Adjuvant bisphosphonates in early breast cancer: consensus guidance for clinical practice from a European Panel.

Bisphosphonates have been studied in randomised trials in early breast cancer to investigate their ability to prevent cancer treatment-induced bone loss (CTIBL) and reduce the risk of disease recurrence and metastasis. Treatment benefits have been reported but bisphosphonates do not currently have regulatory approval for either of these potential indications. This consensus paper provides a review of the evidence and offers guidance to breast cancer clinicians on the use of bisphosphonates in early breast cancer. Using the nominal group methodology for consensus, a systematic review of the literature was augmented by a workshop held in October 2014 for breast cancer and bone specialists to present and debate the available pre-clinical and clinical evidence for the use of adjuvant bisphosphonates. This was followed by a questionnaire to all members of the writing committee to identify areas of consensus. The panel recommended that bisphosphonates should be considered as part of routine clinical practice for the prevention of CTIBL in all patients with a T score of <-2.0 or ≥2 clinical risk factors for fracture. Compelling evidence from a meta-analysis of trial data of >18,000 patients supports clinically significant benefits of bisphosphonates on the development of bone metastases and breast cancer mortality in post-menopausal women or those receiving ovarian suppression therapy. Therefore, the panel recommends that bisphosphonates (either intravenous zoledronic acid or oral clodronate) are considered as part of the adjuvant breast cancer treatment in this population and the potential benefits and risks discussed with relevant patients.

Synergistic effect between denosumab and immune checkpoint inhibitors (ICI)? A retrospective study of 268 patients with ICI and bone metastases.

Background: Bone metastasis is a significant concern in advanced solid tumors, contributing to diminished patient survival and quality of life due to skeletal-related events (SREs). Denosumab (DMAB), a monoclonal antibody targeting the receptor activator of nuclear factor kappa-B ligand (RANKL), is used to prevent SREs in such cases. The RANK/RANKL axis, crucial in immunological processes, has garnered attention, especially with the expanding use of immune checkpoint inhibitors (ICI) in modern oncology. Objective: Our study aims to explore the potential synergistic antitumor effects of combining immunotherapy with denosumab, as suggested by anecdotal evidence, small cohort studies, and preclinical research. Methods: We conducted a retrospective analysis using the IMMUCARE database, encompassing patients receiving ICI treatment since 2014 and diagnosed with bone metastases. We examined overall survival (OS), progression-free survival (PFS) and switch of treatment line based on denosumab usage. Patients were stratified into groups: without denosumab, ICI followed by denosumab, and denosumab followed by ICI. Survival curves and multivariate Cox regression analyses were performed. Results: Among the 268 patients with bone metastases, 154 received treatment with ICI alone, while 114 received ICI in combination with denosumab at some point during their oncological history. No significant differences were observed in overall survival (OS) or progression-free survival (PFS) between patients receiving ICI monotherapy and those receiving ICI with denosumab (p = 0.29 and p = 0.79, respectively). However, upon analyzing patients who received denosumab following ICI initiation (17 patients), a notable difference emerged. The group receiving ICI followed by denosumab exhibited a significant advantage compared to those without denosumab (154 patients) or those receiving denosumab before ICI initiation (72 patients) (p = 0.022). Conclusion: This retrospective investigation supports the notion of potential benefits associated with sequential administration of ICI and denosumab, although statistical significance was not achieved. Future studies, including prospective trials or updated retrospective analyses, focusing on cancers treated with first-line immunotherapy, could provide further insights into this therapeutic approach.

Targeting heat shock protein 27 (HspB1) interferes with bone metastasis and tumour formation in vivo.

BACKGROUND: The small stress heat shock protein 27 (Hsp27) has recently turned as a promising target for cancer treatment. Hsp27 upregulation is associated with tumour growth and resistance to chemo- and radio-therapeutic treatments, and several ongoing drugs inhibiting Hsp27 expression are under clinical trial. Hsp27 is now well described to counteract apoptosis and its elevated expression is associated with increased aggressiveness of several primary tumours. However, its role in the later stage of tumour progression and, more specifically, in the later and most deadly stage of tumour metastasis is still unclear. METHODS/RESULTS: In the present study, we showed by qRT-PCR that Hsp27 gene is overexpressed in a large fraction of the metastatic breast cancer area in 53 patients. We further analysed the role of this protein in mice during bone metastasis invasion and establishment by using Hsp27 genetically depleted MDA-MB231/B02 human breast cancer cell line as a model. We demonstrate that Hsp27 silencing led to reduced cell migration and invasion in vitro and that in vivo it correlated with a decreased ability of breast cancer cells to metastasise and grow in the skeleton. CONCLUSION: Altogether, these data characterised Hsp27 as a potent therapeutic target in breast cancer bone metastasis and skeletal tumour growth.

Serum total periostin is an independent marker of overall survival in bone metastases of lung adenocarcinoma.

More than 35% of lung adenocarcinoma patients have bone metastases at diagnosis and have a poor survival. Periostin, a carboxylated matrix protein, mediates lung cancer cell dissemination by promoting epithelial-mesenchymal transition, and is involved in bone response to mechanical stress and bone formation regulation. This suggests that periostin may be used as a biomarker to predict survival in lung cancer patients. Serum periostin was assessed at diagnosis in a prospective cohort of 133 patients with lung adenocarcinoma of all stages. Patients were divided into localized and bone metastatic groups. Both groups were matched to healthy controls. Survival analysis and Cox proportional hazards models were conducted in the total population and in bone metastatic group. The median serum periostin level was higher in bone metastatic (n = 67; median: 1752 pmol/L) than in the localized group (n = 66; 861 pmol/L; p < 0.0001). Patients with high periostin (>median) had a poorer overall survival in the whole population (33.3 weeks vs. NR; p < 0.0001) and the bone metastatic group (24.4 vs. 66.1 weeks; p < 0.001). In multivariate analysis, patients with high periostin had increased risk of death (HR = 2.09, 95%CI [1.06-4.13]; p = 0.03). This was also found in the bone metastatic group (HR = 3.62, 95%CI [1.74-7.52]; p = 0.0005). Immunohistochemistry on bone metastasis biopsies showed periostin expression in the bone matrix and nuclear and cytoplasmic staining in cancer cells. Serum periostin was an independent survival biomarker in all-stage and in bone metastatic lung adenocarcinoma patients. IHC data suggest that periostin might be induced in cancer cells in bone metastatic niche in addition to bone microenvironment expression.

The RANK-RANKL axis: an opportunity for drug repurposing in cancer?

Drug repurposing offers advantages over traditional drug development in terms of cost, speed and improved patient outcomes. The receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) inhibitor denosumab is approved for the prevention of skeletal-related events in patients with advanced malignancies involving bone, including solid tumours and multiple myeloma. Following improved understanding of the role of RANK/RANKL in cancer biology, denosumab has already been repurposed as a treatment for giant cell tumour of bone. Here, we review the role of RANK/RANKL in tumourigenesis, including effects on tumour initiation, progression and metastasis and consider the impact of RANK/RANKL on tumour immunology and immune evasion. Finally, we look briefly at ongoing trials and future opportunities for therapeutic synergy when combining denosumab with anti-cancer agents such as immune checkpoint inhibitors.

ERRα promotes breast cancer cell dissemination to bone by increasing RANK expression in primary breast tumors.

Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-of-function analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases.

Thrombospondin-1 and -2 messenger RNA expression in normal, benign, and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis, and fibroblastic desmoplasia.

Thrombospondin-1 (TSP1) is a Mr 450,000 extracellular matrix glycoprotein that modulates tumor growth, angiogenesis, and metastasis. Of the five structurally different TSPs described to date, only TSP2 is similar to TSP1 in terms of its molecular architecture, and TSP2 also modulates angiogenesis. Angiogenesis plays a relevant role in the biological aggressiveness of breast cancer, and TSP1 is present in the tumor stroma (termed desmoplasia) of invasive human breast ductal carcinoma not otherwise specified (NOS). The present study was designed to identify and quantify TSP1 and TSP2 mRNAs in normal, benign, and neoplastic human breast tissues using the reverse transcriptase PCR technique. We found that TSP2, like TSP1, was expressed in human breast tissues, and that TSP1 and TSP2 mRNA expression in invasive breast carcinoma NOS was significantly increased compared to that observed in normal and benign tissues. The expression of TSP1 and TSP2 in invasive breast ductal carcinoma NOS did not significantly correlate with any of the prognostic factors studied (tumor size, lymph node status, morphology, and hormone receptor status). However, when our study population was divided according to the quantity of tumor stroma, TSP1 (and possibly TSP2) mRNA expression and microvessel counts in desmoplastic-rich stroma of breast carcinoma NOS were significantly increased compared to those observed in desmoplastic-poor stromata.

Expression of thrombospondin (TSP1) and its receptors (CD36 and CD51) in normal, hyperplastic, and neoplastic human breast.

We have previously shown that thrombospondin (TSP) is present in normal breast secretions, and high levels of TSP are observed in malignant breast secretions and cytosols. Three genes encoding for three distinct TSPs (TSP1, TSP2, TSP3) have recently been described. In this study, using both immunohistochemistry and in situ hybridization, we report on the distribution of TSP1 in normal, hyperplastic, and neoplastic human breast. Its immunolocalization was also compared with that of two known cell surface receptors for TSP1: CD36 and CD51. In nonlactating ducts of normal and hyperplastic breast, TSP1 and CD51 are expressed in the basement membrane and in the basal surface of myoepithelial cells, respectively. In lactating adenomas, both TSP1 and CD51 disappear from the myoepithelial-stromal junction of ducts. However, TSP1 becomes selectively expressed at the apices of secretory epithelial cells of lactating ducts together with CD36, suggesting that the distribution of TSP1 and the appearance of its receptors are dependent on the secretory activity of human mammary ducts. In neoplastic human breast, a strong immunostaining for TSP1 is observed in the basement membrane surrounding in situ carcinomas (preinvasive cancer), and excessive TSP1 deposits are also observed in desmoplasia of invasive ductal carcinomas. TSP1 mRNA is localized in myoepithelial cells surrounding in situ carcinomas and in fibroblasts present in desmoplastic areas. On the other hand, few invasive ductal carcinoma cells (10%) express TSP1, while CD51 is moderately expressed by some neoplastic clusters, and no immunoreactivity is observed for CD36. By contrast, TSP1 is codistributed with CD51 in most of the invasive lobular carcinoma cells (40 to 80%) and with CD36 in a subpopulation (30 to 40%) of these invasive tumor cells. As previously observed with lactating adenomas, it is likely that the coexpression of TSP1 and CD36 is related to the secretory activity of invasive lobular carcinoma cells. The different distribution of TSP1 in invasive ductal versus lobular carcinomas may well reflect biological differences between these two main types of breast carcinoma. In this regard, the coexpression of TSP1 and CD36 may, in part at least, account for the variably invasive behavior of lobular carcinoma cells.

Thrombospondin binds to the surface of human osteosarcoma cells and mediates platelet-osteosarcoma cell interaction.

We have previously shown that thrombospondin (TSP) is synthesized and secreted by human MG-63 osteosarcoma cells. In this study, the secretion and cell surface expression of TSP by two different human osteosarcoma cell lines (MG-63 and TE-85) as well as the involvement of TSP in the platelet-aggregating activity of these tumor cells were studied. Using a sandwich enzyme-linked immunosorbent assay, MG-63 cells secreted 3-fold as much TSP as TE-85 cells at 48 h (0.17 +/- 0.01 (SD) versus 0.06 +/- 0.006 micrograms/10(6) cells, P = 0.007). Binding of exogenous 125I-TSP to MG-63 and TE-85 cells in monolayer indicated that binding was time and concentration dependent, saturable, and inhibited by excess cold TSP. However, despite a similar affinity, MG-63 cells had 10-fold more TSP-binding sites than TE-85 cells (402,394 +/- 130,346 versus 36,748 +/- 7,708 TSP-binding sites/cell; P = 0.002). Similar binding differences of 125I-TSP were observed with both osteosarcoma cell lines in suspension. A fluorescence-activated cell-sorting analysis was used in conjunction with an anti-TSP polyclonal antibody, and binding of endogenous TSP to MG-63 and TE-85 cells in suspension was investigated. Addition of an anti-TSP antibody to MG-63 and TE-85 cells in suspension increased the mean fluorescence intensity 50-fold when compared to an irrelevant antibody. Moreover, the fluorescence intensity of MG-63 cells with an anti-TSP polyclonal antibody was increased by 40% when compared to TE-85 cells. Since TSP was expressed on the surface of osteosarcoma cells, the involvement of this glycoprotein in the platelet-aggregating activity of MG-63 and TE-85 cells was therefore investigated using an anti-TSP polyclonal antibody and two monoclonal antibodies (P10 and MA-II), the epitopes of which lie within the Mr 140,000 non-heparin-binding fragment and the Mr 25,000 heparin-binding fragment of TSP, respectively. Preincubation of MG-63 cells (1 x 10(6) cells/ml) with either an anti-TSP polyclonal antibody (100 micrograms/ml) or monoclonal antibody P10 (15 micrograms/ml) inhibited by 80% other platelet-aggregating activity of these tumor cells, while anti-TSP monoclonal antibody MA-II (15 micrograms/ml) had no effect. In sharp contrast, the anti-TSP polyclonal antibody (100 micrograms/ml) only exhibited a slight inhibitory effect on platelet aggregation induced by TE-85 cells when using a low concentration of tumor cells (0.6 x 10(6) cells/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

Bisphosphonates inhibit prostate and breast carcinoma cell adhesion to unmineralized and mineralized bone extracellular matrices.

The molecular mechanisms by which tumor cells induce osteolytic metastases are likely to involve tumor cell adhesion to bone as well as the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are, therefore, used in the treatment of cancer-associated osteolytic metastases. Here, we investigated the effect of BPs on breast and prostate carcinoma cell adhesion to unmineralized and mineralized bone extracellular matrices. BP pretreatment of tumor cells inhibited tumor cell adhesion to unmineralized and mineralized osteoblastic extracellular matrices in a dose-dependent manner. In contrast, BP did not affect adhesion of normal cells (fibroblasts) to extracellular matrices. The order of potency for four BPs in inhibiting tumor cell adhesion to extracellular matrices was found to be: ibandronate > NE-10244 (antiresorptive active pyridinium analogue of risedronate) > pamidronate > clodronate. BP did not affect [3H]thymidine incorporation by tumor cells, as assessed by a mitogenesis assay, indicating that BP did not exert any cytotoxic effect at concentrations used to inhibit tumor cell adhesion. NE-58051, the inactive pyridylpropylidene analogue of risedronate, had no inhibitory effect on tumor cell adhesion compared to that observed with its active counterpart NE-10244, suggesting that the mechanism of action of BP on tumor cells involved a stereospecific recognition step. Although integrins mediate cell-matrix interactions, BP recognition by tumor cells did not modulate cell surface integrin expression. In conclusion, our results provide evidence for a direct cellular effect of BP in preventing tumor cell adhesion to bone, suggesting that BPs may be useful agents for the prophylactic treatment of patients with cancer that is known to preferentially metastasize to bone.

Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases.

The molecular mechanisms by which tumor cells metastasize to bone are likely to involve invasion, cell adhesion to bone, and the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are, therefore, used in the treatment of patients with osteolytic metastases. However, an added beneficial effect of BPs may be direct antitumor activity. We previously reported that BPs inhibit breast and prostate carcinoma cell adhesion to bone (Boissier et al., Cancer Res., 57: 3890-3894, 1997). Here, we provided evidence that BP pretreatment of breast and prostate carcinoma cells inhibited tumor cell invasion in a dose-dependent manner. The order of potency for four BPs in inhibiting tumor cell invasion was: zoledronate > ibandronate > NE-10244 (active pyridinium analogue of risedronate) > clodronate. In addition, NE-58051 (the inactive pyridylpropylidene analogue of risedronate) had no inhibitory effect, whereas NE-10790 (a phosphonocarboxylate analogue of risedronate in which one of the phosphonate groups is substituted by a carboxyl group) inhibited tumor cell invasion to an extent similar to that observed with NE-10244, indicating that the inhibitory activity of BPs on tumor cells involved the R2 chain of the molecule. BPs did not induce apoptosis in tumor cells, nor did they inhibit tumor cell migration at concentrations that did inhibit tumor cell invasion. However, although BPs did not interfere with the production of matrix metalloproteinases (MMPs) by tumor cells, they inhibited their proteolytic activity. The inhibitory effect of BPs on MMP activity was completely reversed in the presence of an excess of zinc. In addition, NE-10790 did not inhibit MMP activity, suggesting that phosphonate groups of BPs are responsible for the chelation of zinc and the subsequent inhibition of MMP activity. In conclusion, our results provide evidence for a direct cellular effect of BPs in preventing tumor cell invasion and an inhibitory effect of BPs on the proteolytic activity of MMPs through zinc chelation. These results suggest, therefore, that BPs may be useful agents for the prophylactic treatment of patients with cancers that are known to preferentially metastasize to bone.

Role of platelet membrane glycoproteins Ib/IX and IIb/IIIa, and of platelet alpha-granule proteins in platelet aggregation induced by human osteosarcoma cells.

We have previously shown that the platelet-aggregating activity of human MG-63 and HOS osteosarcoma cells depends at least in part upon tumor cell surface-associated thrombospondin, and suggested that platelet-osteosarcoma cell interactions could occur through interactions with specific platelet membrane receptors. In this study, the platelet-aggregating activity of MG-63 and HOS cells was studied by using a variety of platelet disorders. Both osteosarcoma cell lines induced a biphasic platelet aggregation response when added to normal platelet-rich plasma, while the second phase of aggregation was absent when added to gray platelets (deficiency in alpha-granule proteins) and to aspirin-treated platelets. Platelets from two unrelated patients with type I Glanzmann's thrombasthenia (deficiency in glycoprotein (GP) GPIIb/IIIa) did not aggregate at all with osteosarcoma cells. Using giant platelets from three patients with Bernard-Soulier syndrome (deficiency in GPIb/IX), the aggregation response induced by MG-63 and HOS cells was monophasic and reversible when compared to normal-sized platelets and to giant platelets from a patient with May-Hegglin anomaly (no membrane GP defect). Because GPIb serves as a receptor for von Willebrand factor during hemostasis, aggregation experiments were also conducted with the platelet-rich plasma of two patients with a low plasma von Willebrand factor concentration (type I von Willebrand's disease) before and after the infusion of deamino-D-arginine vasopressin. MG-63 and HOS cells induced biphasic platelet aggregation both before and after deamino-D-arginine vasopressin treatment, while the ristocetin-dependent binding of von Willebrand factor to platelets only occurred after deamino-D-arginine vasopressin treatment. Preincubation of normal platelet-rich plasma with monoclonal antibody SZ-2 directed against the von Willebrand binding domain of GPIb did not inhibit the platelet-aggregation activity of osteosarcoma cells, whereas anti-GPIb antibody SZ-2 did inhibit ristocetin-induced platelet agglutination. In addition, anti-GPIX antibodies did not affect platelet-osteosarcoma cell interactions. In conclusion, our data demonstrate that the first phase of the platelet-aggregating activity of human osteosarcoma cells is initiated by the interaction of these tumor cells with platelet membrane GPIIb/IIIa, whereas the second phase, even if plasma von Willebrand factor is deficient, involves platelet membrane GPIb and the participation of platelet alpha-granule proteins in membrane-mediated events, making aggregation irreversible.

Tryptic peptide map analysis of the major human blood platelet membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis.

Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point (pI), apparent molecular weight (Mr), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins(GP) Ia, Ib, IIa, IIb, GP4-4.5 132-135, IIIa, IIIb and IIIc were all different. Glycoproteins with the same Mr but different pI were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins.

Upregulation of the mevalonate pathway by cholesterol depletion abolishes tolerance to N-bisphosphonate induced Vγ9Vδ2 T cell cytotoxicity in PC-3 prostate cancer cells.

Zoledronate (ZOL) inhibits farnesyl pyrophosphate synthase leading to intracellular accumulation of isopentenyl pyrophosphate/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (IPP/ApppI). Cytotoxic Vγ9Vδ2 T cells have been shown to recognize IPP/ApppI in breast cancer cells. Further, human breast cancer cells have been shown to differ remarkably in their ZOL treatment induced IPP/ApppI production and responses to that. In this communication we analysed the responsiveness of prostate cancer cells PC-3 and DU-145, Caki-2 renal carcinoma cells and U87MG glioblastoma cells to ZOL treatment, and the subsequent activation of Vγ9Vδ2 T-cell cytotoxicity. Of the cell lines tested, PC-3 cells were not susceptible to Vγ9Vδ2 T-cell cytotoxicity due to low activity of the mevalonate pathway and low amount of IPP formed. However, the resistance of PC-3 cells to Vγ9Vδ2 T-cell cytotoxicity could be abrogated by upregulation of the mevalonate pathway through cholesterol depletion.

Platelet-osteosarcoma cell interaction is mediated through a specific fibrinogen-binding sequence located within the N-terminal domain of thrombospondin 1.

Approximately 20% of patients with osteosarcoma have metastatic disease in lungs or bones at diagnosis. The requirement of platelets in hematogenous dissemination of metastatic cells is now well established. Tumor cells interact with platelets and induce platelet aggregation. In this respect, metastatic potential of tumor cells correlates with their capacity to aggregate platelets in vitro. We have previously shown that thrombospondin 1 (TSP-1) is synthesized and expressed on the surface of MG-63 osteosarcoma cells and mediates platelet-osteosarcoma cell interaction. However, active sites mimicking the function of TSP-1 during platelet-osteosarcoma cell interaction are not known. In this study, a panel of antibodies directed against the N-terminal and C-terminal domains and type 1, type 2, and type 3 repeats of TSP-1 were first used to delineate the structural requirement for the binding of osteosarcoma cell surface-associated TSP-1 to platelets. A drastic inhibition of the platelet-aggregating activity of MG-63 cells was obtained in the presence of a monoclonal antibody directed against the N-terminal domain of TSP-1. Among a series of 16 synthetic peptides spanning the whole N-terminal domain of TSP-1, only synthetic peptide N12/I encompassing amino acid residues 151-164 of the N-terminal domain of TSP-1 inhibited the platelet-aggregating activity of MG-63 cells. Electron microscopy studies showed that peptide N12/I strongly inhibited platelet-osteosarcoma cell interaction. A polyclonal antibody directed against peptide N12/I specifically bound to the surface of MG-63 cells, recognized TSP-1 and drastically inhibited the platelet-aggregating activity of MG-63 cells. In addition, peptide N12/I specifically bound to fibrinogen and inhibited TSP-1/fibrinogen interaction. Overall, our results provide evidence that a fibrinogen-binding sequence located within the N-terminal domain of TSP-1 mediates the binding of osteosarcoma cell surface-associated TSP-1 to platelet-bound fibrinogen.

Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions.

A very common metastatic site for human breast cancer is bone. The traditional bone metastasis model requires human MDA-MB-231 breast carcinoma cell inoculation into the left heart ventricle of nude mice. MDA-MB-231 cells usually develop osteolytic lesions 3-4 weeks after intracardiac inoculation in these animals. Here, we report a new approach to study the formation of bone metastasis in animals using breast carcinoma cells expressing the bioluminescent jellyfish protein (green fluorescent protein [GFP]). We first established a subclone of MDA-MB-231 cells by repeated in vivo passages in bone using the heart injection model. On stable transfection of this subclone with an expression vector for GFP and subsequent inoculation of GFP-expressing tumor cells (B02/GFP.2) in the mouse tail vein, B02/GFP.2 cells displayed a unique predilection for dissemination to bone. Externally fluorescence imaging of live animals allowed the detection of fluorescent bone metastases approximately 1 week before the occurrence of radiologically distinctive osteolytic lesions. The number, size, and intensity of fluorescent bone metastases increased progressively with time and was indicative of breast cancer cell progression within bone. Histological examination of fluorescent long bones from B02/GFP.2-bearing mice revealed the occurrence of profound bone destruction. Treatment of B02/GFP.2-bearing mice with the bisphosphonate zoledronic acid markedly inhibited the progression of established osteolytic lesions and the expansion of breast cancer cells within bone. Overall, this new bone metastasis model of breast cancer combining both fluorescence imaging and radiography should provide an invaluable tool to study the effectiveness of pharmaceutical agents that could suppress cancer colonization in bone.

Osteonectin is an alpha-granule component involved with thrombospondin in platelet aggregation.

We previously showed that thrombospondin, a major alpha-granule glycoprotein of human platelets, forms a specific complex with osteonectin, a phosphoglycoprotein originally described in bone that is also present in human platelets. The storage organelles and the function of osteonectin in platelets are still unknown. In this study, using electron microscopy in combination with immunogold staining, the major storage organelle for platelet-secreted proteins, the alpha-granules. Furthermore, osteonectin was qualitatively and quantitatively assessed by studying normal platelets and the platelets from a patient with gray platelet syndrome. Gray platelet syndrome is a rare congenital bleeding disorder characterized by a selective deficiency in morphologically recognizable platelet alpha-granules and in the alpha-granule secretory proteins. Binding of an iodinated antiosteonectin monoclonal antibody to gray platelet proteins transferred to nitrocellulose from SDS-polyacrylamide gels showed no band corresponding to osteonectin compared to control platelets. Using a polyclonal antiosteonectin antibody-based radioimmunoassay, gray platelets contained 0.2 +/- 0.03 ng osteonectin per 10(6) platelets, which is only 20% of the normal platelet content of osteonectin (0.93 +/- 0.16 ng per 10(6) platelets). Study of the localization of osteonectin to the surface of human platelets demonstrated that a radioiodinated antiosteonectin polyclonal antibody bound specifically to thrombin-stimulated platelets but not to resting platelets. Binding was concentration-dependent, saturable (1710 +/- 453 binding sites per platelet, Kd = 1 microM), and inhibited by an excess of cold antiosteonectin polyclonal antibody. No binding was observed on the surface of thrombin-stimulated gray platelets. To gain further insights into the role of osteonectin released from activated platelets, the effect of an antiosteonectin polyclonal antibody was tested on the aggregation of washed platelets. F(ab')2 fragments from the antiosteonectin polyclonal antibody inhibited in a dose-dependent manner the aggregation of collagen-stimulated, washed human platelets without affecting collagen-induced platelet serotonin release. To characterize the mechanism through which antiosteonectin F(ab')2 fragments inhibit platelet aggregation, the expression of endogenous thrombospondin (TSP) on the surface of thrombin-activated platelets was studied using 125I-labeled anti-TSP monoclonal antibody P10. The endogenous surface expression of TSP to thrombin-stimulated platelets was significantly inhibited in the presence of antiosteonectin F(ab')2 fragments (6286 +/- 2065 molecules of P10 per platelet) compared to 11,230 +/- 766 molecules of P10 per platelet in the presence of nonimmune F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell attachment and fibrinogen binding properties of platelet and endothelial cell thrombospondin are not affected by structural differences in the 70 and 18 kDa protease-resistant domains.

Structural differences between platelet and endothelial cell thrombospondin (TBSP) were found in two protease-resistant domains (70 and 18 kDa). The 70 kDa fragment is involved in the binding of TBSP to fibrinogen and the 18 kDa fragment in the attachment to various cultured cells. Despite these structural differences, platelet and endothelial cell TBSP bound with the same affinity to fibrinogen and mediated the attachment of smooth muscle cells but not of endothelial cells.

Thrombospondin (TSP1) mediates in vitro proliferation of human MG-63 osteoblastic cells induced by alpha-thrombin.

Thrombospondin (TSP) is a 450-kDa glycoprotein synthesized and secreted by human MG-63 osteoblastic cells. In this study, we have first studied the effect of alpha-thrombin on TSP expression by human MG-63 cells. In situ hybridization indicated that TSP mRNA level in thrombin-treated MG-63 cells was increased when compared to unstimulated cells. As judged by immunofluorescence, thrombin-treatment of MG-63 cells resulted in increased cell surface expression of TSP when compared to quiescent cells. Because thrombin stimulates proliferation of osteoblastic cells, the involvement of TSP in proliferation of thrombin-stimulated osteoblastic cells was then investigated using a serum-free mitogenesis assay. Both alpha-thrombin (0.01 to 0.15 U/ml) and TSP (5 to 600 ng/ml) caused a dose-dependent increase in [3H]thymidine incorporation by MG-63 cells. Proliferation of osteoblastic cells induced by alpha-thrombin or TSP was specifically and totally inhibited by anti-TSP monoclonal antibodies (3-10 micrograms/ml) or by indomethacin (1 microM), an inhibitor of prostaglandin synthesis. Anti-TSP antibodies which inhibited cell proliferation also inhibit TSP expression to the surface of these cells. Our experiments support the existence of a mechanism whereby TSP bound to the cell surface of thrombin-treated MG-63 cells stimulates secretion of prostaglandins which, in turn, allow cell proliferation to proceed.

The growth-supportive effect of thrombospondin (TSP1) and the expression of TSP1 by human MG-63 osteoblastic cells are both inhibited by dexamethasone.

Thrombospondin (TSP) is a 450-kDa extracellular matrix glycoprotein which supports the growth of human MG-63 osteoblastic cells [Abbadia et al., FEBS Lett., 329 (1993) 341-346]. In this study, we describe the effect of the glucocorticoid, dexamethasone, on cell proliferation and TSP expression by MG-63 cells. Using a serum-free mitogenesis assay, dexamethasone (25 to 500 nM) caused a dose-dependent decrease in [3H]thymidine incorporation by MG-63 cells in culture, reaching 40% inhibition of cell proliferation at a concentration of 250 nM. Similarly, the stimulatory effect of TSP (500 ng/ml) on proliferation of MG-63 cells was totally abolished in the presence of dexamethasone (250 nM). In situ hybridization indicated that TSP mRNA level in dexamethasone-treated MG-63 cells decreased compared to quiescent cells. As judged by fluorescence-activated cell sorting analysis, dexamethasone treatment of MG-63 cells resulted in a 50 to 70% decrease in TSP cell surface expression compared to quiescent cells. Secretion of TSP in the culture fluid of dexamethasone-treated MG-63 cells also decreased by 40% while, under similar experimental conditions, a 180% increase in alkaline phosphatase activity was observed in dexamethasone-treated cells. Because glucocorticoids induce osteoporosis in vivo and reduce proliferation of osteoblasts in vitro, our results argue for an important role of TSP during bone formation.