We thank Dov Zipori (The Weizmann Institute of Science, Rehovot, Israel) kindly for providing us the murine bone marrow stromal cell line 14F1.1. This study was supported by grants from the Chinese National Natural Science Foundation (Nos. 81771729) and the Yongchuan Hospital of Chongqing Medical University (Nos. YJQN201330; YJZQN201527).

NOTE: All cells used in this protocol should be grown for at least three generations after thawing from frozen stock cells.
1. Harvest Melanoma Cell-derived Factors
<ol>
	<li>Preparations
	<ol>
		<li>Obtain B16F10 cells and a mouse melanoma cell line. 
		NOTE: For this protocol, a mouse melanoma cell line was obtained from the Stem Cell Bank of the Chinese Academy of Sciences.</li>
		<li>Make a complete medium for the B16F10 cell culture (100 mL). Use Dulbecco&#39;s Mod...

<ol>
	<li>Wang, J., 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=Adipogenic+niches+for+melanoma+cell+colonization+and+growth+in+bone+marrow.">Adipogenic niches for melanoma cell colonization and growth in bone marrow.</a> Laboratory Investigation. 97 (6), 737-745 (2017).</li><li>Trotter, T. N., 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=Adipocyte-Lineage+Cells+Support+Growth+and+Dissemination+of+Multiple+Myeloma+in+Bone.">Adipocyte-Lineage Cells Support Growth and Dissemination of Multiple Myeloma in Bone.</a> The American Journal of Pathology. 186 (11), 3054-3063 (2016).</li><li>Morris, E. V., Edwards, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+bone+marrow+adipocytes+in+bone+metastasis.">The role of bone marrow adipocytes in bone metastasis.</a> Journal of Bone Oncology. 5 (3), 121-123 (2016).</li><li>Diedrich, J. D., 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=Bone+marrow+adipocytes+promote+the+Warburg+phenotype+in+metastatic+prostate+tumors+via+HIF-1alpha+activation.">Bone marrow adipocytes promote the Warburg phenotype in metastatic prostate tumors via HIF-1alpha activation.</a> Oncotarget. 7 (40), 64854-64877 (2016).</li><li>Chkourko Gusky, H., Diedrich, J., MacDougald, O. A., Podgorski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omentum+and+bone+marrow:+how+adipocyte-rich+organs+create+tumour+microenvironments+conducive+for+metastatic+progression.">Omentum and bone marrow: how adipocyte-rich organs create tumour microenvironments conducive for metastatic progression.</a> Obesity Reviews. 17 (11), 1015-1029 (2016).</li><li>Chen, G. 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=High+fat+diet+increases+melanoma+cell+growth+in+the+bone+marrow+by+inducing+osteopontin+and+interleukin+6.">High fat diet increases melanoma cell growth in the bone marrow by inducing osteopontin and interleukin 6.</a> Oncotarget. 7 (18), 26653-26669 (2016).</li><li>Balaban, S., 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=Adipocyte+lipolysis+links+obesity+to+breast+cancer+growth:+adipocyte-derived+fatty+acids+drive+breast+cancer+cell+proliferation+and+migration.">Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive breast cancer cell proliferation and migration.</a> Cancer &amp; Metabolism. 5, 1 (2017).</li><li>Huang, C. K., 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=Adipocytes+promote+malignant+growth+of+breast+tumours+with+monocarboxylate+transporter+2+expression+via+beta-hydroxybutyrate.">Adipocytes promote malignant growth of breast tumours with monocarboxylate transporter 2 expression via beta-hydroxybutyrate.</a> Nature Communications. 8, 14706 (2017).</li><li>Wang, Y. Y., 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=Mammary+adipocytes+stimulate+breast+cancer+invasion+through+metabolic+remodeling+of+tumor+cells.">Mammary adipocytes stimulate breast cancer invasion through metabolic remodeling of tumor cells.</a> JCI Insight. 2 (4), 87489 (2017).</li><li>Wang, C., Gao, C., Meng, K., Qiao, H., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipocytes+stimulate+invasion+of+breast+cancer+MCF-7+cells+by+secreting+IGFBP-2.">Human adipocytes stimulate invasion of breast cancer MCF-7 cells by secreting IGFBP-2.</a> PLoS One. 10 (3), 0119348 (2015).</li><li>Nieman, K. M., 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=Adipocytes+promote+ovarian+cancer+metastasis+and+provide+energy+for+rapid+tumor+growth.">Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.</a> Nature Medicine. 17 (11), 1498-1503 (2011).</li><li>Herroon, M. K., 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=Bone+marrow+adipocytes+promote+tumor+growth+in+bone+via+FABP4-dependent+mechanisms.">Bone marrow adipocytes promote tumor growth in bone via FABP4-dependent mechanisms.</a> Oncotarget. 4 (11), 2108-2123 (2013).</li><li>Tabe, Y., 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=Bone+Marrow+Adipocytes+Facilitate+Fatty+Acid+Oxidation+Activating+AMPK+and+a+Transcriptional+Network+Supporting+Survival+of+Acute+Monocytic+Leukemia+Cells.">Bone Marrow Adipocytes Facilitate Fatty Acid Oxidation Activating AMPK and a Transcriptional Network Supporting Survival of Acute Monocytic Leukemia Cells.</a> Cancer Research. 77 (6), 1453-1464 (2017).</li><li>Wen, Y. 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=Adipocytes+activate+mitochondrial+fatty+acid+oxidation+and+autophagy+to+promote+tumor+growth+in+colon+cancer.">Adipocytes activate mitochondrial fatty acid oxidation and autophagy to promote tumor growth in colon cancer.</a> Cell Death &amp; Differentiation. 8 (2), 2593 (2017).</li><li>Liu, Z., 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=Mature+adipocytes+in+bone+marrow+protect+myeloma+cells+against+chemotherapy+through+autophagy+activation.">Mature adipocytes in bone marrow protect myeloma cells against chemotherapy through autophagy activation.</a> Oncotarget. 6 (33), 34329-34341 (2015).</li><li>Ye, 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=Leukemic+Stem+Cells+Evade+Chemotherapy+by+Metabolic+Adaptation+to+an+Adipose+Tissue+Niche.">Leukemic Stem Cells Evade Chemotherapy by Metabolic Adaptation to an Adipose Tissue Niche.</a> Cell Stem Cell. 19 (1), 23-37 (2016).</li><li>Templeton, Z. S., 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=Breast+Cancer+Cell+Colonization+of+the+Human+Bone+Marrow+Adipose+Tissue+Niche.">Breast Cancer Cell Colonization of the Human Bone Marrow Adipose Tissue Niche.</a> Neoplasia. 17 (12), 849-861 (2015).</li><li>Daquinag, A. C., Souza, G. R., Kolonin, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+engineering+in+three-dimensional+levitation+tissue+culture+system+based+on+magnetic+nanoparticles.">Adipose tissue engineering in three-dimensional levitation tissue culture system based on magnetic nanoparticles.</a> Tissue Engineering Part C: Methods. 19 (5), 336-344 (2013).</li><li>Emont, M. P., 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=Using+a+3D+Culture+System+to+Differentiate+Visceral+Adipocytes+In+Vitro.">Using a 3D Culture System to Differentiate Visceral Adipocytes In Vitro.</a> Endocrinology. 156 (12), 4761-4768 (2015).</li><li>Katt, M. E., Placone, A. L., Wong, A. D., Xu, Z. S., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Tumor+Models:+Advantages,+Disadvantages,+Variables,+and+Selecting+the+Right+Platform.">In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform.</a> Frontiers in Bioengineering and Biotechnology. 4, 12 (2016).</li><li>Edmondson, R., Broglie, J. J., Adcock, A. F., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+systems+and+their+applications+in+drug+discovery+and+cell-based+biosensors.">Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors.</a> ASSAY and Drug Development Technologies. 12 (4), 207-218 (2014).</li><li>Herroon, M. K., Diedrich, J. D., Podgorski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+3D-Culture+Approaches+to+Study+Interactions+of+Bone+Marrow+Adipocytes+with+Metastatic+Prostate+Cancer+Cells.">New 3D-Culture Approaches to Study Interactions of Bone Marrow Adipocytes with Metastatic Prostate Cancer Cells.</a> Frontiers in Endocrinology (Lausanne). 7, 84 (2016).</li><li>Lee, J. M., 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+three-dimensional+microenvironment+alters+protein+expression+and+chemosensitivity+of+epithelial+ovarian+cancer+cells+in+vitro.">A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro.</a> Laboratory Investigation. 93 (5), 528-542 (2013).</li><li>Imamura, Y., 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=Comparison+of+2D-+and+3D-culture+models+as+drug-testing+platforms+in+breast+cancer.">Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer.</a> Oncology Reports. 33 (4), 1837-1843 (2015).</li><li>Birgersdotter, A., Sandberg, R., Ernberg, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+perturbation+in+vitro--a+growing+case+for+three-dimensional+(3D)+culture+systems.">Gene expression perturbation in vitro--a growing case for three-dimensional (3D) culture systems.</a> Seminars in Cancer Biology. 15 (5), 405-412 (2005).</li><li>Wang, W., 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=3D+spheroid+culture+system+on+micropatterned+substrates+for+improved+differentiation+efficiency+of+multipotent+mesenchymal+stem+cells.">3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells.</a> Biomaterials. 30 (14), 2705-2715 (2009).</li><li>Dirat, 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=Cancer-associated+adipocytes+exhibit+an+activated+phenotype+and+contribute+to+breast+cancer+invasion.">Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion.</a> Cancer Research. 71 (7), 2455-2465 (2011).</li><li>Scott, M. A., Nguyen, V. T., Levi, B., James, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+methods+of+adipogenic+differentiation+of+mesenchymal+stem+cells.">Current methods of adipogenic differentiation of mesenchymal stem cells.</a> Stem Cells and Development. 20 (10), 1793-1804 (2011).</li><li>Zipori, D., Toledo, J., von der Mark, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+among+stromal+cell+lines+from+mouse+bone+marrow+disclosed+in+their+extracellular+matrix+composition+and+interactions+with+normal+and+leukemic+cells.">Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells.</a> Blood. 66 (2), 447-455 (1985).</li><li>Maridas, D. E., Rendina-Ruedy, E., Le, P. T., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Culture,+and+Differentiation+of+Bone+Marrow+Stromal+Cells+and+Osteoclast+Progenitors+from+Mice.">Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.</a> Journal of Visualized Experiments. (131), e56750 (2018).</li><li>Iguchi, T., Niino, N., Tamai, S., Sakurai, K., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+Quantification+of+Plasma+MicroRNA+Levels+in+Cynomolgus+Monkeys,+Using+Quantitative+Real-time+Reverse+Transcription+PCR.">Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR.</a> Journal of Visualized Experiments. (132), e56850 (2018).</li><li>Bozec, A., Hannemann, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Regulation+of+Adipocyte+Numbers+in+Adult+Organisms+Through+Differentiation+and+Apoptosis+Homeostasis.">Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis.</a> Journal of Visualized Experiments. (112), e53822 (2016).</li><li>Shafat, M. S., 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=Leukemic+blasts+program+bone+marrow+adipocytes+to+generate+a+protumoral+microenvironment.">Leukemic blasts program bone marrow adipocytes to generate a protumoral microenvironment.</a> Blood. 129 (10), 1320-1332 (2017).</li><li>Gazi, E., 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=Direct+evidence+of+lipid+translocation+between+adipocytes+and+prostate+cancer+cells+with+imaging+FTIR+microspectroscopy.">Direct evidence of lipid translocation between adipocytes and prostate cancer cells with imaging FTIR microspectroscopy.</a> The Journal of Lipid Research. 48 (8), 1846-1856 (2007).</li><li>Brown, M. D., 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=Influence+of+omega-6+PUFA+arachidonic+acid+and+bone+marrow+adipocytes+on+metastatic+spread+from+prostate+cancer.">Influence of omega-6 PUFA arachidonic acid and bone marrow adipocytes on metastatic spread from prostate cancer.</a> British Journal of Cancer. 102 (2), 403-413 (2010).</li><li>Hardaway, A. L., Herroon, M. K., Rajagurubandara, E., Podgorski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+adipocyte-derived+CXCL1+and+CXCL2+contribute+to+osteolysis+in+metastatic+prostate+cancer.">Marrow adipocyte-derived CXCL1 and CXCL2 contribute to osteolysis in metastatic prostate cancer.</a> Clinical &amp; Experimental Metastasis. 32 (4), 353-368 (2015).</li><li>Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+metastasis+in+the+elderly.">Spinal metastasis in the elderly.</a> European Spine Journal. 12, 202-213 (2003).</li><li>Wagner, M., Bjerkvig, R., Wiig, H., Dudley, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+adipocyte+specification+and+necrosis+augment+tumor-associated+inflammation.">Loss of adipocyte specification and necrosis augment tumor-associated inflammation.</a> Adipocyte. 2 (3), 176-183 (2013).</li><li>Bochet, 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=Cancer-associated+adipocytes+promotes+breast+tumor+radioresistance.">Cancer-associated adipocytes promotes breast tumor radioresistance.</a> Biochemical and Biophysical Research Communications. 411 (1), 102-106 (2011).</li><li>Hirano, T., 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=Enhancement+of+adipogenesis+induction+by+conditioned+media+obtained+from+cancer+cells.">Enhancement of adipogenesis induction by conditioned media obtained from cancer cells.</a> Cancer Letters. 268 (2), 286-294 (2008).</li><li>Gordeev, A. A., Chetverina, H. V., Chetverin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+arrangement+of+eukaryotic+cells+in+merged+hydrogels+combines+the+advantages+of+3-D+and+2-D+cultures.">Planar arrangement of eukaryotic cells in merged hydrogels combines the advantages of 3-D and 2-D cultures.</a> Biotechniques. 52 (5), 325-331 (2012).</li></ol>

The authors have nothing to declare.

Cocultures with inserts have been widely used to study cell-to-cell interactions. The 2D coculture system is an effective way to observe how the two parts crosstalk in vitro, by which we here showed two different cancer cell-driven effects on bone marrow adipocytes. Many labs have utilized this method to investigate the crosstalk between adipocytes and cancer cells6,12,27,39.
<p cl...

Bone metastases are widespread among advanced cancer patients, but a curative treatment is still unavailable. Beyond specializing in storing energy as fat, adipocytes can support tumor growth and metastasis in bone marrow and other organs1,2,3,4,5,6. Moreover, adipocytes play an essential role in regulating cancer cell biology7,8,9,10 and metabolism4,11,12,13,14,15,16, as well as in bone metastasis1,4,12. In the bone marrow niche, adipocytes can also affect the biological behavior of cancer cells4,6,17. The interplay between bone marrow adipocytes and cancer cells with osteotropism is significant for an understanding of bone metastasis. However, little is known.
Based on the current studies, various methods are applied to adipocytes, including two- or three-dimensional (2/3D) and ex vivo cultures17,18,19,20,21. Recently, Herroon et al. designed a new 3D-culture approach to study interactions of bone marrow adipocytes with cancer cells22. Although the 3D coculture is optimal for mimicking physiological interactions between adipocytes and cancer cells in vivo, it suffers from poor reproducibility22,23. In comparison to a 2D coculture system, a 3D coculture system may provide different cellular phenotypes, such as cell morphology21,22,24,25,26. Moreover, the ex vivo culture of isolated cancellous bone tissue fragments can lead to a robust outgrowth of adipocytes from cultured bone marrow cells17.
In contrast to these previous models, however, the 2D cell culture model remains a classic, reliable, and easy technique for quickly scanning candidate molecules and the phenotypes changed in either adipocytes or cancer cells in vitro1,4,6,12,15,27. To better understand the crosstalk between bone marrow adipocytes and melanoma cells, we provide a detailed protocol for a 2D coculture system of bone marrow adipocytes with melanoma cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:684px;" width="685">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:18px;width:215px;">DMEM</td>
			<td style="width:140px;">Invitrogen Inc.</td>
			<td style="width:143px;">11965092</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Fetal Bovine Serum</td>
			<td style="width:140px;">Invitrogen Inc.</td>
			<td style="width:143px;">16000&#8211;044</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Phosphate Buffered Saline</td>
			<td style="width:140px;">Invitrogen Inc.</td>
			<td style="width:143px;">14190-144</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Insulin</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">91077C</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">3-isobutyl-1-methyl-xanthine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">I5879</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Dexamethasone</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">D4902</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Oil Red o</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">O0625</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">24-well plate</td>
			<td style="width:140px;">Corning</td>
			<td style="width:143px;">CLS3527</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Transwell insert</td>
			<td style="width:140px;">Millipore</td>
			<td style="width:143px;">MCHT24H48</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Penicillin/Streptomycin</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:143px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">isopropanol</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">I9516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">0.25% trypsin</td>
			<td style="width:140px;">Thermo Scientific</td>
			<td style="width:143px;">25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">hemocytometer</td>
			<td style="width:140px;">Bio-Rad</td>
			<td style="width:143px;">1450016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Culture incubator</td>
			<td style="width:140px;">Thermo Scientific</td>
			<td style="width:143px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">50ml falcon</td>
			<td style="width:140px;">Corning</td>
			<td style="width:143px;">CLS430828</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Clean Bench</td>
			<td style="width:140px;">Thermo Scientific</td>
			<td style="width:143px;">-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Microscopy</td>
			<td style="width:140px;">Olympus</td>
			<td style="width:143px;">-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">200 &#956;L pipet tips</td>
			<td style="width:140px;">BeyoGold</td>
			<td style="width:143px;">FTIP620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">1000 mL pipet tips</td>
			<td style="width:140px;">BeyoGold</td>
			<td style="width:143px;">FTIP628</td>
			<td></td>
		</tr>
	</tbody>
</table>

dual effects of melanoma cell-derived factors on bone marrow adipocytes differentiation

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are available for studying the significant crosstalk; however, a two-dimensional transwell system for coculture remains a classic, reliable, and easy way for this crosstalk study. Here, we present a detailed protocol that shows the coculture of bone marrow adipocytes and melanoma cells. Nevertheless, such a coculture system could not only contribute to the study of cell signal transductions of cancer cells induced by bone marrow adipocytes, but also to the future mechanistic study of bone metastasis which may reveal new therapeutic targets for bone metastasis.

In the bone marrow, adipocytes can appear in the tumor microenvironment1,13,33,34,35 at an early stage for supporting tumor progression through soluble factors or activating osteoclastogenesis6,12,36, especially in the context of obesi...

Watch this Scientific Journal Video about Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation at JoVE.com

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

This method can help answer key questions in the bone metastasis research field, such as what changes are induced in the bone marrow adipocyte growth and their development in a cancer microenvironment. The big advantages of this technique are that it is easy to perform and that the candidate molecules can be quickly obtained. This method can provide insights into the crosstalk between bone marrow adipocytes and the melanomal cells.It can also be applied to other cell types, such as osteoblasts. Begin by seeding two times 10 to the fifth B16F10 melanoma cells in a 100-millimeter dish with 10 milliliters of DMEM supplemented with 10%FBS, for a 24-hour incubation at 37 degrees Celsius, and 5%CO2. The next day, wash the about 80%confluent cell culture two times with five milliliters of PBS per wash, and starve the cells with 10 milliliters of serum-free culture medium in the cell culture incubator for 18 to 24 hours.The next day, use a pipette to transfer the conditioned supernatant into a 50-milliliter conical tube for centrifugation, and load the medium into a syringe equipped with a 0.45 micron-pore filter. Then, strain the solution into a sterile container to remove any remaining cell debris, and store the conditioned medium at minus 20 degrees Celsius for up to one month. To induce bone marrow adipocytes, culture mouse F14 1.1 bone marrow stromal cells in a 60-millimeter dish with complete DMEM to an 80%confluency.At the appropriate stage of culture, harvest the cells with 1.5 milliliters of a 0.25%trypsin solution for one to two minutes at 37 degrees Celsius. When the cells have detached, arrest the enzymatic reaction with five milliliters of medium supplemented with 10%FBS, and adjust the cells to a one to two times 10 to the fifth cells-per-milliliter concentration with fresh serum-supplemented culture medium. Seed five times 10 to the fourth cells in 0.5 milliliters of adipogenic induction medium per well in a 24-well plate, and return the cells to the cell culture incubator for another two days, until they reach about 90%confluency.Then, replace the supernatant with one milliliter of adipogenic maintenance medium per well, and continue to culture the cells until adipocyte maturation. When the cells look like small to large soap bubbles under light microscopy, wash these mature adipocytes two times with PBS, followed by fixation in 0.2 milliliters of 3.7%formaldehyde for one hour. Rinse the fixed cells in 60%isopropanol, and allow the wells to dry completely.Add 0.2 milliliters of Oil Red O working solution to the cells for a 30 to 120-minute incubation at room temperature, and wash the cells with water to remove any unbound dye for imaging of the adipocytes by light microscopy. Alternatively, upon their maturation, add 0.5 milliliters of ready-to-use RNA isolation reagent to each adipocyte culture well, and use the appropriate primers to confirm the adipocyte-specific gene signature of the cells. Then, run the reaction in a thermal cycler using the indicated settings.To set up a bone marrow-derived adipocyte melanoma cell co-culture, first add 150 microliters of serum-free DMEM to the inner chambers of 0.4 micron-pore size membrane inserts, and place each insert into one well of a 24-well plate containing 500 microliters of serum-free medium per well. Dilute B16F10 melanoma cells from an 80%cell culture to an experimentally-appropriate concentration in fresh serum-free medium, and replace the medium in the inner chamber of each insert with 150 microliters of cells. Next, replace the adipogenic maintenance medium supernatant in each well of a 90%confluent mature bone marrow adipocyte culture with 600 microliters of complete DMEM, and transfer the melanoma cell-containing inserts to each well of the mature adipocyte culture plate.When all of the inserts have been transferred, place the plate in the cell culture incubator. After two days, remove the inserts and wash the bottom of each well two times with 500 microliters of PBS per wash. Then, stain the adipocytes with the Oil Red O working solution, or analyze the adipocyte gene expression by quantitative PCR, as demonstrated.Mature adipocytes differentiated in tumor-conditioned medium contain lipid droplets, allowing the differentiated adipocytes to be easily identified by Oil Red O staining. The adipocyte-specific gene signature of these cells can also be confirmed by quantitative PCR. A reduction in lipid storage is observed in adipocytes co-cultured in the presence of high numbers of melanoma cells, as well as a de-differentiation in adipocyte phenotype, as evidenced by a decreased adipocyte-specific gene expression in these formerly mature cells.After watching this video, you should have a good understanding of how to design and set up a 2D co-culture system for studying the interactions between two different cell types.

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Cancer Research

6.1K Görüntüleme.  Shanghai Jiao Tong University. This method can help answer key questions in the bone metastasis research field, such as what changes are induced in the bone marrow adipocyte growth and their development in a cancer microenvironment. The big advantages of this technique are that it is easy to perform and that the candidate molecules can be quickly obtained. This method can provide insights into the crosstalk between bone marrow adipocytes and the melanomal cells.It can also be applied to other cell types, such as ost...