In vitro studies of human prostatic epithelial cells: attempts to identify distinguishing features of malignant cells

Recent advances in culture techniques have enabled routine establishment and propagation of epithelial cells derived from normal and malignant tissues of the human prostate. Comparative studies of the responses of normal and cancer-derived cell populations to various growth and differentiation factors in vitro were undertaken to examine the possibility that cancer cells might respond differentially. Clonal growth assays in serum-free medium demonstrated that optimal proliferation of normal as well as cancer cell strains was generally dependent on the presence of cholera toxin, epidermal growth factor, pituitary extract, hydrocortisone, insulin, and high levels of calcium in the culture medium, and on the use of collagen-coated dishes. Only one cancer strain responded aberrantly to epidermal growth factor and hydrocortisone. Putative differentiation factors (transforming growth factor-beta and vitamin A) inhibited the growth of all normal and cancer strains. The origin of a cancer-derived cell strain that responded similarly to normal strains was verified by positive labeling with a prostate cancer-specific antibody, validating the conclusion from these studies that normal and cancer prostatic epithelial cells are not distinguishable on the basis of responses to the tested factors.     

Normal and malignant prostate epithelial cells differ in their response to hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived factor in the development and progression of prostate cancer.     

Perlecan Knockdown in Metastatic Prostate Cancer Cells Reduces Heparin-binding Growth Factor Responses in vitro and Tumor Growth in vivo

Perlecan (Pln) is a major heparan sulfate proteoglycan (HSPG) of extracellular matrices and bone marrow stroma. Pln, via glycosaminoglycans in domains I and V, acts as a co-receptor for delivery of heparin binding growth factors (HBGFs) that support cancer growth and vascularization. Specifically, glycosaminoglycans bind HBGFs and activate HBGF receptors, including those for FGF-2 and VEGF-A. The contribution of Pln to prostate cancer growth was tested using a ribozyme approach to knockdown Pln expression levels. Transfection into the androgen-independent, bone targeted prostate cancer line, C4-2B, and efficient stable knockdown of Pln was demonstrated by quantitative PCR, immunohistochemistry and immunoblotting. Three individually isolated subclones with 75–80% knockdown in Pln mRNA, protein expression and secretion into ECM were used to study in vitro growth responses to FGF-2 and VEGF-A. While cells with normal Pln levels responded to both HBGFs, knockdown cells responded poorly. All lines responded to serum growth factors and IGF-I. Anchorage-independent growth assays showed reduced colony size and cohesiveness by all Pln deficient subclones compared to parental C4-2B cells. In vivo effects of Pln knockdown were measured by inoculating knockdown and control ribozyme transfected cell lines into athymic mice. A reduced growth rate, smaller tumor size, diminished vascularization and failure to elevate serum PSA characterized mice bearing Pln knockdown C4-2B cells. Poor vascularization correlated with reduced levels of VEGF-A secreted by Pln knockdown lines. We conclude that Pln is an essential ECM component involved in growth responses of metastatic prostate cancer cells to HBGFs deposited in local and metastatic microenvironment.     

Effects of Growth Factors on Cytokine Production in Serum-Free Cultures of Human Thymic Epithelial Cells

Human thymic epithelial cells (TEC) of medullary phenotype were cultured for 14 days in a growth factor-defined scrum-free medium. The effects of added growth factors on cell numbers and the production of cytokines were investigated by separate exclusion of the various growth factors from the medium. We found that hydrocortisone stimulated cell proliferation but inhibited the differentiation of TEC and significantly reduced the production of interleukin-lα, interleukin-6 and granulocyte-macrophage colony stimulating factor. Insulin was found to enhance the differentiation of TEC and the production of the three cytokines. Transfcrrin and choleratoxin were found to inhibit cell proliferation, but they did not affect produetion of the cytokines. Exclusion of epidermal growth factor, however, leads to cell death. We conclude that it is essential to exclude hydrocortisone from the medium to optimize production of cytokines, and that transferrin and choleratoxin seem to be unnecessary constituents in serum-free cultures of human TEC.     

Primary culture of human oral epithelial cells. Growth requirements and expression of differentiated characteristics

Epithelial cell cultures were initiated from explants of normal human oral mucosa. Growth parameters, cell type, and degree of maturation/cytodifferentiation were assessed by morphological and surface topographical criteria (light and scanning electron microscopy) together with immunofluorescence studies with a panel of antibodies to cytokeratins and extracellular matrix components. The effects of different media formulations were compared. Whereas stromal cell over-growth soon became apparent in media containing 10% serum, in low serum (0.5%) media containing insulin, hydrocortisone, epidermal growth factor (EGF), and/or cholera toxin (CT), epithelial growth was maintained with minimal or absent stromal cell contamination. Cell proliferation, maturation, and differentiation were modulated by EGF and CT: cultures maintained on EGF showed optimal growth but cells typically displayed only limited differentiation. By contrast, CT promoted considerably more cytodifferentiation but at the expense of proliferative capacity. Both factors together were complementary, resulting in maintenance of cells of a more mature phenotype of high proliferative capacity. Cytokeratins of normal oral epithelium in situ demonstrated characteristic changes in patterns of expression associated with differentiation. In culture, proliferative epithelial cells expressed keratins typical of the basal layer, whereas the most differentiated cells were identified by their strong reactivity with antibodies to epidermal keratins. Less mature cells showed expression of keratins associated with nonstratified epithelia. In cultures maintained with CT but no EGF, there was a tendency for weaker expression of basal type keratins, further suggesting that these cells were maintaining a more differentiated phenotype. Extracellular matrix components (fibronectin, laminin, collagen type IV) were not expressed by any epithelial cells in culture. Irrespective of medium composition, cultures did not survive beyond 100 days (5 or 6 subcultivations) before undergoing an irreversible 'crisis' of growth arrest and onset of degenerative changes.     

Induction of Proliferation and Differentiation of Leukaemic B Cells from Patients with Chronic Lymphocytic Leukaemia by Anti-μ and Conditioned Medium

We investigated the growth and differentiation of leukaemia B cells from patients with B-cell chronic lymphocytic leukaemia (CLL) in response to proliferation and differentiation factors present in conditioned medium and to anti-immunoglobulin antibodies. Highly purified E-rosette negative (E-) B cells from 5 out of 15 patients with CLL exhibited moderate proliferative responses to 10 or 50 micrograms/ml of F(ab)'2 fragments of rabbit anti-human mu-chain specific antibody. Conditioned medium (CM), derived by stimulating human peripheral blood mononuclear leucocytes with PHA, induced significant proliferative responses of purified E- cells in 13 out of 14 patients examined. The extent of these proliferative responses varied substantially, and was in the range of 2.6- to 91-fold. Stimulation of purified E- cells from patients with CLL with both anti-mu and CM resulted in significant proliferation in all 15 patients examined. These responses were significantly higher than those induced by CM alone (P less than 0.02). Synergism between CM and anti-mu in inducing proliferative responses was observed in 11 out of 15 patients. Largely leukaemic B cell populations expressing on the cell surface more than one immunoglobulin heavy-chain isotype, exhibited significantly higher (P less than 0.009) proliferative response to CM and anti-mu than those expressing IgM only. Highly purified E-peripheral blood or tonsil lymphocytes from all normal donors examined responded by proliferation to anti-mu alone or to CM alone. Synergism in inducing proliferative responses was also observed when the cells were stimulated with both CM and anti-mu. In addition to inducing proliferative responses, culture with CM of purified E-rosette negative, largely leukaemic, B cells from patients with CLL for 6 days at 37 degrees C resulted in differentiation into immunoglobulin synthesizing and secreting cells. Synthesis and secretion of IgM were observed in 7 out of 10 patients examined. A switch to IgG production was observed in three patients. Morphological examination of E- cells from patients with CLL after treatment with CM demonstrated that these cells were differentiated into plasma-like cells. These results suggest that leukaemic B cells from patients with CLL can be induced to proliferate and differentiate in response to growth and differentiation factors derived by mononuclear leucocytes, in a manner similar to that of normal B cells.     

Culture of conducting airway epithelial cells in serum-free medium

Summary Reproducible techniques for the isolation and culture of conducting airway epithelial cells from various species including human are described. Tissues recovered after necropsy or surgery are treated with 0.1% protease at 4°C overnight. The epithelial lining cells are liberated from adventitia by flushing with ice-cold minimal essential medium containing 10% fetal bovine serum. The resulting suspension of single cells and cell clusters is centrifuged and then plated on type 1 collagen gel substratum in a serum-free F12 medium supplemented with insulin, transferrin, epidermal growth factor, cholera toxin, hydrocortisone (or dexamethasone), bovine hypothalamus extract, and retinol. Increased calcium concentration in the medium to 1 to 3 mM augments in vitro mucous cell differentiation. Both a squamouslike and mucociliary differentiation of the cultured epithelium can be identified by biochemical, immunohistologic, and morphologic means. The polarity of epithelial cell cultures is promoted by use of biphasic culture methods, in which epithelial cells are fed basally and are in direct contact with the air phase.     

Characterization of human thymic epithelial cells grown in serum-free medium

Thymic epithelial cells have a critical influence on T-cell differentiation. In order to characterize these cells in humans, a serum-free growth medium was developed for their long-term culture. Important components of this medium included transferrin, epidermal growth factor, prostaglandin E1, and selenious acid. The presence of a keratin cytoskeleton, tonofilaments, and desmosomes confirmed the epithelial nature of these cells. Indirect immunofluorescence study of these epithelial cells demonstrated the presence of Ia and B-2 microglobulin antigens. The availability of highly enriched thymic epithelial cultures should simplify the functional characterization of this cell.     

Enrichment of prostate cancer stem cells from primary prostate cancer cultures of biopsy samples

This study was to enrich prostate cancer stem cells (PrCSC) from primary prostate cancer cultures (PPrCC). Primary prostate cancer cells were amplified in keratinocyte serum-free medium with epidermal growth factor (EGF) and bovine pituitary extract (BPE), supplemented with leukemia inhibitory factor (LIF), stem cell factor (SCF) and cholera toxin. After amplification, cells were transferred into ultra-low attachment dishes with serum-free DMEM/F12 medium, supplemented with EGF, basic fibroblast growth factor (bFGF), bovine serum albumin (BSA), insulin, and N2 nutrition. Expression of cell-type-specific markers was determined by RT-qPCR and immunostaining. Tumorigenicity of enriched PrCSC was determined by soft agar assay and xenograft assay in NOD/SCID mice. Biopsy samples from 19 confirmed prostate cancer patients were used for establishing PPrCC, and 18 cases (95%) succeeded. Both basal marker (CK5) and luminal markers (androgen receptor and CK8) strongly co-expressed in most of PPrCC, indicating their basal epithelial origin. After amplification under adherent culture condition in vitro, transient amplifying cells were the dominant cells. Sphere formation efficiency (SFE) of passaged PPrCC was about 0.5%, which was 27 times lower than SFE of LNCaP (13.67%) in the same condition. Compared with adherent cells from PPrCC, prostasphere from PPrCC showed up regulated stem cell markers and increased tumorigenic potential in soft-agar assay. However, spheroid cells from PPrCC prostasphere failed to initiate tumor in xenograft assay in 6 months. Thus, PPrCC can be established and amplified from prostate cancer biopsy samples. Our modified sphere culture system can enrich PrCSC from PPrCC.     

Maintenance of normal human breast organoids within rat mammary fat pads in organ culture

Summary Normal human breast organoids, derived by collagenase digestion of reduction mammaplasty tissue specimens, have been cultured in vitro for up to 28 days after injection into organ cultures of virgin rat mammary fat pads. The culture medium was serum-free Waymouth's MB 752/ 1 with hormonal additives. The rat mammary tissue responded well to growth-promoting and lactogenic stimuli in the culture medium, in agreement with previous investigations. Using immunohistochemistry casein was identified in rat epithelia exposed to lactogenic medium. Human organoids in culture remained viable but did not show hormone-responsiveness. Electron microscopy confirmed the presence of both luminal epithelial cells and myoepithelial cells.The serum-free culture of normal human breast organoids in a three-dimensional matrix provides a system in which to study factors controlling growth and differentiation.     

Prostate cancer tends to metastasize in the bone-mimicking microenvironment via activating NF-kB signaling

Prostate cancer preferentially metastasizes to the bone. However, the underlying molecular mechanisms are still unclear. To explore the effects of a bone-mimicking microenvironment on PC3 prostate cancer cell growth and metastasis, we used osteoblast differentiation medium (ODM; minimal essential medium alpha supplemented with L-ascorbic acid) to mimic the bone microenvironment. PC3 cells grown in ODM underwent epithelial-mesenchymal transition and showed enhanced colony formation, migration, and invasion abilities compared to the cells grown in normal medium. PC3 cells grown in ODM showed enhanced metastasis when injected in mice. A screening of signaling pathways related to invasion and metastasis revealed that the NF-kB pathway was activated, which could be reversed by Bay 11-7082, a NF-kB pathway inhibitor. These results indicate that the cells in different culture conditions manifested significantly different biological behaviors and the NF-kB pathway is a potential therapeutic target for prostate cancer bone metastasis.     

Assay of growth factors for prostate epithelial cells

Summary The maintenance and proliferation of isolated prostate epithelial cells is androgen independent, but requires multiple other hormones and hormonelike growth factors. Methods are described for isolation and characterization of epithelial cells from normal rat prostate and the androgen-responsive transplantable Dunning R3327 rat tumor. Pure tumor-derived cell lines can be established by serial culture techniques. The normal primary and serially cultured cell lines are then used to assay growth factors. Cell proliferation is quantitated by computerized videometry.     

Interleukin-6 Is an Autocrine Growth Factor in Human Prostate Cancer

Prostate cancer is the most common cancer in American men and the second leading cause of cancer deaths in this group. We have found that interleukin (IL)-6 protein concentrations are increased approximately 18-fold in clinically localized prostate cancers when compared to normal prostate tissue. Normal and neoplastic prostatic epithelial cells in culture, with the exception of LNCaP cells, secrete IL-6. Addition of exogenous IL-6 to primary epithelial cells in culture or the LNCaP prostate cancer cell line leads to phosphorylation of Stat-3 and increases in net cell proliferation. The concentration of IL-6 receptor is increased eightfold in the prostate cancer tissues and is increased in the cancer cells by immunohistochemistry. The increased expression of IL-6 receptor is correlated with increased proliferation of prostate cancer cells in vivo as assessed by Ki67 immunohistochemistry. These findings strongly support the hypothesis that IL-6 acts as a significant autocrine growth factor in vivo for primary, androgen-dependent prostate cancers.     

Self Renewal of Embryonic Stem Cells in the Absence of Feeder Cells and Exogenous Leukaemia Inhibitory Factor

Abstract

To evaluate the role of leukaemia inhibitory factor (LIF) for maintaining pluripotent embryonic stem (ES) cells in culture, we established several exogenous LIF-independent ES cell lines by continuous passaging in culture. The newly established ES cells, Kli and CBli, sustained their growth and remained undifferentiated in LIF-deficient medium. Analysis of chimaeric animals, produced with the p-galactosidase transgenic Kli ES cells, revealed that LDF-independent ES cells can contribute to all embryonic germ layers. There was no detectable LIF protein in ES cell conditioned medium, and no upregulation of LIF mRNA was found. The addition of neutralising anti-LIF antibodies was not sufficient to abrogate the self renewal of the Kli ES cells. These studies suggest that the signalling pathway involving diffusible LIF can be bypassed for maintaining the pluripotency in culture, and indicate a considerable heterogeneity in growth factor dependence and differentiation of different ES cells.     

Adherence of Staphylococcus aureus to cultured epidermal cells during differentiation

The adherence of clinical isolates of Staphylococcus aureus to cultured mouse epidermal cells was studied. Adherence of the isolates to the cells varied from strain to strain. When epidermal cell differentiation was induced by raising the calcium concentration in the medium, three out of 10 strains tested adhered better to calcium-induced differentiated cells than to undifferentiated cells, and one strain demonstrated higher adherence to undifferentiated cells than to differentiated cells. No significant difference between the adherences to both types of epidermal cells was observed with the other six strains. No relationship was observed between adherence and surface hydrophobicity with bacterial cells. Lipoteichoic acid and N-acetyl sugars caused limited inhibition of adherence. The adherence assay method employed in this study is useful for investigating the effects of epidermal cell differentiation on bacterial adherence in vitro.     

Primary culture of rabbit gallbladder epithelial cells in collagen gel matrix

Primary culture of gallbladder epithelial cells obtained from normal rabbits was attempted in collagen gel matrix for up to 6 weeks. Fluid medium containing 0.02% ethylenediaminetetraacetic acid and 0.25% trypsin was poured into the gallbladder lumen. The pellets obtained by centrifugation of recovered fluid contained many isolated epithelial cells and a few small cell clumps. These pellets were dispersed and embedded inside collagen gel matrix and cultured in William's medium E supplemented with fetal calf serum and epidermal growth factor. The three-dimensional outgrowth from individual cells and small cell clumps consisted predominantly of spherical cystic masses 2-4 days later. These cysts contained mucin and were covered by a single layer of cuboidal or low columnar epithelial cells. Electron microscopy revealed the epithelial arrangement of cells lining the cyst walls, and these cells were similar to gallbladder epithelial cells in vivo. These epithelial cells showed active mucin secretion into the cystic cavities. Cytokeratin was diffusely present in the cytoplasm. This isolation and culture system provides a reproducible and consistent method for sustained growth of normal gallbladder epithelial cells from normal tissue in primary culture and seems valuable for investigating pathologic conditions of the gallbladder.     

Paracrine communication between malignant and non-malignant prostate epithelial cells in culture alters growth rate,matrix protease secretion and in vitro invasion

Epithelial cancer cell invasion is facilitated by stromal cells, immune cells, endothelial cells and other epithelial cells. We have used two human papilloma immortalized prostate cell lines, CA-HPV-10 from a carcinoma and PZ-HPV-7 cells from normal prostatic epithelium to study cell–cell influences on growth, gelatinase secretion, invasion and responses to TGFβ1. We found that co-culture with CA-10 carcinoma cells stimulates proliferation of the PZ-7 epithelial line. TGFβ1 inhibited growth of both lines, but while inhibitory effects on the CA-10 cells diminished after removal of the peptide, inhibition of PZ-7 was lasting. Interestingly, the TGFβ-induced growth inhibition in PZ-7 cells could be partially reversed by co-culture with CA-10 cells. Co-culture with CA cells in a 3-chamber invasion assay also promoted invasion of PZ cells. CA-10 invasion was enhanced by co-culture with TGFβ1-treated-PZ-7 cultures and this enhancement was associated with TGFβ1-induced secretion of matrix metalloproteinase-9. Our observations suggest that interaction between prostate cancer cells and prostate epithelial cells may promote proliferation of the epithelial cell population and produce a paracrine source of MMP-9 which may facilitate early cancer cell invasion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Establishment of primary cell cultures: Experiences with 155 cell strains

Summary Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: (1) Disintegration, (2) culture media supplemented with basal additions, (3) special supplements (growth factors, hormones), and (4) attachment factors. The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times. Procedures for how to obtain a relatively high plating efficiency (approx. 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion. If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin. (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases). Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases). The media were supplemented with the basal additions indicated. (3) In approx. 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth. (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures. The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.Abbreviations EGF epidermal growth factor - FGF fibroblast growth factor - GHL glycyl-L-histidyl-L-lysine - PAN pancreozymin - E estrogen - PR progesterone Supported by the Deutsche Forschungsgemeinschaft, grant Di 276/1–2, SFB 232, Hamburger Landesverband zur Krebsbekämpfung und Krebsforschung, and Hamburger Stiftung zur Förderung der Krebsbekämpfung     

Tumour-stroma interactions between metastatic prostate cancer cells and fibroblasts

Previous work has shown the importance of tumour-stroma interactions for prostate cancer development at the primary site. The aim of the present study was to find out whether evidence can be found for a tumour-stroma cross- talk also between metastatic prostate cancer cell lines and non-prostatic stromal fibroblasts which are encountered by metastatic cells at most sites. We addressed this issue in cell culture systems using 3 metastatic human prostate cancer cell lines (LnCaP, PC-3 and DU-145) on the one hand, and a human fibroblast line (HFF, human foreskin fibroblasts) on the other. We incubated fibroblasts with tumour cell- and tumour cells with fibroblast-conditioned media and evaluated several parameters important for the establishment of metastases such as cell proliferation, migration and expression of matrix degrading proteases. We also determined in the conditioned media the concentrations of several growth factors and cytokines which might be responsible for the observed effects. We found that media conditioned by all 3 metastatic prostate cancer cell lines stimulated fibroblast proliferation which corresponds to fibrous stroma induction in vivo. DU-145 cell conditioned media induced in fibroblasts expression of mmp-1 mRNA known to be important for tumour invasion. ELISA assays revealed that tumour cells secrete bFGF, PDGF and TNFalpha known to stimulate fibroblast proliferation and/or MMP-1 expression. Cultivation of DU-145 carcinoma cells in fibroblast conditioned medium resulted in an enhanced proliferation and anchorage-independent growth of this cell line in soft agar. Fibroblast conditioned medium also increased migration of PC-3 cells in the wound assay and slightly augmented mmp-1 expression. KGF (able to stimulate proliferation of normal and neoplastic prostate epithelial cells) was secreted by fibroblasts at higher concentrations than by all 3 tumour cell lines. In addition, fibroblasts secreted TNFalpha, bFGF, PDGF, HGF and also VEGF, the most important factor for tumour vascularization. Our results provide evidence that tumour-stroma interactions do not only exist at the primary site but also between metastatic prostate cancer cell lines and their fibroblastic microenvironment. These interactions, which are mediated through secreted factors, affect several steps of the metastatic cascade including proliferation, anchorage-independent growth, migration and the secretion of matrix-degrading proteases.