Project description:The primary goal of this study was to assess differences in gene expression between prostate cancer cell lines and normal prostate epithelial and stromal cells in primary culture. Experiment Overall Design: 9 samples were analyzed with 1 replicate each. Samples included 6 prostate cancer cell lines, 1 normal prostate epithelial cell system in primary culture, and 2 normal prostate stromal cell systems in primary culture.
Project description:The primary goal of this study was to assess differences in gene expression between prostate cancer cell lines and normal prostate epithelial and stromal cells in primary culture.
Project description:Organotypic in vitro culture is useful to model mammalian disease in numerous tissues. Normal epithelial differentiation and carcinogenesis both undergo in vivo regulation by stroma, but current culture methods exclude stroma. To mimic this in vivo environment, we developed and characterized a human 3D prostate organoid co-culture model that incorporates prostate stroma. Primary prostate stromal cells supported increased organoid formation and expressed growth factors and WNT-related genes involved in epithelial differentiation. Organoid branching occurred distal to physical contact with stromal cells, demonstrating non-random branching. Tumoroids derived from primary prostate cancer maintained differential expression of the prostate cancer marker AMACR only in the presence of stroma. Stroma-induced phenotypes were similar in all patients examined, yet maintained inter-patient heterogeneity in the degree of response. Addition of stroma to in vitro organoid culture recapitulated the in vivo microenvironment by inducing organization of benign organoids into branching structures and preserving prostate cancer phenotypes.
Project description:Full title: Comprehensive Characterization of Three-Dimensional Models for Prostate Cancer Growth and Invasion in Laminin-rich Extracellular Matrix Prostate Cancer (PrCa) cells undergo acinar morphogenesis and spheroid formation in three-dimensional (3D) culture, supported by laminin-rich extracellular matrix (lrECM, Matrigel). We developed miniaturized 3D model systems that facilitate investigation of morphogenesis and invasion of normal and PrCa cell lines in lrECM. Primary and non-transformed cell lines formed round structures with strong cell-cell contacts and epithelial polarization, lumen and a complete basal lamina (BL). In contrast, most PrCa cell lines formed either defective, “mass” spheroids with incomplete BL, or invasive “stellate” structures. The bioinformatic analyses of genome-wide mRNA expression data revealed massive alteration of key functional and signaling pathways in 3D cultures, with lipid and steroid metabolism, epigenetic reprogramming, and differentiation-related transcription factors induced across all cell lines by lrECM. In invasive cells, AKT, PI3Kinase, mTOR, and hedgehog signaling pathways were most highly activated, validated by small molecule inhibitors compounds specifically targeting key regulatory molecules. Compounds against AKT and PI3kinase pathways were significantly more effective in invasive cells, compared to mass or round/normal phenotype spheroids, and monolayer culture. A severe morphologic conversion was observed in PC-3 and PC-3M cells, transforming initially round, normal-appearing epithelial spheroids into rapidly invading cell masses. Markers for EMT (epithelial-mesenchymal transition) were highly expressed already in early stage, round spheroids prior to invasive conversion, and were not further increased in invasive cells. This indicates that PrCa cells can display extraordinary plasticity. EMT may be involved in providing a metastable genotype that allows morphological transformation, but is not be required for invasive processes themselves. Total RNA was obtained from non-transformed prostate epithelial cells and prostate cancer cells cultured in monolayer and three-dimensional laminin-rich extracellular matrix (growth factor-reduced Matrigel).
Project description:Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. The goals and objectives of this study were 1.) to characterize and validate the molecular identity of human primary epithelial and stromal/mesenchymal breast cells maintained long-term in novel ex vivo culture conditions in serum free medium. 2.) To analyze changes in gene expression profiles of normal human primary epithelial and stromal/mesenchymal breast cells upon long-term ex vivo co-culture when compared to corresponding monocultures 3.) To study the dynamic reciprocity between normal human primary epithelial and stromal/mesenchymal breast cells. 4.) To identify critical molecular pathways and biomarkers controlling epithelial and/or stromal cell growth and quiescence. Human primary epithelial progenitor cells and mesenchymal stem cells bearing fluorescent tags were either co-cultured in novel ex vivo culture conditions on ECM coated meshes in serum free medium (M5) or cultured as monocultures in the same conditions for 30 days. The cultures were then dissociated and epithelial and stromal/mesenchymal cells from either co-cultures or monocultures separated by FACS. Gene expression profiling of epithelial or stromal/mesenchymal cells was performed. Clean gene expression profiles from three different epithelial and stromal/mesenchymal cell extracts either grown in co-cultures or monocultures were successfully obtained.
Project description:The understanding of common prostatic disorders has been restricted by both cellular heterogeneity and the scarcity of established cell lines, although organoid technology, based on primary cultures, promises much for the future. In particular, little is known about the aetiology of benign prostatic hyperplasia (BPH), for which culture of single cell types might not accurately not reflect the stromal and epithelial overgrowths observed in tissues. To address the applicability of primary cell culture models of prostate disease, we compared cell type-specific mRNA expression patterns in BPH tissues and primary basal cells cultured from the same transurethral biopsies.
Project description:Prostate primary epithelial and stromal cells were isolated from normal donorhuman prostate tissue and patient prostate tumors (>Gleason 8) using in vitroselective media conditions and differential substrate adhesion properties.Tumor cells specific to the prostate epithelial cell component attached tomatrigel while epithelial cells from donors preferentially attached to gelatinsubstrate. The prostate tumor cells did not proliferate over time in culturebut cells of normal epithelial phenotype in comparison to the donor tissueeventually emerged from this cell compartment. The epithelial componentof both donor and tumor specimens produced stem cell colonies thatexpressed OCT4 in growth factor reduced media but subsequently formedembryoid bodies in hanging drop cultures which differentiated into multiplegerm line lineages under the influence of growth factors. To determineif prostate tumor-derived stem cells were capable of forming tumors invivo, we compared the behavior of the donor and tumor stem cells aftertransplantation as tissue recombinants under the renal capsule of SCID micein the presence of rat urogenital mesenchyme (rUGM). Stem cells from bothdonor and tumor sources produced glandular structures that secreted prostatespecific antigen (PSA) while no glandular structures were formed from tissue recombinants of normal epithelial or tumor epithelial cells plus rUGM or fromrUGM alone. Serial transplantation of the stem cell recombinants from tumor specimens yielded subrenal structures reflecting an abundance of glandswith morphological features typical of Prostatic Intraepithelial Neoplasia(PIN). In order to determine whether intrinsic differences existed amongthe donor-derived and tumor-derived stem cells, array based microRNAexpression profiling was performed on all cell types obtained after in vitro isolation. Sample Labeling Key: PC-Prostate Cancer PD-Prostate Donor (Normal Tissue) DNE-Donor Normal Epithelial NS-Donor Normal Stem TNE-Tumor Normal Epithelial TE-Tumor Epithelial (TNE and TE samples with corresponding numbers are matched pairs) TS-Tumor Stem Cell S-Stromal PC 73 TNE was run in all 6 batches as a control for batch effect. The number in parentheses corresponds with the batch the sample was run in.
Project description:Prostate primary epithelial and stromal cells were isolated from normal donorhuman prostate tissue and patient prostate tumors (>Gleason 8) using in vitroselective media conditions and differential substrate adhesion properties.Tumor cells specific to the prostate epithelial cell component attached tomatrigel while epithelial cells from donors preferentially attached to gelatinsubstrate. The prostate tumor cells did not proliferate over time in culturebut cells of normal epithelial phenotype in comparison to the donor tissueeventually emerged from this cell compartment. The epithelial componentof both donor and tumor specimens produced stem cell colonies thatexpressed OCT4 in growth factor reduced media but subsequently formedembryoid bodies in hanging drop cultures which differentiated into multiplegerm line lineages under the influence of growth factors. To determineif prostate tumor-derived stem cells were capable of forming tumors invivo, we compared the behavior of the donor and tumor stem cells aftertransplantation as tissue recombinants under the renal capsule of SCID micein the presence of rat urogenital mesenchyme (rUGM). Stem cells from bothdonor and tumor sources produced glandular structures that secreted prostatespecific antigen (PSA) while no glandular structures were formed from tissuerecombinants of normal epithelial or tumor epithelial cells plus rUGM or fromrUGM alone. Serial transplantation of the stem cell recombinants from tumorspecimens yielded subrenal structures reflecting an abundance of glands with morphological features typical of Prostatic Intraepithelial Neoplasia(PIN). In order to determine whether intrinsic differences existed amongthe donor-derived and tumor-derived stem cells, array based microRNAexpression profiling was performed on all cell types obtained after in vitro isolation. Sample Labeling Key: PC-Prostate Cancer PD-Prostate Donor (Normal Tissue) DNE-Donor Normal Epithelial NS-Donor Normal Stem TNE-Tumor Normal Epithelial TE-Tumor Epithelial (TNE and TE samples with corresponding numbers are matched pairs) TS-Tumor Stem Cell S-Stromal PC 73 TNE was run in all 6 batches as a control for batch effect. The number in parentheses corresponds with the batch the sample was run in.
Project description:The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G1 to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate, but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in Normal Prostate Fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1 overexpressing fibroblasts (NPF cyclin D1) resulted in permanent malignant transformation of epithelial cells (BPH-1 NPF-cyclin D1 cells) similar to that seen with Carcinoma Associated Fibroblasts (CAFs). Microarray analysis showed that the expression profiles between CAFs and NPF cyclin D1 cells were highly concordant including cyclin D1 upregulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue-specific. Keywords: cyclin D1; stromal-epithelial interactions; prostate cancer; cDNA microarray
Project description:To conduct comparative transcriptomic analyses on normal or malignant prostate epithelial cells in response to tissue contextual changes, we cultured immortalized prostate epithelial cells or prostate cancer cells as cell monolayers or three-dimensional organoids and profiled their transcriptomes in respective culture contexts.