Name: dual effects of melanoma cell-derived factors on bone marrow adipocytes differentiation
Uploaded: 2018-08-23T12:30:00
Description: Watch this Scientific Journal Video about Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation at JoVE.com

我々 は骨髄間質細胞をご提供するため親切ドヴ ・ Zipori (ワイツマン科学研究所、レホヴォト、イスラエル) に感謝 14F1.1 ラインします。本研究は、中国国家自然科学基金 (第 81771729)、永川病院の重慶医科大学 (Nos からの補助金によって支えられました。YJQN201330;YJZQN201527)。

注: このプロトコルで使用されているすべてのセルは冷凍ストック細胞から解凍後少なくとも 3 世代にわたる栽培にする必要があります。
1. 悪性黒色腫細胞由来因子を収穫します。
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準備
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<li>B16F10 細胞およびマウスのメラノーマ細胞株を取得します。 注: このプロトコル マウス黒色腫細胞株は中国科学院の幹細胞バンクから得られました。</li>
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	<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. 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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. 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挿入して共培養系は、細胞間相互作用の研究に広く使用されています。2次元培養システムは、どのように二つの我々 はここで骨髄脂肪細胞の 2 つの異なった癌セル駆動の効果を示した生体外クロストーク、部分を観察する効果的な方法です。多くのラボでは、脂肪細胞と癌細胞6,12,27,39間?...

骨転移は進行癌患者の間で広まっているが、根治療法はまだ使用できません。脂肪としてエネルギーを格納することに特化したを超えて脂肪細胞は骨髄や他臓器1,2,3,4,5,6で腫瘍の成長と転移をサポートできます。また、脂肪細胞はがん細胞生物学7,8,9,10と代謝4,11,12 の調節に重要な役割を果たしてください。 ,13,14,15,16、よくように骨転移1,4,12として。骨髄ニッチで脂肪細胞は癌細胞4,6,17の生物学的態度も影響します。骨髄脂肪細胞と癌 osteotropism 細胞間の相互作用は骨転移の理解のために重要です。しかし、少しは知られています。
現在の研究に基づいて、さまざまな方法は、2 または 3 次元 (2 D)/前のヴィヴォ文化17,18,19,20,21を含む脂肪細胞に適用されます。最近、Herroonらは、癌細胞22骨髄脂肪細胞の相互作用を研究する新しい 3 D 文化のアプローチを設計されています。3 D 共が生理学的な模倣するため最適な脂肪細胞と癌との間の相互作用が生体内で細胞、それは再現性に乏しく22,23から苦しんでいます。2次元培養システムと比較して、3 D の共培養系細胞形態21,22,24,25,26などの別の細胞表現型があります。さらに、孤立した海綿骨の組織片の前のヴィヴォ文化は、培養した骨髄細胞17から脂肪細胞の堅牢な副産物につながります。
これらの以前のモデルとは対照的しかし、2次元細胞培養モデル古典的な信頼性、および簡単な手法のままさっと候補分子と表現型の脂肪細胞やがんの変更細胞の in vitro1、 4,6,12,15,27.骨髄脂肪細胞と悪性黒色腫細胞間クロストークを理解、悪性黒色腫細胞の骨髄脂肪細胞の 2次元培養システムの詳細なプロトコルを提供します。

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	<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

骨髄脂肪細胞と腫瘍細胞間クロストークは、骨転移の過程において重要な役割を再生可能性があります。さまざまな方法があります; 重要なクロストークを勉強ただし、共の二次元 transwell システムは古典的な信頼性の高い、このクロストークの研究のための簡単な方法。ここでは、骨髄の脂肪細胞と悪性黒色腫細胞の培養を示す詳細なプロトコルを提案する.それにもかかわらず、このような培養システム可能性がありますだけでなく骨髄脂肪細胞によるがん細胞の細胞シグナル伝達の研究に貢献、骨の新しい治療上のターゲットを明らかにすることが骨転移の研究機構の将来にも転移。

骨髄、脂肪細胞が腫瘍微小環境1,13,33,34,35に可溶性因子を介して腫瘍の進行をサポートするための初期段階で表示できるか破骨細胞形成6,12,36, 特に肥満6,</...

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

ここでは、提案する、信頼性が高く、簡単な二次元 (2 D) 共培養系における骨髄脂肪細胞の悪性黒色腫細胞由来因子の二重効果を明らかにする腫瘍細胞と骨髄脂肪細胞間の相互作用の分化と骨転移機構の解明のための古典的な方法をまたポーズします。

デュアル骨髄脂肪細胞分化に及ぼす悪性黒色腫細胞由来因子

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 ...

Research

JoVE Journal

Cancer Research

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

単球/マクロファージにおける腫瘍微小環境の採用を検討する癌細胞株における遺伝子発現の条件付きのノックダウン

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

サンプルおよび測定可能な残存病変と急性骨髄性白血病の白血病幹細胞を測定するプロセス骨髄に対する包括的なプロトコル

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

A 3D Organotypic Melanoma Spheroid Skin Model

黒色腫切片 3 D 回転楕円体の皮膚モデル

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

NOG マウスの多発性骨髄腫, 骨髄異種のマルチ モーダルな発光とポジトロニック放出断層レントゲン写真撮影/計算断層イメージング

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

A Melanoma Patient-Derived Xenograft Model

黒色腫患者由来異種移植片モデル

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

免疫不全マウスにおける肝直交細胞性ヒトうveal黒色腫異種移植プラットフォームの生成

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

高分解能共焦点顕微鏡による3D腫瘍スフェロイド侵入に対する移動性阻害剤の効果の特徴

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

固形腫瘍移植による骨原発性骨腫瘍と骨転移のモデリング

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

トランスクリプトーム解析を用いた無秩序発がん性転写因子の構造機能関係のマッピング

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

注射器と針を用いた循環腫瘍細胞に及ぼす血行力学的ストレスのモデル化

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed ...