Name: development of a human preclinical model of osteoclastogenesis from peripheral blood monocytes co-cultured with breast cancer cell lines
Uploaded: 2017-09-13T15:00:00
Description: Watch this Scientific Journal Video about Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines Video at JoVE.com

We would like to thank Yibin Kang for providing the SCP2 cell line and Cristiano Verna for editorial assistance.

Human osteoclasts were differentiated from PBMCs of healthy donors who gave written informed consent to take part in the study. The study protocol was approved by the local Ethics Committee, in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
1. Osteoclast Differentiation
NOTE: Collect peripheral blood or buffy coats from healthy human donors in EDTA. Do not use less than 20 mL of peripheral blood. Buffy coats are preferable because...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+bone+metastases:+it's+all+about+the+neighborhood.">Breast cancer bone metastases: it's all about the neighborhood.</a> Cell. 154 (5), 957-959 (2013).</li><li>Ell, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-induced+osteoclast+miRNA+changes+as+regulators+and+biomarkers+of+osteolytic+bonemetastasis.">Tumor-induced osteoclast miRNA changes as regulators and biomarkers of osteolytic bonemetastasis.</a> Cancer Cell. 24 (4), 542-556 (2013).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VCAM-1+promotes+osteolytic+expansion+of+indolent+bone+micrometastasis+of+breast+cancer+by+engaging+&#945;4&#946;1-positive+osteoclast+progenitors.">VCAM-1 promotes osteolytic expansion of indolent bone micrometastasis of breast cancer by engaging &#945;4&#946;1-positive osteoclast progenitors.</a> Cancer Cell. 20 (6), 701-714 (2011).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteogenic+niche+promotes+early-stage+bone+colonization+of+disseminated+breast+cancer+cells.">The osteogenic niche promotes early-stage bone colonization of disseminated breast cancer cells.</a> Cancer Cell. 27 (2), 193-210 (2015).</li><li>Liverani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF-1+blockade+impairs+breast+cancer+osteoclastogenic+potential+in+co-culture+systems.">CSF-1 blockade impairs breast cancer osteoclastogenic potential in co-culture systems.</a> Bone. 66, 214-222 (2014).</li><li>Mercatali, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+everolimus+in+an+in+vitro+model+of+triple+negative+breast+cancer+and+osteoclasts.">The effect of everolimus in an in vitro model of triple negative breast cancer and osteoclasts.</a> Int J Mol Sci. 1 (11), e1827 (2016).</li><li>Glantschnig, H., Fisher, J. E., Wesolowski, G., Rodan, G. A., Reszka, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-CSF,+TNFalpha+and+RANK+ligand+promote+osteoclast+survival+by+signaling+through+mTOR/S6+kinase.">M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase.</a> Cell DeathDiffer. 10 (10), 1165-1177 (2003).</li><li>Sugatani, T., Hruska, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt1/Akt2+and+mammalian+target+of+rapamycin/Bim+play+critical+roles+in+osteoclast+differentiation+and+survival,+respectively,+whereas+Akt+is+dispensable+for+cell+survival+in+isolated+osteoclast+precursors.">Akt1/Akt2 and mammalian target of rapamycin/Bim play critical roles in osteoclast differentiation and survival, respectively, whereas Akt is dispensable for cell survival in isolated osteoclast precursors.</a> J Biol Chem. 280 (5), 3583-3589 (2005).</li><li>Bertoldo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+bone+metastatic+cancer:+role+of+the+mTOR+pathway.">Targeting bone metastatic cancer: role of the mTOR pathway.</a> Biochim Biophys Acta. 1845 (2), 248-254 (2014).</li><li>Kang, Y., Siegel, P. M., Shu, W., Drobnjak, M., Kakonen, S. M., Cord&#243;n-Cardo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multigenic+program+mediating+breast+cancer+metastasis+to+bone.">A multigenic program mediating breast cancer metastasis to bone.</a> Cancer Cell. 3 (6), 537-549 (2003).</li><li>Simone, V., Ciavarella, S., Brunetti, O., Savonarola, A., Cives, M., Tucci, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everolimus+restrains+the+paracrine+pro-osteoclast+activity+of+breast+cancer+cells.">Everolimus restrains the paracrine pro-osteoclast activity of breast cancer cells.</a> BMC Cancer. 14 (15), 692 (2015).</li></ol>

Preclinical in vitro models studying the mechanisms of crosstalk between cancer cells and bone cells are needed to identify mechanisms of bone metastasis that can be used to create new therapeutic strategies. We developed a fully human in vitro model of osteoclastogenesis from human peripheral blood (Figure 3). During optimization of the methodology, a number of critical points were identified and resolved. The first concerned the quantity of mononuclear cells to seed. The ...

Bone is a common site of metastasis for different types of primary tumors such as prostate, lung and breast cancer, with 20 - 25% of patients developing bone metastases during the course of disease1,2,3. In particular, 70% of breast cancer patients carry evidence of bone metastasis at death4. Tumor and stromal cell interaction is essential for cancer progression in both primary cancer and secondary lesions. In the bone microenvironment, osteolytic bone metastases from breast cancer depend on the establishment of a pathological vicious cycle occurring between cancer cells, bone cells, and the bone microenvironment. Cancer cells disrupt bone balance, increasing bone resorption5,6,7.
In normal and pathological conditions, osteoclasts are the cells responsible for bone resorption, whereas osteoblasts, in depositing new matrix, are responsible for new bone formation8. Osteoclast activity is regulated by osteoblasts through the expression of RANKL, which binds to its receptor RANK on the pre-osteoclast surface, inducing pre-osteoclast fusion, a necessary process for differentiation into mature osteoclasts. The induction of osteoclastogenesis increases bone resorption. A large number of in vivo studies have significantly improved our understanding of bone metastasis formation9,10,11. Breast cancer cells from the primary tumor and in the bone microenvironment perturb bone homeostasis, promoting osteoclastogenesis and bone resorption8. In this scenario, all the molecular interactions that occur between cancer cells and osteoclasts are of crucial importance. As already mentioned, the mechanism of bone metastasis formation has been elucidated in in vivo mice models. However, in addition to the need for the approval of all in vivo animal experiments by the Ethics Committee, there are several other drawbacks to performing in vivo experiments including high costs and time-consuming methods. Several authors have combined preclinical in vivo and in vitro models of osteoclastogenesis using a murine line of pre-osteoclasts called RAW246.79,10,11. The drawbacks of this model stem from the fact that the cells are already committed to becoming pre-osteoclasts and are not of human origin. For these reasons, translational research could greatly benefit from the availability of in vitro fully human preclinical models to study bone cancer cell interactions.
We optimized a method of osteoclastogenesis in vitro starting from human peripheral blood samples12,13. Osteoclasts derive from monocytes, which are present, albeit to a small degree, in peripheral blood samples. Mononuclear cells are first separated from the erythrocytes and granulocytes present in whole blood by Ficoll density gradient; they are then selected thanks to their ability to adhere to plastic substrate, unlike lymphocytes. After seeding, cells are cultured for 14 days. MCSF and RANKL are the GFs required by monocytes to differentiate first into macrophages and then into osteoclasts14,15. MCSF is needed for the entire duration of the assay, whereas RANKL is used to induce the differentiation process in the late stages of osteoclastogenesis. In the early phase of differentiation, MCSF helps monocytes proliferate and survive14,15. During the second part of osteoclastogenesis, cells fuse together and mature as osteoclasts, showing the characteristic distribution of Actin F in rings and expressing specific markers such as tartrate-resistant acid phosphatase (TRAP) and calcitonin receptor (CTR)14,15. Our method consists of adding MCSF to the monocyte culture for the first 7 days of the experiment and a combination of MCSF and RANKL from days 7 to 14. At the end of the experiment, osteoclastogenesis is analyzed by counting the differentiated cells, as detailed below.
The monocyte cultures induced to differentiate by GFs form the basis of our preclinical model. We optimized a co-culture system without GFs to better understand the osteoclastogenic power of breast cancer cells. We first developed a model of indirect co-cultures by adding a medium (80% &#945; -Minimal Essential Medium (&#945;-MEM) and 20% conditioned medium collected from a culture of breast cancer cells that were about 90% confluent to cells undergoing differentiation12. The conditioned medium (not collected under serum deprivation conditions) was collected after 24 hours and mixed with fresh medium at a proportion of 1:4. The conditioned medium induced significant osteoclast differentiation with respect to the negative control. However, as the information on the reciprocal interaction between cancer cells and bone cells is lost when using indirect co-cultures, we improved our system by carrying out direct co-cultures. We seeded cancer cells in 0.4 &#181;M inserts and placed them in wells where mononuclear cells were plated. Using this method, cells share the same medium and exchange secreted proteins. We thus created a fully human preclinical model of osteoclastogenesis induced by cancer cells13.
This system is extremely versatile and can be used for different research purposes, e.g., in pharmacological studies investigating the role of drugs in bone metastasis. Our model makes it possible to study the efficacy and mechanisms of action of bone-targeted therapies and/or antitumor drugs in the bone microenvironment in the presence of cancer cells13. Designing the experiments with the correct controls, i.e., cancer cells and osteoclasts cultured individually, makes it easier to understand the impact of the co-culture on drug activity. This approach becomes even more interesting when the drug being studied targets both cancer cells and osteoclasts, e.g., everolimus16. This model can also be used to identify new pathways of interaction between cancer cells and bone cells.

<table>
	<tbody>
		<tr>
			<td>&#945;MEM</td>
			<td>Euroclone</td>
			<td>BE12-169F</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Life-technologies</td>
			<td>ECB3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life-technologies</td>
			<td>ECS0180DPR</td>
			<td></td>
		</tr>
		<tr>
			<td>hMCSF</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td>Storage indications must be respected</td>
		</tr>
		<tr>
			<td>hRANKL</td>
			<td>Peprotech</td>
			<td>310-01</td>
			<td>Storage indications must be respected</td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma Aldrich</td>
			<td>387 A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphocyte separation media</td>
			<td>Biowest</td>
			<td>L0560-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood cell lysing buffer</td>
			<td>SIgma</td>
			<td>11814389001 
			ROCHE</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>EuroClone</td>
			<td>COD. ECB3052D</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4% aqueous solution, EM grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231 cell line</td>
			<td>ATCC</td>
			<td>CRM-HTB-267</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF7</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Corning</td>
			<td>3470-Clear</td>
			<td>These inserts are for 24-well plates; 
			6.5 mm, 0.4 &#956;M; 
			pore size</td>
		</tr>
	</tbody>
</table>

development of a human preclinical model of osteoclastogenesis from peripheral blood monocytes co-cultured with breast cancer cell lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

A method was optimized to easily differentiate osteoclasts from human peripheral blood monocytes. The monocyte culture was cultured with cancer cells, confirming (as described in the literature18) that cancer cells are capable of sustaining osteoclastogenesis in bone metastases. Osteoclasts differentiated from cancer cells and GFs are shown in Figure 1. An osteoclast-like cell is a cell with 4 or more nuclei and is positive for TRAP st...

Watch this Scientific Journal Video about Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines Video at JoVE.com

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines Video (Video) | JoVE

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

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