Prostate cancer stem cells.

Despite the discovery over 60 years ago by Huggins and Hodges that prostate cancers respond to androgen deprivation therapy, hormone-refractory prostate cancer remains a major clinical challenge. There is now mounting evidence that solid tumours originate from undifferentiated stem cell-like cells coexisting within a heterogeneous tumour mass that drive tumour formation, maintain tumour homeostasis and initiate metastases. This review focuses upon current evidence for prostate cancer stem cells, addressing the identification and properties of both normal and transformed prostate stem cells.

Primary prostatic epithelial cell binding to human bone marrow stroma and the role of alpha2beta1 integrin.

Prostate cancer selectively metastasises to the bone. To investigate the importance of prostate epithelial cell adhesion to bone marrow cells in this process we examined the binding of human primary prostatic epithelial cells (PEC) to human bone marrow stromal cultures (BMS). We found that PEC derived from both malignant and benign tissue showed greater adhesion to BMS than to benign prostatic fibroblasts (median difference was 340% and 200% respectively), skin fibroblasts or plastic tissue culture plates. Adhesion to BMS grown from the bone marrow of patients with prostatic skeletal metastases was no different from those grown from normal bone marrow. The role of integrin molecules in these cell interactions was determined. Collagen type I and fibronectin were found to increase PEC adhesion whereas vitronectin and laminin did not. Inhibition studies demonstrated that although there was heterogeneity between samples, antibodies against the integrins alpha2 and beta1 consistently inhibited PEC binding to BMS. This result was more marked for PEC derived from malignant tissue. However studies investigating the effects of disintegrins and anti-alpha3 and anti-alpha5 integrins indicated that for a percentage of patients these integrins and RGD (arginine, glycine, aspartamine)-dependent binding pathways were also involved. In summary, the results indicate that BMS are adherent to primary PEC derived from both malignant and benign tissue. The integrin alpha2beta1 is a major contributor to this interaction.

Scatter factor influences the formation of prostate epithelial cell colonies on bone marrow stroma in vitro.

Prostate cancer metastases form selectively in the bone marrow. Previously we demonstrated motility was important for the formation of primary prostatic epithelial cell colonies in bone marrow stroma (BMS) co-culture. In this study we looked at the influence of motility factors on the colony formation of epithelial cells derived from benign (bPEC) or malignant (mPEC) prostate tissue. After 7 days co-culture we found that anti-scatter factor consistently inhibited prostate epithelial cell colony formation on BMS (7/7 mPEC and 4/7 bPEC samples showed significant inhibition). Antibodies against bFGF and 5T4 did not significantly affect colony formation. Addition of fibroblast conditioned media (derived from benign prostates) to co-cultures stimulated the colony formation of bPEC (170%) and mPEC (252%). This stimulation was eliminated by depletion of SF from the conditioned media. Immunohistochemical staining found c-Met expression in 5/6 bPEC cultures and 7/9 mPEC cultures. When grown in BMS co-culture expression of c-Met was positive in 3/6 bPEC and 2/7 mPEC samples. In conclusion, scatter factor influences the in vitro formation of prostate epithelial cell colonies on BMS co-culture.

[Electron microscopic observations on Bacteroides thetaiotaomicron].

Three strains of B. thetaiotaomicron have been isolated from caeca of BALB/c-nu/+ mice of SPF. These Bacteroides are obligately anaerobic, Gram-negative, non-spore-forming and non-motil without flagella rods. A characteristic of staining is deep at ends and stainless at the medium of a rod. The isolation and identification of these strains has been reported in 1987. This paper introduced only the results of EM observations. Under the SEM, the unstained area of rods is always showing a concavity, which is just a nucleoid in sections under the TEM. Many lamellar corpuscles have been found in cell plasma. Some of them have been secreted out of the cells. The chemical properties and physiological functions of them are unknown.

Treatment with tirofiban for acute coronary syndrome (ACS): a systematic review and network analysis.

OBJECTIVE: To assess the efficacy of tirofiban in comparison to usual care or other GPIIb/IIIa antagonists (eptifibatide and abciximab). Results were analysed by drug administration with planned percutaneous coronary intervention (PCI) or as medical management without planned PCI, and separately for STEMI or NSTE ACS patients. RESEARCH DESIGN AND METHODS: A systematic review was performed of randomized controlled trials of tirofiban, abciximab, eptifibatide or usual care given to patients with acute coronary syndrome. Nine databases were searched up to March 2010. Pair-wise meta-analysis was used to combine all available direct comparisons; indirect comparisons and network analysis were performed when this was not possible. The primary outcome was MACE (major adverse cardiac event). RESULTS: The search yielded 8, 119 records and 50 trials were included (total number of patients = 52,958). Compared to usual care, high and medium-dose tirofiban (25 and 10 µg/kg/min) administered with planned PCI reduced MACE at 30 days for patients with STEMI (RR 0.67, 95% CI 0.45, 0.99; RR 0.28, 95% CI 0.10, 0.80), but was not effective as a medical management. Medium-dose tirofiban (10 µg/kg/min) administered with planned PCI or low dose (0.4 µg/kg/min) as medical management reduced the risk of MACE for patients with NSTE ACS (RR 0.39, 95% CI 0.21, 0.75; RR 0.58, 95% CI 0.41, 0.83) in comparison to usual care, but at the expense of increased thrombocytopenia (RR 3.26, 95% CI 1.31, 8.13). Evidence from RCTs and network analysis indicated tirofiban and abciximab were equally effective and safe. Comparing tirofiban and eptifibatide treatment by indirect and network analysis produced inconclusive results. CONCLUSIONS: Tirofiban was more effective than usual care for STEMI and NSTE ACS patients receiving planned PCI, and NSTE ACS patients receiving medical management. Tirofiban and abciximab were equally effective. Comparisons of tirofiban and eptifibatide were inconclusive.

Polarized fluid movement and not cell death, creates luminal spaces in adult prostate epithelium.

There are two predominant theories for lumen formation in tissue morphogenesis: cavitation driven by cell death, and membrane separation driven by epithelial polarity. To define the mechanism of lumen formation in prostate acini, we examined both theories in several cell lines grown in three-dimensional (3D) Matrigel culture. Lumen formation occurred early in culture and preceded the expression of cell death markers for apoptosis (active caspase 3) and autophagy (LC-3). Active caspase 3 was expressed by very few cells and inhibition of apoptosis did not suppress lumen formation. Despite LC-3 expression in all cells within a spheroid, this was not associated with cell death. However, expression of a prostate-secretory protein coincided with lumen formation and subsequent disruption of polarized fluid movement led to significant inhibition of lumen formation. This work indicates that lumen formation is driven by the polarized movement of fluids and proteins in 3D prostate epithelial models and not by cavitation.

Stimulation of human prostate cancer cell lines by factors present in human osteoblast-like cells but not in bone marrow.

Secondary deposits of prostate tumours are frequently found in the skeleton where they produce osteoblastic lesions. In this study both osteoblast-like cells and bone marrow from the proximal femur have been cultured to determine whether or not they can release factors which could support the growth of secondary prostate tumours. Media conditioned by both osteoblast-like cells (OBCM) and bone marrow were examined for their potential to stimulate prostate carcinoma cell lines. Whilst the results obtained demonstrated that OBCM could enhance the growth of both the hormone sensitive (LNCaP) and hormone unresponsive (PC-3 and DU-145) prostate carcinoma cell lines, no proliferative effect could be shown on cell lines derived from cancers of the breast, bladder, and liver. Significantly, media conditioned by either bone marrow or human skin fibroblasts also had no effect on the growth of prostate carcinoma cell lines. This study supports the possibility that the proliferation of prostate cancer cells at secondary skeletal sites, in vivo, may be due to osteoblast derived factors.

Effect of positioning on discomfort from intramuscular injections in the dorsogluteal site.

An intramuscular injection into a relaxed muscle is believed to result in less discomfort than an injection into a contracted muscle. When the femur is internally rotated, the gluteus maximus muscle is relaxed. The hypothesis that a dorsogluteal injection with the femur internally rotated will cause less discomfort than when the femur is externally rotated was tested in 44 surgical patients who received two injections of preoperative medication. Each patient received an injection of a narcotic medication and one of diazepam. All possible combinations of the factors--position (internal and external rotation), order of injection (first or second injection), and medication (narcotic or diazepam)--were determined, and patients were randomly assigned to one of these conditions. Patients rated their perceived discomfort after each injection on a five-point scale. The hypothesis was supported by discomfort ratings from injections of both types of medications, although diazepam injections caused significantly more discomfort than injections of narcotics. Older patients tended to report less discomfort from diazepam injections than younger patients. Sex, order of injection, and nurse administering the injection did not significantly influence discomfort ratings.

In vitro modelling of epithelial and stromal interactions in non-malignant and malignant prostates.

To study the effects of stromal epithelial cell interactions on prostate cancer metastasis, we have used primary human prostatic stromal cells derived from malignant and non-malignant tissues and established epithelial cell lines from normal (PNT1a and PNT2-C2) and tumour (PC-3, DU145 and LNCaP) origins. The effects of stromal cells on epithelial cell growth were studied in direct and indirect (using culture inserts) co-culture and by exposure to stromal cell-conditioned medium (assessed by MTT assay). The influence of stromal cells on epithelial cell invasion was measured using matrigel invasion chambers and on epithelial cell motility using time lapse microscopy. Results indicated that epithelial cell line growth was similarly unaffected or inhibited by stromal cells derived from malignant (n = 8) or non-malignant tissue (n = 8). In contrast, PNT2-C2 and PC-3 cells were found to be the least and the most invasive and motile epithelia respectively. Stromal cultures enhanced the invasion of both epithelial cells, but no differences were observed between the use of malignant and non-malignant tissues. All stromal cultures modestly stimulated PNT2-C2 motility but displayed a greater stimulation of PC-3 cell motility, while stromal cells derived from malignant tissue stimulated PNT2-C2 and PC-3 cell motility more than stromal cultures from non-malignant tissues.

Investigation of mammary epithelial cell-bone marrow stroma interactions using primary human cell culture as a model of metastasis.

A model has been established using primary human cell culture to study the cell biology of breast cancer metastasis to bone marrow. Mammary epithelia were obtained in single cell suspension from tumour (macroscopically involved), benign (macroscopically uninvolved) and normal (reduction mammoplasty) breast tissue as well as from locally involved lymph nodes. Stromal layers were generated from long-term cultures of human bone marrow or from mammary fibroblasts derived from normal or malignant tissue. The interaction between epithelia and stroma has been studied in terms of adhesion of the epithelia to the stroma and their subsequent growth in co-culture. Our results show that when assayed up to 9 hr after plating, epithelial cells from malignant tissue (14 primary tumours and 9 metastases in lymph nodes) displayed a significant preference for adhesion to bone marrow stroma compared with mammary fibroblasts. In contrast, epithelial cells from 4 normal and 2 of 4 benign samples showed no significant preferential adherence. Subsequent co-culture of mammary epithelia with each of the 3 stromal layers revealed that under serum-free, in vitro conditions, bone marrow stromal layers did not provide an advantageous environment for colony growth, in contrast to their ability to provide a preferential substratum for adhesion.

Production and response of human prostate cancer cell lines to granulocyte macrophage-colony stimulating factor.

Prostate cancer selectively metastasises to skeletal sites, where it normally produces osteoblastic lesions. This study investigated whether haematopoietic growth factors known to be present in the bone environment could be involved in the survival and proliferation of prostate skeletal metastases. To evaluate this hypothesis we investigated the effects of recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF), recombinant granulocyte colony-stimulating factor (rG-CSF), recombinant erythropoietin (rEPO) and recombinant interleukin-3 (rIL-3) on the growth of 3 human prostate cancer cell lines. Two hormone-insensitive cell lines, PC-3 and DU145, were significantly stimulated by rGM-CSF and rEPO in serum-free medium but their growth was unaffected by incubation with rIL-3 or rG-CSF. A hormone-sensitive cell line, LNCaP, was stimulated only by rGM-CSF. To investigate further the involvement of GM-CSF in prostate cancer, the presence of GM-CSF protein in the 3 prostate cancer cell lines was examined by immunohistochemistry, and analysis of cell line conditioned media was carried out by ELISA and Western blotting. These techniques demonstrated that GM-CSF-like material was produced by both DU145 and PC-3 cells but not by LNCaP. The results from ELISA found that media conditioned by DU145 and PC-3 cells contained 1.7 and 2.5 pg GM-CSF/micrograms protein, respectively, whereas no GM-CSF was detectable in the LNCaP conditioned media. Our results were also confirmed by Western blot analysis demonstrating one single band for DU145 and PC-3 conditioned media which co-migrated along with the standard rGM-CSF band. No bands were associated with the LNCaP conditioned media. The presence of GM-CSF gene transcripts in DU145 and PC-3 cells was established by reverse transcription and polymerase chain reaction of total RNA.

Prostate epithelial cell lines form spheroids with evidence of glandular differentiation in three-dimensional Matrigel cultures.

Normal (PNT2-C2) and metastatic (PC-3) prostate cell lines were grown in Matrigel to observe the effects on morphology and phenotype in comparison to monolayer culture. In monolayer cultures, PNT2-C2 showed typical round/cuboidal epithelial morphology, with tight cell associations, whereas in Matrigel they formed smooth spheroids, tightly packed with cells. In both monolayer and Matrigel, PNT2-C2 had a differentiated luminal epithelial phenotype with high expression of cytokeratin 8, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), E-cadherin and desmoglein. In contrast, PC-3 cells possessed an epithelial/mesenchyme morphology in monolayer with loose cell to cell contact and pseudopodial extensions. Immunohistochemical phenotyping indicated the cells were undifferentiated, expressing high levels of vimentin, beta1 integrin, CD44 and low expression of cytokeratin 8. In Matrigel they formed smooth and irregular spheroids, which had a lumen surrounded by a single cell layer. Matrigel also influenced the expression of PSA, PSMA and CD44. These results indicate that Matrigel culture can induce morphological differentiation of prostate cancer cells which initially had a basal phenotype.

Experimental prostate epithelial morphogenesis in response to stroma and three-dimensional matrigel culture.

To reproduce the structural and functional differentiation of human prostatic acini in vivo, prostatic epithelial and stromal cells derived from human primary cultures were cocultured in Matrigel. In the absence of stroma and serum, epithelial spheroids composed of solid masses of stratified and cuboidal cells formed. Outer cells of the spheroid expressed cytokeratins 1, 5, 10, and 14, whereas the inner cells expressed cytokeratin 18. The addition of 2% serum induced formation of a lumen surrounded by a layer of one or two cuboidal and columnar epithelial cells. The further addition of stromal cultures, dihydrotestosterone, and estrogen induced polarization of the epithelium and increased spheroid-forming efficiency. Epithelium expressed either cytokeratin 18 alone or additionally cytokeratins 1, 5, 14, and 10. All spheroid epithelium expressed prostate-specific antigen and prostate-specific membrane antigen. Androgen receptor was only detected in the presence of stroma, serum, and hormones. Thus, development of a functional and morphologically correct prostate gland in vitro is dependent on extracellular matrix, steroid hormones, and factors from stromal cells and serum.

In vitro effects of recombinant human megakaryocyte growth and development factor on primary human tumour colony growth.

A study was made of the in vitro effects of recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on breast, prostate, cervix and colon cancers. Tumour growth was assessed using a soft agar clonogenic assay. Stimulation of megakaryocyte numbers was seen in liquid culture following 10 ng/ml PEG-rHuMGDF. The same concentration led to no significant difference in colony-forming efficiencies for 22 of 25 tumour samples. A significant reduction in colony-forming efficiencies was seen for the remaining 3 specimens. These results suggest that PEG-rHuMGDF will not stimulate tumour growth in vivo.

Role of proteolytic enzymes in human prostate bone metastasis formation: in vivo and in vitro studies.

Prostate cancers ability to invade and grow in bone marrow stroma is thought to be due in part to degradative enzymes. The formation of prostate skeletal metastases have been reproduced in vitro by growing co-cultures of prostatic epithelial cells in bone marrow stroma. Expression of urokinase plasminogen activator, matrix metalloproteinase 1 and 7 by prostatic epithelial cells were identified using immunocytochemistry. Also, in vivo tissue sections from human prostatic bone marrow metastases were stained. To establish the role of these enzymes on colony formation, inhibitory antibodies directed against urokinase plasminogen activator, matrix metalloproteinase 1 and matrix metalloproteinase 7 were added into primary prostatic epithelial cells and bone marrow stroma co-cultures. All prostatic epithelial cell cultures stained positively for matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator. Generally prostatic epithelial cells derived from malignant tissues showed increased staining in comparison to epithelia derived from non-malignant tissue. In agreement with in vitro co-cultures, the in vivo tissue sections of prostate bone marrow metastases showed positive staining for all three enzymes. Inhibition studies demonstrated that blocking matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator function reduced the median epithelial colony area significantly in bone marrow stroma co-cultures in vitro. Using a human ex-vivo model we have shown that matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator play an important role in the establishment of prostatic epithelial cells within bone marrow.

Interactions of human prostatic epithelial cells with bone marrow endothelium: binding and invasion.

Prostate cancer shows a propensity to form secondary tumours within the bone marrow. Such tumours are the major cause of mortality in this disease. We have developed an in vitro system to study the binding of prostate epithelial cells to bone marrow endothelium (BME) and stroma (BMS). The metastatic prostate cancer cell line, PC3 (derived from a bone metastasis), was seeded onto confluent layers of BME and its binding characteristics compared to human umbilical vein endothelial cells (HUVEC), lung endothelium (Hs888Lu) and BMS. The PC3 cell line showed significantly increased binding to BME (P< 0.05) compared to endothelium derived from HUVEC and lung or BMS with maximal binding occurring at 1 h. Following pre-incubation with a beta1 integrin antibody PC3 binding to BME was inhibited by 64% (P< 0.001). Antibodies directed against the integrins beta4, alpha2, alpha4, alpha5 and the cellular adhesion molecules P-selectin, CD31, VCAM-1 and sialy Lewis X showed no effect on blocking PC3 binding. Primary prostatic epithelial cells from both malignant (n = 11) and non-malignant tissue (n = 11) also demonstrated equivalent levels of increased adhesion to BME and BMS compared to HUVEC, peaking at 24 h. Further studies examined the invasive ability of prostate epithelial cells in response to bone marrow endothelium using Matrigel invasion chamber assays. In contrast to the previous results, malignant cells showed an increase (1000 fold) in invasive ability, whilst non-malignant prostate epithelia did not respond. We have shown that both malignant and non-malignant prostate epithelial cells can bind at equivalent levels and preferentially to primary human bone marrow endothelium in comparison to controls. However, only malignant prostate epithelia show increased invasive ability in response to BME.

Interaction of prostate epithelial cells from benign and malignant tumor tissue with bone-marrow stroma.

BACKGROUND: Metastases of prostate cancer form selectively within the skeleton. To understand this metastatic spread, we studied the ability of prostate epithelial cells to grow and proliferate within the bone marrow, using primary coculture. METHODS: Prostate epithelia and fibroblasts were prepared from men with benign prostatic hyperplasia (n = 13) and cancer of the prostate (n = 10). Confluent cultures of bone-marrow stroma or fibroblast controls were prepared in 96-well plates, and identical plates were treated with detergent to expose the extracellular matrix of the cells. Epithelial cells were seeded onto either cells or matrix, and their growth characteristics were determined by counting increases in colony size and number over time. Further experiments evaluated the effects on epithelial growth when cells were exposed to media conditioned by these stroma, using an MTT assay. RESULTS: Results showed that for epithelial cells derived from malignant (or benign) tissue, the median value of the total area of colonies formed on bone-marrow stroma was 2.1 (benign, 2.6) mm2, in contrast to 0.3 (benign, 0.4) mm2 or 0.25 (benign, 0) mm2 when these cells were cocultured with fibroblasts from benign or malignant prostates, respectively. Statistics indicated that growth was significantly greater on bone-marrow stroma than on control stroma (P < 0.005). However, no significant stimulation of epithelial cell growth was seen when these epithelial cells were cultured on extracellular matrix from bone-marrow stroma or when exposed to bone-marrow stroma-conditioned media in comparison to fibroblast controls. No statistical differences were found between the formation of colonies from malignant tissue in comparison to benign. CONCLUSIONS: This system allows the investigation of bone-marrow stroma colonization by primary prostate epithelial cells, and could be developed for the study of metastasis.