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 they have a greater abundance of mononuclear cells. Perform all the following steps in a sterile tissue culture hood.
<ol>
	<li>Dilute EDTA whole blood 1:1 with 1x phosphate-buffered saline (PBS). Re-suspend well.</li>
	<li>Layer on lymphocyte separation media (blood PBS: lymphocyte separation media, 2:1) in 50 mL tubes.
	<ol>
		<li>Pipette 30 mL of blood diluted in PBS into a 50 mL tube.</li>
		<li>Gently underlay 15 mL of lymphocyte separation media into the bottom of the 50-mL tube.</li>
	</ol>
	</li>
	<li>Centrifuge at 757 &#215; g for 10 min at room temperature (no brakes).</li>
	<li>Collecting the mononuclear cells:
	<ol>
		<li>Collect the white layer of mononuclear cells with a 5 mL pipette without agitating and place it in a new 50 mL tube.</li>
		<li>Dilute the collected PBMCs with 20 mL of PBS.</li>
	</ol>
	</li>
	<li>Washing the PBMCs:
	<ol>
		<li>Centrifuge at 225 x g for 5 min at room temperature.</li>
		<li>Discard the supernatant by inverting the tube. Repeat once.</li>
	</ol>
	</li>
	<li>RBC lysis: 
	NOTE: If the cell pellet is red, treat cells with Red Blood cell lysing buffer solution (see Table of Materials).
	<ol>
		<li>Put the 50 mL tube on ice.</li>
		<li>Add 5 mL of Red Blood cell lysing buffer.</li>
		<li>Wait 3 - 5 min to complete the erythrocyte lysis. The time varies according to the degree of erythrocyte contamination.</li>
		<li>Stop the reaction by adding 20 mL of PBS.</li>
		<li>Centrifuge at 225 x g for 5 min at room temperature. Discard the supernatant by inverting the tube. 
		NOTE: Optional: If the RBC lysis is performed, wash the PBMCs as described below.</li>
	</ol>
	</li>
	<li>Wash PBMCs:
	<ol>
		<li>Add 20 mL of PBS and centrifuge at 225 x g for 5 min at room temperature.</li>
		<li>Discard the supernatant by inverting the tube.</li>
	</ol>
	</li>
	<li>Re-suspend the cells in complete &#945;-MEM (supplemented with 1% glutamine (initial concentration 100X) and 10% fetal bovine serum).</li>
	<li>Count the cells with a Neubauer chamber.
	<ol>
		<li>Dilute the sample 1:100 in glacial acetic acid and use 10 &#181;L of the dilution to count cells.</li>
		<li>Count at least twice and calculate the mean of replicates to get the total mean number of cells collected.</li>
	</ol>
	</li>
	<li>Plate cells:
	<ol>
		<li>Plate cells at a concentration of 750,000 PBMCs per cm2.</li>
		<li>Store plated cells in an incubator at 37 &#176;C in a 5% CO2 atmosphere. 
		NOTE: Design the experiment to include negative controls, which are cells that are treated with normal complete &#945;-MEM without MCSF or RANKL GFs. Remember to perform each condition in at least 3 technical replicates. Perform at least 3 biological replicates.</li>
	</ol>
	</li>
	<li>Change the medium supplemented with GFs:
	<ol>
		<li>Wait about 3 h after the cell seeding and then remove the medium by pipette (200 - 1,000 &#181;L) to discard debris, unattached cells, and erythrocytes. 
		NOTE: Be careful when changing the medium to avoid cell detachment.</li>
		<li>Add new complete &#945;-MEM supplemented with 20 ng/mL of MCSF (500 &#181;L/well). 
		NOTE: When co-culturing with cancer cells, seed the cancer cells the day after the PBMC seeding.</li>
	</ol>
	</li>
	<li>Change the media every 2 - 3 days (&#945;-MEM supplemented with 20 ng/mL of MCSF):
	<ol>
		<li>Discard the medium by a 200 - 1,000 &#181;L pipette.</li>
		<li>Add new fresh medium by a 200 - 1,000 &#181;L pipette.</li>
	</ol>
	</li>
	<li>6-7 days after cell seeding, add 20 ng/mL of RANKL to complete &#945;-MEM+MCSF.</li>
	<li>Change the medium (complete &#945;-MEM+MCSF+RANKL) every 2-3 days:
	<ol>
		<li>Discard the medium with a 200 - 1,000 &#181;L pipette.</li>
		<li>Add new medium with a 200 - 1,000 &#181;L pipette. Stop induction of differentiation 14 days after cell seeding.</li>
		<li>Discard medium and wash twice with PBS.</li>
	</ol>
	</li>
	<li>Fix cells with paraformaldehyde:
	<ol>
		<li>Leave cells to dry.</li>
		<li>Add 4% paraformaldehyde (see Table of Materials).</li>
		<li>Incubate for 20 min at room temperature.</li>
	</ol>
	</li>
	<li>Discard paraformaldehyde and wash twice with 500 &#181;L of PBS. 
	CAUTION: Paraformaldehyde is toxic; please wear gloves, mask and eyeglasses.</li>
	<li>Allow cells to dry.</li>
	<li>Perform TRAP staining according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Count osteoclast-like cells manually under the microscope at magnification 10X; NA 0.30. 
	NOTE: Osteoclast-like cells are defined as TRAP+ polycarion cells with at least 4 nuclei.</li>
</ol>
2. Cancer Cell Cultures
NOTE: The experiments can be performed with different types of breast adenocarcinoma cell lines such as SCP217, an osteotropic cell line originating from the triple negative human breast cancer cell line (MDA-MB-231), and the hormonal receptor-positive cell line MCF7.
<ol>
	<li>Culture the cells as a monolayer in 75-cm2 flasks.
	<ol>
		<li>Seed 1,000,000 cancer cells in a 75-cm2 flask.</li>
		<li>Store the cells at 37 &#176;C in complete &#945;-MEM medium supplemented with 1% glutamine and 10% fetal bovine serum in a 5% CO2 atmosphere.</li>
	</ol>
	</li>
	<li>Detach the cells at a confluency of about 90%:
	<ol>
		<li>Discard the medium, wash with PBS, and add 2 - 3 mL of trypsin 1X in PBS.</li>
		<li>Incubate cells for 3-5 min at 37 &#176;C.</li>
		<li>Collect the detached cells from the 75-cm2 flask by a 10 mL pipette, adding 8 mL of complete medium to stop the enzymatic reaction.</li>
		<li>Centrifuge at 225 x g for 5 min.</li>
	</ol>
	</li>
	<li>Wash the cancer cells:
	<ol>
		<li>Add 10 mL of PBS.</li>
		<li>Centrifuge at 600 x g for 5 min.</li>
	</ol>
	</li>
	<li>Count cancer cells with a Neubauer chamber.
	<ol>
		<li>Count cancer cells at least twice and calculate the mean of replicates to get the total mean number of cells collected.</li>
	</ol>
	</li>
</ol>
3. Direct Co-Cultures of Cancer Cells a nd Monocytes Undergoing Differentiation towards Osteoclasts
<ol>
	<li>Seed the cancer cells on inserts (pore diameter 0.4 &#181;m):
	<ol>
		<li>Seed the cancer cells at a concentration of 4,000 cells per cm2.</li>
		<li>Incubate at 37 &#176;C and 5% CO2. 
		NOTE: Seed the cancer cells the day after PBMC seeding for the co-culture experiment in 0.4 &#181;m inserts.</li>
	</ol>
	</li>
	<li>Wait for 24 h to allow the cells to adhere.</li>
	<li>Place the seeded insert culture over the mononuclear cultures to start the co-culture (see section 1.10 of the protocol).</li>
	<li>Change the media every 2 - 3 days with complete &#945;-MEM.
	<ol>
		<li>Discard the medium with a 200 - 1,000 &#181;L pipette.</li>
		<li>Add 500 &#181;L of fresh medium. 
		NOTE: The primary aim of the co-culture is to understand the osteoclastogenic power of cancer cells, therefore, the GFs are not added to &#945;-MEM. In each experiment of co-culture, include negative controls (see note in step 1.10 of the protocol) and positive controls of osteoclasts differentiated by RANKL and MCSF (no cancer cells). Cancer cells seeded in the inserts not placed over mononuclear cells are used as a negative control of the co-culture.</li>
	</ol>
	</li>
	<li>Stop the co-culture and use cancer cells for downstream analysis:
	<ol>
		<li>13 days after PBMC seeding and 12 days after cancer cell seeding, place the insert on a new plate.</li>
		<li>Stop cancer cell culture on the basis of which downstream analyses are to be performed (see Discussion).</li>
	</ol>
	</li>
	<li>On day 14, stop osteoclast differentiation and fix cells, as reported in step 1.15.</li>
	<li>Perform TRAP staining immediately or within 14 - 30 days. 
	NOTE: Optional staining: To confirm that the TRAP+ polycarion cells are osteoclasts, researchers can perform additional staining to detect CTR, an osteoclast-specific marker. The detection of F-actin rings is another osteoclast-specific staining assay12,13,14.</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+course+of+bone+metastases+from+breast+cancer.">The clinical course of bone metastases from breast cancer.</a> Br J Cancer. 55 (1), 61-66 (1987).</li><li>Ibrahim, T., Mercatali, L., Amadori, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+emergency+in+oncology:+bone+metastases+in+breast+cancer+patients.">A new emergency in oncology: bone metastases in breast cancer patients.</a> Oncol Lett. 6 (2), 306-310 (2013).</li><li>Swartz, M. A., 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+microenvironment+complexity:+emerging+roles+in+cancer+therapy.">Tumor microenvironment complexity: emerging roles in cancer therapy.</a> Cancer Res. 72 (10), 2473-2480 (2012).</li><li>Roodman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+bone+metastasis.">Mechanisms of bone metastasis.</a> N Eng J Med. 350 (12), 1655-1664 (2004).</li><li>Patel, L. R., Camacho, D. F., Shiozawa, Y., Pienta, K. J., Taichman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cancer+cell+metastasis+to+the+bone:+a+multistep+process.">Mechanisms of cancer cell metastasis to the bone: a multistep process.</a> Future Oncol. 7 (11), 1285-1297 (2011).</li><li>Chen, Y. C., Sosnoski, D. M., Mastro, A. M. <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+metastasis+to+the+bone:+mechanisms+of+bone+loss.">Breast cancer metastasis to the bone: mechanisms of bone loss.</a> Breast Cancer Res. 12 (6), 215 (2010).</li><li>Guise, T. 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>

The authors have no conflicts of interest to disclose.

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>&amp;alpha;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&lt;br/&gt; ROCHE</td><td></td></tr><tr><td>Trypsin</td><td>EuroClone</td><td>COD.&lt;br/&gt; 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;&lt;br/&gt; 6.5 mm, 0.4 &amp;mu;M;&lt;br/&gt; 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...

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

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.

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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7.1K Views.  IRCCS. Dit protocol beschrijft de ontwikkeling van een in vitro menselijke preklinische model van osteoclastogenesis uit perifeer bloed monocyten gekweekt met borst kanker cellijnen na te bootsen de kanker cel-osteoclast interactie. Het model kan worden gebruikt om verdere van ons begrip van metastase biomineralisatie en therapeutische opties te verbeteren.

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

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Developmental Biology

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Medicine

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Immunology and Infection

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Biology

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

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

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