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Corning osteo assay surface for the study of bone resorption
Abstracts / Bone 48 (2011) S22–S55
cells results in hybrids with new characteristics, increasing heterogeneity within tumour populations. Methods: Strains of the human prostate cancer cell line PC-3 were generated expressing either GFP or RFP, selectable with different antibiotics. These cells were grown in direct contact to allow them opportunity to fuse. Co-cultures were treated with both antibiotics and cells with dual resistance/fluorescence allowed to form clones. These populations were isolated and their growth in vitro (monolayer, semi-solid agar) and in vivo in bone (intratibial injection) compared to control non-fused strains. Results: The frequency of spontaneous fusions that formed growing clones was low: ~1 in 200,000 interacting cells. The hybrids contained single nuclei, were significantly larger than non-fused strains and contained ~150 identifiable chromosomes. The growth rates of these cells in monolayer were similar to non-fused strains but they were very significantly more clonogenic (5 fold) in semi-solid agar. In vivo, the hybrid cells formed more tumours than non-fused strains and these lesions appeared earlier. Conclusion: This data supports our hypothesis and suggests that a low frequency of spontaneous fusion contributes to the development of phenotypes able to initiate tumours in bone. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.131
P-76 Corning osteo assay surface for the study of bone resorption A.F. Faruqi*, H. Rao, J. Causer, J. Beltzer Corning Life Sciences, Corning Inc, Corning, NY, USA Bone related diseases such as osteoporosis, multiple myeloma, and bone metastasis from prostate and breast cancer tumors represent a significant challenge for both research and drug development. In vitro assays that can measure both the formation and function of osteoclasts are critical for furthering our understanding of the biology of normal bone remodeling. Current systems for assaying osteoclast activity using bone or dentine slices are time consuming and difficult to quantify. An inorganic coating has been developed on standard polystyrene multiwell plates by Corning Life Sciences that can be used for the study of bone cell biology. The Corning Osteo Assay Surface (COAS) has been evaluated for the research of osteoclast differentiation and bone resorption and the co-culture of osteoclasts and osteoblasts. Additionally, the co-culture of RAW 264.7 cells with prostate cancer PC3 cells and breast cancer MBAMB231 cells has been demonstrated on the surface. We have also compared these plates to dentine discs using cultures of primary human osteoclast precursors and found that osteoclast differentiation and activity can be assessed rapidly and accurately on COAS whereas use of dentine discs remains problematic. In summary, both osteoclast differentiation and activity can be readily measured with COAS, which provides consistent and reproducible results in assays using osteoclast precursors and also provides a platform for bone related drug screening. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.132
P-77 A new Β Crosslaps® (CTX-I) assay on the IDS-ISYS automated analyser Z. Seres*, S. Middlemist, C. Dixon, D. Laurie, A.K. Barnes, M. Garrity R & D, IDS Ltd, Boldon, UK Type I collagen accounts for more than 90% of the organic matrix of bone and is synthesized primarily in bone during renewal of the skeleton. Small peptide fragments from Type I collage degradation
S47
are secreted into the bloodstream. The degradation products of Cterminal telopeptides of Type I collagen, cross-linked by pyridinoline (Pyr), are called Crosslaps® or CTX-I. Measurement of CTX-I is useful in the monitoring of bone resorption status and in the response to anti resorption therapies in postmenopausal women and osteopenia patients. CTX-I is also an important diagnostic marker for early detection of bone metastasis in breast and prostate carcinoma patients. CTX-I is measured by a two-site chemiluminescent immunoassay. The acridinium-conjugated tracer and biotinylated capture antibodies form a capture antibody–antigen–signal antibody complex with CTX-I peptide complex. This immuno-complex is then immobilized by addition of streptavidin-coated magnetic particles. Unbound reagent and sample are removed by a washing step. Bound CTX-I is then measured in a highly sensitive luminometer with the measured signal directly proportional to the concentration of CTX-I in the sample. The CTX-I assay has a range of 0.05–6.0 ng/ml. LoB, LoD and LoQ were measured at 0.012, 0.026 and 0.030 ng/ml respectively. Intra-assay CV is 4.8% (0.227 ng/mL), 2.8% (0.890 ng/mL) and 4.1% (2.403 ng/mL), interassay CV is 9.3% (0.212 ng/ml), 4.3% (0.877 ng/ml) and 3.3% (2.290 ng/ mL). Excellent linearity was observed across the full clinical decision range with an observed/expected value of 99.8%. Correlation to the IDS Serum Crosslaps® ELISA (n= 299) was good with a gradient of 0.94, an intercept of −0.02 and an R2 of 0.93. Time to first result is 40 min. Sample volume is 45 μL. This assay has potential to provide accurate data for monitoring of CTX-I. Conflict of interest statement: All authors are employees of IDS. doi:10.1016/j.bone.2010.10.133
P-78 Twist 1 expression in an osteotropic breast cancer cell line promotes bone metastasis formation in mouse M. Croseta,*, D. Goëhriga, E. Bonnelyea, S. Ansieaub, A. Puisieuxb, P. Clézardina a INSERM U664 Faculté Laennec, INSERM, Lyon, France b INSERM U590 Centre Léon Bérard, INSERM, Lyon, France Metastatic breast cancer cells arrest, survive and grow out in the bone microenvironment by acquiring a bone cell pseudo-phenotype named osteomimicry. The transcription factor Twist-1, overexpressed in high-grade breast carcinomas contributes to the invasion and dissemination of tumor cells by promoting an epithelial–mesenchyme transition and/or by escaping the oncogenic-induced failsafe program. To investigate the potential of Twist-1 to interfere with the bone metastasis formation we used a model of MDA-BO2 breast cancer cells previously selected for their osteomimicry, which form osteolytic metastases only to bone when inoculated to the arterial circulation of mice. Twist-1 has been overexpressed in MDA-BO2 using the tet-Offregulated expression system in which the Twist-1 expression is repressed by doxycycline. The intravenous inoculation of MDA-BO2 expressing high (H-BO2), moderate (M-BO2) and nul (Wtype-BO2) levels of Twist-1 led to metastasis formation only in bone without affecting the specific tropism of these cells to bone. However osteolytic lesions increased from 4.13 ± 0.32 mm2 in mice inoculated with WtypeBO2 to 8.21 ± 0.52 and 7.53 ± 0.84 mm2 in mice inoculated with H-B02 and M-BO2, respectively. These lesions decreased to 3.22 ± 0.38 mm2, in mice inoculated with H-B02 and treated with doxycycline. The injection of H-BO2 in the mouse mammary gland induced a rapid tumor onset (5 weeks) wheras the tumor appearance was strongly delayed after MBO2 injection (9 weeks) or by doxycycline treatment. However, Twist-1 did not affect the rate of tumor cell proliferation measured by the mitotic index Ki-67 nor did it reduce tumor angiogenesis in vivo, as assessed by
cells results in hybrids with new characteristics, increasing heterogeneity within tumour populations. Methods: Strains of the human prostate cancer cell line PC-3 were generated expressing either GFP or RFP, selectable with different antibiotics. These cells were grown in direct contact to allow them opportunity to fuse. Co-cultures were treated with both antibiotics and cells with dual resistance/fluorescence allowed to form clones. These populations were isolated and their growth in vitro (monolayer, semi-solid agar) and in vivo in bone (intratibial injection) compared to control non-fused strains. Results: The frequency of spontaneous fusions that formed growing clones was low: ~1 in 200,000 interacting cells. The hybrids contained single nuclei, were significantly larger than non-fused strains and contained ~150 identifiable chromosomes. The growth rates of these cells in monolayer were similar to non-fused strains but they were very significantly more clonogenic (5 fold) in semi-solid agar. In vivo, the hybrid cells formed more tumours than non-fused strains and these lesions appeared earlier. Conclusion: This data supports our hypothesis and suggests that a low frequency of spontaneous fusion contributes to the development of phenotypes able to initiate tumours in bone. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.131
P-76 Corning osteo assay surface for the study of bone resorption A.F. Faruqi*, H. Rao, J. Causer, J. Beltzer Corning Life Sciences, Corning Inc, Corning, NY, USA Bone related diseases such as osteoporosis, multiple myeloma, and bone metastasis from prostate and breast cancer tumors represent a significant challenge for both research and drug development. In vitro assays that can measure both the formation and function of osteoclasts are critical for furthering our understanding of the biology of normal bone remodeling. Current systems for assaying osteoclast activity using bone or dentine slices are time consuming and difficult to quantify. An inorganic coating has been developed on standard polystyrene multiwell plates by Corning Life Sciences that can be used for the study of bone cell biology. The Corning Osteo Assay Surface (COAS) has been evaluated for the research of osteoclast differentiation and bone resorption and the co-culture of osteoclasts and osteoblasts. Additionally, the co-culture of RAW 264.7 cells with prostate cancer PC3 cells and breast cancer MBAMB231 cells has been demonstrated on the surface. We have also compared these plates to dentine discs using cultures of primary human osteoclast precursors and found that osteoclast differentiation and activity can be assessed rapidly and accurately on COAS whereas use of dentine discs remains problematic. In summary, both osteoclast differentiation and activity can be readily measured with COAS, which provides consistent and reproducible results in assays using osteoclast precursors and also provides a platform for bone related drug screening. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.132
P-77 A new Β Crosslaps® (CTX-I) assay on the IDS-ISYS automated analyser Z. Seres*, S. Middlemist, C. Dixon, D. Laurie, A.K. Barnes, M. Garrity R & D, IDS Ltd, Boldon, UK Type I collagen accounts for more than 90% of the organic matrix of bone and is synthesized primarily in bone during renewal of the skeleton. Small peptide fragments from Type I collage degradation
S47
are secreted into the bloodstream. The degradation products of Cterminal telopeptides of Type I collagen, cross-linked by pyridinoline (Pyr), are called Crosslaps® or CTX-I. Measurement of CTX-I is useful in the monitoring of bone resorption status and in the response to anti resorption therapies in postmenopausal women and osteopenia patients. CTX-I is also an important diagnostic marker for early detection of bone metastasis in breast and prostate carcinoma patients. CTX-I is measured by a two-site chemiluminescent immunoassay. The acridinium-conjugated tracer and biotinylated capture antibodies form a capture antibody–antigen–signal antibody complex with CTX-I peptide complex. This immuno-complex is then immobilized by addition of streptavidin-coated magnetic particles. Unbound reagent and sample are removed by a washing step. Bound CTX-I is then measured in a highly sensitive luminometer with the measured signal directly proportional to the concentration of CTX-I in the sample. The CTX-I assay has a range of 0.05–6.0 ng/ml. LoB, LoD and LoQ were measured at 0.012, 0.026 and 0.030 ng/ml respectively. Intra-assay CV is 4.8% (0.227 ng/mL), 2.8% (0.890 ng/mL) and 4.1% (2.403 ng/mL), interassay CV is 9.3% (0.212 ng/ml), 4.3% (0.877 ng/ml) and 3.3% (2.290 ng/ mL). Excellent linearity was observed across the full clinical decision range with an observed/expected value of 99.8%. Correlation to the IDS Serum Crosslaps® ELISA (n= 299) was good with a gradient of 0.94, an intercept of −0.02 and an R2 of 0.93. Time to first result is 40 min. Sample volume is 45 μL. This assay has potential to provide accurate data for monitoring of CTX-I. Conflict of interest statement: All authors are employees of IDS. doi:10.1016/j.bone.2010.10.133
P-78 Twist 1 expression in an osteotropic breast cancer cell line promotes bone metastasis formation in mouse M. Croseta,*, D. Goëhriga, E. Bonnelyea, S. Ansieaub, A. Puisieuxb, P. Clézardina a INSERM U664 Faculté Laennec, INSERM, Lyon, France b INSERM U590 Centre Léon Bérard, INSERM, Lyon, France Metastatic breast cancer cells arrest, survive and grow out in the bone microenvironment by acquiring a bone cell pseudo-phenotype named osteomimicry. The transcription factor Twist-1, overexpressed in high-grade breast carcinomas contributes to the invasion and dissemination of tumor cells by promoting an epithelial–mesenchyme transition and/or by escaping the oncogenic-induced failsafe program. To investigate the potential of Twist-1 to interfere with the bone metastasis formation we used a model of MDA-BO2 breast cancer cells previously selected for their osteomimicry, which form osteolytic metastases only to bone when inoculated to the arterial circulation of mice. Twist-1 has been overexpressed in MDA-BO2 using the tet-Offregulated expression system in which the Twist-1 expression is repressed by doxycycline. The intravenous inoculation of MDA-BO2 expressing high (H-BO2), moderate (M-BO2) and nul (Wtype-BO2) levels of Twist-1 led to metastasis formation only in bone without affecting the specific tropism of these cells to bone. However osteolytic lesions increased from 4.13 ± 0.32 mm2 in mice inoculated with WtypeBO2 to 8.21 ± 0.52 and 7.53 ± 0.84 mm2 in mice inoculated with H-B02 and M-BO2, respectively. These lesions decreased to 3.22 ± 0.38 mm2, in mice inoculated with H-B02 and treated with doxycycline. The injection of H-BO2 in the mouse mammary gland induced a rapid tumor onset (5 weeks) wheras the tumor appearance was strongly delayed after MBO2 injection (9 weeks) or by doxycycline treatment. However, Twist-1 did not affect the rate of tumor cell proliferation measured by the mitotic index Ki-67 nor did it reduce tumor angiogenesis in vivo, as assessed by