them extremely challenging to manage. Many of these diseases, including Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) have been explored in vitro. The main purpose is to generate patient-specific iPS cell lines from the somatic cells that carry mutations or genetic instabilities for the aim of studying their differentiation potential and behavior. This new technology will pave the way for future development in the field of stem cell research anticipating its use in clinical settings and in regenerative medicine in order to treat various human diseases, including neurological and neurodegenerative diseases.

Molecular epidemiology and antimicrobial resistance of Salmonella species from clinical specimens and food Items in Lebanon.

INTRODUCTION: Foodborne illnesses can be due to a wide range of bacteria, one of the most common being Salmonella. In this study, PulseNet International was implemented in Lebanon to identify circulating pathogens at the species and strain levels, determine antimicrobial resistance, and link food sources and clinical cases during outbreaks. METHODOLOGY: Clinical and food Salmonella isolates received from the Epidemiological Surveillance Unit, Ministry of Public Health (ESUMOH) and the Lebanese Agriculture Research Institute (LARI) between 2011 and 2014 were identified to the species level using API 20E. Serotyping was carried out using the Kauffman and White scheme. Antimicrobial susceptibility to a panel of antimicrobials was tested by the disc diffusion method. The DNA fingerprinting patterns were determined using Pulsed-Field Gel Electrophoresis (PFGE) followed by BIONUMERICS analysis. RESULTS: 290 clinical and 49 food isolates were identified to be Salmonella. The serotyping of the isolates revealed the prevalence of ten serotypes in the clinical isolates and seven serotypes within the food isolates; S. Enteritidis and S. Typhimurium being the two most common. Antimicrobial susceptibility test showed resistance to tested antimicrobials among both clinical and food isolates. PFGE results showed a wide range of pulsotypes by the different serovars. These pulsotypes were then used to confirm the linkage of two outbreaks to their food sources. CONCLUSION: This study calls out to set and implement food safety regulations and emphasizes the importance of surveillance through a "farm-to-fork" approach in identifying widely circulating food borne pathogens.

Assessment of combination therapy in BALB/c mice injected with carbapenem-resistant Enterobacteriaceae strains.

Monotherapeutic options for carbapenem resistant infections are limited. Studies suggest that combination therapy may be associated with better outcomes than monotherapies. However, this is still controversial. This study assessed, the efficacy of combination therapy against carbapenem resistant Enterobacteriaceae harboring singly various extended spectrum beta lactamase or carbapenemase encoding genes. Thus, four isolates harboring either bla CTXM-15, bla CTXM-15 and bla OXA-48, bla NDM-1, or bla KPC-2 genes were selected for testing. Minimal inhibitory concentration was determined by broth dilution method. Gene transcript levels on single and combined treatments were done in vitro and in vivo by qRT-PCR. Assessment of treatments was done in BALB/c mice according to a specific protocol. As such, the qRT-PCR revealed a significant decrease of transcript levels in all isolates upon using rifampicin or tigecycline, singly or in combination with colistin. However, variable levels were obtained using colistin singly or in combination with meropenem or fosfomycin. In vivo assessment showed that all combinations used were effective against isolates harboring bla CTXM-15, bla OXA-48, and bla NDM-1. Conversely, the most significant combination against the isolate harboring bla KPC-2 gene was colistin with either carbapenem, fosfomycin, or kanamycin. As a conclusion, combination therapy selected based on the type of carbapenemase produced, appeared to be non-toxic and might be effective in BALB/c mice. Therefore, the use of a rationally optimized combination therapy might lead to better results than monotherapy, however, clinical trials are needed for human consumption.

Corrigendum: Escherichia coli Isolated from Urinary Tract Infections of Lebanese Patients between 2005 and 2012: Epidemiology and Profiles of Resistance.

[This corrects the article on p. 26 in vol. 2, PMID: 4415468.].

Prevalence of Clostridium difficile toxinotypes in infected patients at a tertiary care center in Lebanon.

INTRODUCTION: Due to the increase in the incidence of Clostridium difficile associated diseases at a tertiary care center in Lebanon, this study was undertaken to determine the prevalent C. difficile toxinotypes. METHODOLOGY: The immunocard method was used to test for toxins A and B in 88 collected stool samples, followed with API 20A to confirm for C. difficile. PCR amplification of the triose phosphate isomerase (tpi) gene, the toxin encoding genes tcdA, and tcdB, followed by toxinotyping, were performed on recovered isolates and stool specimens. RESULTS: Out of the 88 stool samples obtained, 30 (65.2%) were Immunocard positive, culture and or tpi positive for C. difficile. Of the 30 isolates, 4 were PCR negative for the tcdA and tcdB genes (A-B-), and 26 were PCR positive for the tcdA and / or tcdB genes with 4 being A+B+, 1 A+B-, and 21 A-B+. The results of toxinotyping showed that 2 isolates belonged to toxinotype 0, 4 to toxinotype XI, 2 to toxinotype XII, 1 to toxinotype XVI, 1(A+B-) and twenty (A-B+) designated as toxinotype 0-like. C. difficile was detected in 65.2% of patients' stools with prevalence of toxinotype 0-like. CONCLUSION: Identification of toxinotypes of C. difficile is important to determine the virulence potential of strains and control their spread.