Role of prostate stem cells and treatment strategies in benign prostate hyperplasia.

Benign prostate hyperplasia (BPH) is a progressive disease with a direct correlation between incidence and age. Since the treatment and management of BPH involve harmful side effects and decreased quality of life for the patient, the primary focus of research should be to find better and longer-lasting therapeutic options. The mechanisms regulating prostate stem cells in development can be exploited to decrease prostate growth. BPH is defined as the overgrowth of the prostate, and BPH is often diagnosed when lower urinary tract symptoms (LUTS) of urine storage or voiding symptoms cause patients to seek treatment. While multiple factors are involved in the hyperplastic growth of the stromal and epithelial compartments of the prostate, the clonal proliferation of stem cells is considered one of the main reasons for BPH initiation and regrowth of the prostate after therapies for BPH fail. Several theories explain possible reasons for the involvement of stem cells in the development, progression, and pathogenesis of BPH. The aim of the current review is to discuss current literature on the fundamentals of prostate development and the role of stem cells in BPH. This review examines the rationale for the hypothesis that unregulated stem cell properties can lead to BPH and therapeutic targeting of stem cells may reduce treatment-related side effects and prevent the regrowth of the prostate.

Comparison of SureSelect and Nextera Exome Capture Performance in Single-Cell Sequencing.

BACKGROUND: Advances in single-cell sequencing provide unprecedented opportunities for clinical examination of circulating tumor cells, cancer stem cells, and other rare cells responsible for disease progression and drug resistance. On the genomic level, single-cell whole exome sequencing (scWES) started to gain popularity with its unique potentials in characterizing mutational landscapes at a single-cell level. Currently, there is little known about the performance of different exome capture kits in scWES. Nextera rapid capture (NXT; Illumina, Inc.) has been the only exome capture kit recommended for scWES by Fluidigm C1, a widely accessed system in single-cell preparation. RESULTS: In this study, we compared the performance of NXT following Fluidigm's protocol with Agilent SureSelectXT Target Enrichment System (AGL), another exome capture kit widely used for bulk sequencing. We created DNA libraries of 192 single cells isolated from spheres grown from a melanoma specimen using Fluidigm C1. Twelve high-yield cells were selected to perform dual-exome capture and sequencing using AGL and NXT in parallel. After mapping and coverage analysis, AGL outperformed NXT in coverage uniformity, mapping rates of reads, exome capture rates, and low PCR duplicate rates. For germline variant calling, AGL achieved better performance in overlap with known variants in dbSNP and transition-transversion ratios. Using calls from high coverage bulk sequencing from blood DNA as the golden standard, AGL-based scWES demonstrated high positive predictive values, and medium to high sensitivity. Lastly, we evaluated somatic mutation calling by comparing single-cell data with the matched blood sequence as control. On average, 300 mutations were identified in each cell. In 10 of 12 cells, higher numbers of mutations were identified using AGL than NXT, probably caused by coverage depth. When mutations are adequately covered in both AGL and NXT data, the two methods showed very high concordance (93-100% per cell). CONCLUSIONS: Our results suggest that AGL can also be used for scWES when there is sufficient DNA, and it yields better data quality than the current Fluidigm's protocol using NXT.

Gene Expression in Single Cells Isolated from the CWR-R1 Prostate Cancer Cell Line and Human Prostate Tissue Based on the Side Population Phenotype.

Defining biological signals at the single cell level can identify cancer initiating driver mutations. Techniques to isolate single cells such as microfluidics sorting and magnetic capturing systems have limitations such as: high cost, labor intense, and the requirement of a large number of cells. Therefore, the goal of our current study is to identify a cost and labor effective, reliable, and reproducible technique that allows single cell isolation for analysis to promote regular laboratory use, including standard reverse transcription PCR (RT-PCR). In the current study, we utilized single prostate cells isolated from the CWR-R1 prostate cancer cell line and human prostate clinical specimens, based on the ATP binding cassette (ABC) transporter efflux of dye cycle violet (DCV), side population assay. Expression of four genes: ABCG2; Aldehyde dehydrogenase1A1 (ALDH1A1); androgen receptor (AR); and embryonic stem cell marker, Oct-4, were determined. Results from the current study in the CWR-R1 cell line showed ABCG2 and ALDH1A1 gene expression in 67% of single side population cells and in 17% or 100% of non-side population cells respectively. Studies using single cells isolated from clinical specimens showed that the Oct-4 gene is detected in only 22% of single side population cells and in 78% of single non-side population cells. Whereas, AR gene expression is in 100% single side population and non-side population cells isolated from the same human prostate clinical specimen. These studies show that performing RT-PCR on single cells isolated by FACS can be successfully conducted to determine gene expression in single cells from cell lines and enzymatically digested tissue. While these studies provide a simple yes/no expression readout, the more sensitive quantitative RT-PCR would be able to provide even more information if necessary.

Impact of devascularization and tissue procurement on cell number and RNA integrity in prostatectomy tissue.

BACKGROUND: Minimizing the time between tissue devascularization in robot-assisted laparoscopic radical prostatectomy (RALP) and tissue procurement should produce the highest quality tissue for research study. This study examines the relationship between intra-operative time and two indicators of tissue integrity: number of epithelial cells per gram of tissue and RNA integrity numbers (RINs). The study also compares the RIN values of tissue obtained intra-operatively by biopsy, before and after devascularization, to those from RALP specimen tissue, obtained through the routine research tissue procurement process. METHODS: Prostate tissues from two series of patients were analyzed. In the first, tissue from 18 patients undergoing RALP was analyzed for number of epithelial cells per gram of tissue. In the second, RIN values of tissue from 46 patients involved in a clinical study were analyzed. RIN values were assessed from RALP specimen tissue as well as tissue removed intra-operatively by biopsy, before and after devascularization. RESULTS: Time from RALP to tissue procurement was not significantly associated with number of epithelial cells per gram of tissue or with RIN values. RINs of biopsy tissue obtained intra-operatively before and after devascularization were similar. However, the RIN values of tissue from RALP specimens were significantly higher than those of biopsy tissue obtained either before or after devascularization. CONCLUSIONS: Tissue quality, defined by number of epithelial cells or RIN values, was not affected by time between devascularization and procurement. Obtaining tissue from intra-operative biopsies, either before or after devascularization, is not necessary and actually produced lower RINs than found in tissue from RALP specimens, obtained through the routine research tissue procurement process.

Interleukin-6 and JAK2/STAT3 signaling mediate the reversion of dexamethasone resistance after dexamethasone withdrawal in 7TD1 multiple myeloma cells.

We previously reported the establishment and characteristics of a DXM-resistant cell line (7TD1-DXM) generated from the IL6-dependent mouse B cell hybridoma, 7TD1 cell line. After withdrawing DXM from 7TD1-DXM cells over 90 days, DXM significantly inhibited the cell growth and induced apoptosis in the cells (7TD1-WD) compared with 7TD1-DXM cells. Additionally, IL-6 reversed while IL-6 antibody and AG490 enhanced the effects of growth inhibition and apoptosis induced by DXM in 7TD1-WD cells. Our study demonstrates that 7TD1-DXM cells become resensitized to DXM after DXM withdrawal, and IL-6 and JAK2/STAT3 pathways may regulate the phenomenon.

Human prostate side population cells demonstrate stem cell properties in recombination with urogenital sinus mesenchyme.

Stem cell enrichment provides a tool to examine prostate stem cells obtained from benign and malignant tissue. Functional assays can enrich stem cells based on common stem cell phenotypes, such as high ATP binding cassette (ABC) transporter mediated efflux of Hoechst substrates (side population assay). This functional assay is based upon mechanisms that protect cells from environmental insult thus contributing to the survival and protection of the stem cell population. We have isolated and analyzed cells digested from twelve clinical prostate specimens based on the side population assay. Prostate stem cell properties of the isolated cells were tested by serial recombination with rat urogenital mesenchyme. Recombinants with side population cells demonstrate an increase in the frequency of human ductal growth and the number of glands per recombinant when compared to recombinants with non-side population cells. Isolated cells were capable of prostatic growth for up to three generations in the recombination assay with as little as 125 sorted prostate cells. The ability to reproducibly use cells isolated by fluorescence activated cell sorting from human prostate tissue is an essential step to a better understanding of human prostate stem cell biology. ABC transporter G2 (ABCG2) was expressed in recombinants from side population cells indicating the side population cells have self-renewal properties. Epithelial cell differentiation of recombinants was determined by immunohistochemical analysis for expression of the basal, luminal, and neuroendocrine markers, p63, androgen receptor, prostate specific antigen, and chromogranin A, respectively. Thus, the ABCG2 expressing side population demonstrates multipotency and self-renewal properties indicating stem cells are within this population.

Aldehyde dehydrogenase and ATP binding cassette transporter G2 (ABCG2) functional assays isolate different populations of prostate stem cells where ABCG2 function selects for cells with increased stem cell activity.

INTRODUCTION: High expression of aldehyde dehydrogenase1A1 (ALDH1A1) is observed in many organs and tumors and may identify benign and cancer stem cell populations. METHODS: In the current study, the stem cell characteristics were determined in cells isolated from human prostate cell lines and clinical prostate specimens based upon the ALDEFLUOR™ assay. Cells isolated based on the ALDEFLUOR™ assay were compared to cells isolated based on ATP binding cassette transporter G2 (ABCG2) activity using the side population assay. To test for stem cell characteristics of self-renewal and multipotency, cells with high and low ALDH1A1 activity, based on the ALDEFLUOR™ assay (ALDHHi and ALDH Low), were isolated from prostate clinical specimens and were recombined with rat urogenital sinus mesenchyme to induce prostate gland formation. RESULTS: The percentage of ALDH Hi cells in prostate cell lines (RWPE-1, RWPE-2, CWR-R1, and DU-145) was 0.5 to 6%, similarly in non-tumor and tumor clinical specimens the percentage of ALDH Hi cells was 0.6 to 4%. Recombinants using ALDH Hi cells serially generated prostate tissue up to three generations with as few as 250 starting cells. Immunohistochemical analysis of the recombinants using ALDHHi cells contained prostatic glands frequently expressing androgen receptor (AR), p63, chromogranin A, ALDH1A1, ABCG2, and prostate specific antigen (PSA), compared to their ALDH Low counterparts. Inhibition of ALDH resulted in the reduction of sphere formation capabilities in the CWR-R1, but not in the RWPE-2 and DU-145, prostate cell lines. ABCG2 inhibition resulted in a more robust decrease of sphere formation in androgen sensitive cell lines, CWR-R1 and RWPE-2, but not androgen insensitive DU-145. ALDH1A1 expression was enriched in ALDH Hi cells and non-side population cells. ABCG2 expression was only enriched in side population cells. CONCLUSIONS: The percentage of ALDHHi cells in prostate cell lines and prostate tissue was consistently higher compared to cells with high ABCG2 activity, identified with the side population assay. The expression of the stem and differentiation markers indicates the ALDH Hi recombinants contained cells with self-renewal and multipotency activity. When the two assays were directly compared, cells with the side population phenotype demonstrated more stem cell potential in the tissue recombination assay compared to ALDH Hi cells. The increased stem cell potential of side population cells in the tissue recombination assay and the decrease in sphere formation when ABCG2 is inhibited indicates that the side population enriches for prostate stem cells.

Apoptotic resistance exhibited by dexamethasone-resistant murine 7TD1 cells is controlled independently of interleukin-6 triggered signaling.

Interleukin-6 (IL6)-mediated signaling is known to play a role in pathogenesis and resistance in several cancers like multiple myeloma (MM). In this report we used the IL6-dependent 7TD1 murine B-cell hybridoma as an in vitro model to study the interactions between IL6-signaling pathways and the development of dexamethasone resistance. Though in initial stages, 7TD1 cells grew IL6-dependent and were sensitive to dexamethasone-induced apoptosis, chronic exposure to dexamethasone led to a dexamethasone-resistant phenotype (7TD1-Dxm) that grew independent of exogenous IL6. While IL6-mediated JAK/STAT3 and PI3K/AKT signaling was important for proliferation of both cell lines, as shown in proliferation assays using the respective pathway inhibitors, AG490 and LY294002, the resistant cells were insensitive to induction of apoptosis using the same. STAT3 was constitutively phosphorylated in resistant cells and inhibition of its dimerization induced apoptosis but did not alter their insensitivity to dexamethasone. Our results suggest a role of entities downstream of IL6-mediated JAK/STAT3 signaling in development of dexamethasone resistance by 7TD1-Dxm cells.