Isolation and characterization of a new cell line from a renal carcinoma bone metastasis (original) (raw)
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Anticancer …, 2006
Background: The contribution of angiogenesis to renal carcinoma bone metastases is virtually unknown. Materials and Methods: The effect of a cell line from a renal carcinoma bone metastasis (CRBM) was compared in vitro with the primitive renal adenocarcinoma line ACHN, by evaluating the influence on the ability of bone endothelial cells to activate osteoclasts. Results: The ACHN-conditioned medium produced a significant expression of macrophage-colony-stimulating factor mRNA. The conditioned medium from ACHN, CRBM, or from endothelial cells previously stimulated with the neoplastic cellconditioned media, had no direct effect on osteoclast differentiation from blood precursors (PBMC), such as CRBM and ACHN co-cultured with PBMC. However, PBMC cocultured with endothelial cells previously stimulated with the CRBM-conditioned medium showed significantly higher levels of tartrate-resistant acid phosphatase. Conclusion: It is possible that the bone metastatic line CRBM releases factors that induce endothelial cells to favor osteoclast differentiation. Renal cell carcinoma (RCC) bone metastases are osteolytic and rich in vessels. The process of new capillary formation from pre-existing vessels is essential for tumor growth and metastasis. Endothelial cells, after stimulation with interleukin-1 or tumor necrosis factor • (TNF•), produce bone-resorbing cytokines that may also play a role in bone metastasis. The expression of osteoprotegerin and the receptor activator of nuclear factor κB ligand (RANK-L) mRNAs was demonstrated in human microvascular 3065
Molecular Pathway for Cancer Metastasis to Bone
Journal of Biological Chemistry, 2003
SPARC by the integrins induces VEGF production to support bone remodeling and neovascularization to nourish the metastatic tumor. EXPERIMENTAL PROCEDURES Cell Lines and Materials-The LNCaP lineage-derived human prostate cancer cell line, LNCaP-C4-2, is androgen-independent and highly tumorigenic, with a proclivity to metastasize to bone (33). All human prostate cancer cell lines LNCaP, LNCaP-C4-2, PC3, and lacZ-transfected CWR22R (H-clones), kindly provided by Dr. Lloyd A. Culp (Case Western Reserve University, Cleveland, OH), were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Recombinant SPARC was expressed in insect cells and purified as described previously (34). Platelet-derived purified SPARC was obtained from Hematologic Technologies, Inc. (Essex Junction, VT). Fibronectin was purchased from Roche Diagnostics, and collagen type I was obtained from Calbiochem. Antibodies against ␣ v  3 (LM609), ␣ v  5 (P1F6), ␣ 5  1 (JBS5), and ␣ 2  1 (BHA2.1) integrins were obtained from Chemicon (Temecula, CA). Anti-VEGF and anti-VEGFR-2 were obtained from R&D Systems (Minneapolis, MN). Anti-platelet-derived growth factor (PDGF) was obtained from Sigma, and WOW-1 was provided by Dr. Shattil, La Jolla, CA. cRGDfV peptide was obtained from Peninsula Laboratories Inc. (San Carlos, CA). Preparation of Bone Extracts-SPARC-null and control mice of the same genetic background were generated as described previously (23). The SPARC-null mice do not contain detectable amounts of SPARC mRNA or protein. Bones excised from 6-week-old male wild-type and SPARC-null littermates (from pelvic region and limb) (1 g) were crushed and extracted for 24 h at 4°C, as described previously (4). Extracts were centrifuged at 25,000 ϫ g for 30 min, and the supernatant fractions were dialyzed against phosphate-buffered saline. Cell Migration Assays-Cell migration assays on Transwell plates (8-m pore size) were performed as described previously (27). Ligand (recombinant or purified SPARC or bone extract, fibronectin, and collagen) was diluted to a selected concentration, and 10 l of this solution was placed on the lower surface of a polycarbonate filter and air-dried. Cell migration was performed in the presence of blocking antibodies against ␣ v  3, ␣ v  5, ␣ 5  1 , and ␣ 2  1 , neutralizing antibodies to VEGF, VEGFR-2, and PDGF (20 g/ml each) or cRGDfV peptides (20 M). Flow Cytometry-WOW-1, a Fab fragment, reacts selectively with activated ␣ v  3 and ␣ v  5 (29). Its binding to cells was assessed by flow cytometry, as described previously (16). In selected experiments, cells were preincubated for 5 min in the presence of an inhibitor (anti-VEGF or anti-VEGFR-2 neutralizing antibodies or VEGFR2/Fc chimera; 20 g/ml each). WOW-1 Fab was then added at a final concentration of 30 g/ml, followed by the addition of Alexa 488 goat anti-mouse IgG (Molecular Probes, Eugene, OR) at 15 g/ml. After 30 min, the cells were washed and analyzed by flow cytometry. Specific binding of WOW-1 Fab was defined as that which could be inhibited by 10 mM EDTA. Flow cytometry was performed with a FACScan instrument, and the data were analyzed with the CellQuest software program (version 1.2). Proliferation Assays-These assays were performed as described (35). Briefly, cells were maintained in 1% serum for 20 h prior to experiments. Trypsinized cells were distributed into 96-well microtiter plates (2 ϫ 10 5 cells/well) in the presence or absence of inhibitors. The cells were labeled with 1 Ci of [ 3 H]thymidine per well. After 24-48 h, the cells were washed, and the radioactivity was precipitated with trichloroacetic acid and quantified by scintillation counting. Soft Agar Assays-LNCaP-C4-2 cells were trypsinized, washed, and resuspended in 0.4% Bacto-agar (Difco, Detroit, MI) prepared in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were plated on the top of 0.7% agar in the presence or absence of VEGFR-2/Fc chimera or anti-VEGFR-2 blocking antibodies (10 g/ml each). After 5 days, the colonies were photographed, and the number of colonies per power field was quantified 7 days after plating. Relative Quantitative Real Time PCR-Total RNA from two prostate cancer cell lines (LNCaP and LNCaP-C4-2) was prepared with RNeasy mini kits (Qiagen, Valencia, CA). Cells were plated in wells, which were left uncoated (control) or were coated with SPARC (200 ng/well), anti-␣ v  5 integrin antibodies (P1F6), or control nonimmune IgG (10 g/ml each). In selected experiments cRGDfV (20 M) was added to the cells; after specific time periods of incubation at 37°C, RNA was isolated. Real time PCR was performed using SYBR Green PCR core reagents (PerkinElmer Life Sciences) in an ABI Prism 7700 sequence detection system (PerkinElmer Life Sciences). The forward primer was 5Ј-GAG-GAGTCCAACATCACCATGC-3Ј, located on exon 3; the reverse primer
Bone Metastasis from Renal Cell Carcinoma
International Journal of Molecular Sciences, 2016
About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastasis that are often osteolytic and cause substantial morbidity, such as pain, pathologic fracture, spinal cord compression and hypercalcemia. The presence of bone metastasis in RCC is also associated with poor prognosis. Bone-targeted treatment using bisphosphonate and denosumab can reduce skeletal complications in RCC, but does not cure the disease or improve survival. Elucidating the molecular mechanisms of tumor-induced changes in the bone microenvironment is needed to develop effective treatment. The "vicious cycle" hypothesis has been used to describe how tumor cells interact with the bone microenvironment to drive bone destruction and tumor growth. Tumor cells secrete factors like parathyroid hormone-related peptide, transforming growth factor-β and vascular endothelial growth factor, which stimulate osteoblasts and increase the production of the receptor activator of nuclear factor κB ligand (RANKL). In turn, the overexpression of RANKL leads to increased osteoclast formation, activation and survival, thereby enhancing bone resorption. This review presents a general survey on bone metastasis in RCC by natural history, interaction among the immune system, bone and tumor, molecular mechanisms, bone turnover markers, therapies and healthcare burden.
Cancer Research, 2008
Approximately 30% of patients with renal cell carcinoma (RCC) develop bone metastasis, which is characterized by extensive osteolysis leading to severe bone pain and pathologic fracture. Although the mechanism of RCC-induced osteolysis is unknown, studies of bone metastasis have shown that tumor-induced changes in bone remodeling are likely mediated by alterations in the bone microenvironment. Here, we report the discovery of a novel osteoclast stimulatory factor secreted by RCC bone metastasis (RBM). Through microarray analysis, we found expression of the chemokine, macrophage inflammatory protein-1D (MIP-1D), to be increased in RBM versus patient-matched primary RCC tissues and confirmed this finding by quantitative reverse transcription-PCR (qRT-PCR) and ELISA (P < 0.05). Furthermore, MIP-1D expression in RBM tissues was significantly (P < 0.001) higher than in human bone marrow, suggesting a potential alteration of the bone microenvironment. The receptors for MIP-1D, CCR1 and CCR3, were expressed in both osteoclast precursors and mature, bone-resorbing osteoclasts as shown by qRT-PCR and Western analysis. In functional studies, MIP-1D stimulated chemotaxis of two osteoclast precursor cell types: murine bone marrow mononuclear cells (BM-MNC) and RAW 264.7 cells. Furthermore, MIP-1D treatment of murine calvaria caused increased bone resorption as determined by measurement of released calcium. Correspondingly, MIP-1D significantly enhanced osteoclast formation and activity in response to RANKL in both BM-MNC and RAW 264.7 cells. Taken together, these data suggest that MIP-1D expression is increased in RBM relative to RCC and bone marrow, and may promote RBM-induced osteolysis by stimulating the recruitment and differentiation of osteoclast precursors into mature osteoclasts.
Tumor-bone cellular interactions in skeletal metastases
Journal of musculoskeletal & neuronal interactions, 2004
Human tumor cells inoculated into the arterial circulation of immunocompromised mice can reliably cause bone metastases, reproducing many of the clinical features seen in patients. Animal models permit the identification of tumor-produced factors, which act on bone cells, and of bone-derived factors. Local interactions stimulated by these factors drive a vicious cycle between tumor and bone that perpetuates skeletal metastases. Bone metastases can be osteolytic, osteoblastic, or mixed. Parathyroid hormone-related protein, PTHrP, is a common osteolytic factor, while vascular endothelial growth factor and interleukins 8 and 11 also contribute. Osteoblastic metastases can be caused by tumor-secreted endothelin-1, ET-1. Other potential osteoblastic factors include bone morphogenetic proteins, platelet-derived growth factor, connective tissue growth factor, stanniocalcin, N-terminal fragments of PTHrP, and adrenomedullin. Osteoblasts are the main regulators of osteoclasts, and stimulatio...
Neoplasia (New York, N.Y.), 2017
Bone metastasis is common in renal cell carcinoma (RCC), and the lesions are mainly osteolytic. The mechanism of bone destruction in RCC bone metastasis is unknown. We used a direct intrafemur injection of mice with bone-derived 786-O RCC cells (Bo-786) as an in vivo model to study if inhibition of osteoblast differentiation is involved in osteolytic bone lesions in RCC bone metastasis. We showed that bone-derived Bo-786 cells induced osteolytic bone lesions in the femur of mice. We examined the effect of conditioned medium of Bo-786 cells (Bo-786 CM) on both primary mouse osteoblasts and MC3T3-E1 preosteoblasts and found that Bo-786 CM inhibited osteoblast differentiation. Secretome analysis of Bo-786 CM revealed that BIGH3 (Beta ig h3 protein), also known as TGFBI (transforming growth factor beta-induced protein), is highly expressed. We generated recombinant BIGH3 and found that BIGH3 inhibited osteoblast differentiation in vitro. In addition, CM from Bo-786 BIGH3 knockdown cells...
Effects of Human Tumor Cell Lines on Local New Bone Formation In Vivo
Calcified Tissue International, 1997
Although some tumors cause osteolytic lesions, there are some that stimulate new bone formation. This is an important phenomenon because the responsible mechanisms probably represent an aberration of normal physiological bone formation, and identifying the factors involved in the process may lead to new therapies for various bone diseases. To clarify our understanding of the potential mechanism responsible, we compared and quantitated the extent of new bone formation stimulated by human tumors (HeLa, Hep-2, AV-3, FL, WISH and KB), some of which have osteogenic activity in vivo [2]. Tumor cells were injected over the calvaria of nude mice to examine formation of new bone. The tumor cells produced three histologically distinct patterns of new bone growth: (1) WISH and KB stimulated appositional bone growth adjacent to periosteal bone surfaces; (2) HeLa and Hep2 induced new bone growth over calvarial surface even when distant from the tumor mass; (3) FL stimulated bone formation adjacent to periosteum as well as ectopic bone formation in sites distant from bone. All tumors except AV3 induced mean new bone thickness >100 m, and Hep-2 cells produced bone 330 m thick. PCR and Northern blot analysis of mRNA isolated from cultured tumor cells revealed that all cell lines expressed mRNA for TGF, (fibroblast growth factor) FGF-1, FGF-2, and IGF-I, and most cell lines produced mRNA for PDGF. Only FL expressed large amounts of mRNA for BMP2. In serum-free conditioned media from Hep2 and HeLa cells purified by heparin affinity chromatography, we have identified FGF-1, FGF-2, and PDGF by immunodetection with specific antibodies. Our results show that new bone growth caused by these tumors is likely due to the production of bone growth factors by the tumor cells, and that the overall effects on bone may be due to several factors working in concert.
Vascular endothelial growth factor acts as an osteolytic factor in breast cancer metastases to bone
The Women's Oncology Review, 2005
Vascular endothelial growth factor (VEGF) is a proangiogenic cytokine that is expressed highly in many solid tumours often correlating with a poor prognosis. In this study, we investigated the expression of VEGF and its receptors in bone metastases from primary human breast tumours and further characterised its effects on osteoclasts in vitro. Breast cancer metastases to bone were immunohistochemically stained for VEGF, its receptors VEGFR1 and 2 (vascular endothelial growth factor receptor 1 and 2), demonstrating that breast cancer metastases express VEGF strongly and that surrounding osteoclasts express both VEGFR1 and VEGFR2. RAW 264.7 cells (mouse monocyte cell line) and human peripheral blood mononuclear cells (PBMCs) were cultured with VEGF, RANKL and M-CSF. VEGF and RANKL together induced differentiation of multinucleated, tartrate-resistant acid phophatase (TRAP)-positive cells in similar numbers to M-CSF and RANKL. The PBMCs were also able to significantly stimulate resorption of mineralised matrix after treatment with M-CSF with RANKL and VEGF with RANKL. We have shown that VEGF in the presence of RANKL supports PBMC differentiation into osteoclast-like cells, able to resorb substrate. Vascular endothelial growth factor may therefore play a role in physiological bone resorption and in pathological situations. Consequently, VEGF signalling may be a therapeutic target for osteoclast inhibition in conditions such as tumour osteolysis.
International Journal of Oncology, 2007
Bone is a common site of osteolytic and richly vascularized metastases of renal cell carcinoma (RCC) and Interferon (IFN)-• based therapies have been considered for the treatment of patients affected by this disease. The effects of IFN-• on metastatic RCC patients have been related to its immunomodulatory, and cytotoxic activity on tumor cells, but there could be an effect also on tumor induced osteoclast differentiation and bone angiogenesis. When osteoclasts obtained from human peripheral blood mononuclear cells, cultured in the presence of receptor activator of nuclear factor-κB (RANKL) and macrophage-colony stimulating factor (M-CSF), were treated with IFN-•, the expression of bone tartrate resistant acid phosphatase (TRACP) type 5b was reduced, as well as calcium-phosphate resorption activity and expression of pro-osteoclatic transcription factor c-Fos. IFN-• modulation of angiogenesis was studied by analysis of proliferation, survival, and migration of a bone endothelial cell line (BBE), and by the analysis of pro-angiogenic factor expression in RCC cell lines. IFN-• inhibited bone endothelial cell proliferation and the expression of FGF-2, while the vascular endothelial growth (VEGF) did not show any significant variation. Moreover, IFN-• inhibited the migration induced by the RCC through the impairment of fibroblast growth factor-2 (FGF-2) secretion. These data demonstrate multiple activities of IFN-• on renal cancer-induced bone disease, in addition to its recognized role as a cytotoxic and immunomodulatory agent, because they indicate its ability to reduce bone resorption and to impair tumor-associated angiogenesis, and they also suggest the use of IFN-• to treat skeletal metastases of other carcinomas.