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

我们要感谢宜宾康提供 SCP2 细胞系和罗纳尔多佛娜的编辑协助。

人破骨细胞与 PBMCs 的健康献血者有区别, 他们书面知情同意参与研究。根据1964《赫尔辛基宣言》中规定的道德标准, 该研究方案得到地方道德委员会的批准. 
 1. 破骨细胞分化 注意: 在 EDTA 中收集健康的人类捐献者的外周血或棕黄色大衣。不要使用少于20毫升的外周血。巴比大衣更可取, 因为它们有更丰富的单核细胞。在无菌组织培养罩中执行以下步骤. </p...

<ol>
	<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=Bone+and+cancer:+the+osteoncology.">Bone and cancer: the osteoncology.</a> Clin Cases Miner Bone Metab. 10 (2), 121-123 (2013).</li><li>Coleman, R. E., Rubens, R. 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>

临床前体外模型研究肿瘤细胞与骨细胞之间的串扰机制, 需要确定骨转移的机制, 以建立新的治疗策略。我们开发了一个完全人的体外模型的人外周血破 (图 3)。在方法优化过程中, 确定和解决了一些关键点。第一个是关于单个核细胞数量的种子。通过显微镜计数获得的细胞数量只是一个粗略的定量, 因为它很难区分淋巴细胞和单核细胞, 后者是破实验中的靶体细?...

骨是一种常见的转移为不同类型的原发性肿瘤, 如前列腺癌, 肺癌和乳腺癌, 20-25% 的病人发展骨转移在疾病过程中1,2,3。特别是, 70% 的乳腺癌患者携带骨转移的证据在死亡4。肿瘤和基质细胞的相互作用是癌症进展的主要癌症和继发性病变。在骨骼微环境中, 乳腺癌的骨骨转移依赖于肿瘤细胞、骨细胞和骨微环境之间的病理恶性循环的建立。肿瘤细胞破坏骨平衡, 增加骨吸收5,6,7。
在正常和病理条件下, 破骨细胞是负责骨质吸收的单元, 而骨细胞, 在沉积新的基质, 负责新骨形成8。破骨细胞的活性通过 RANKL 的表达来调节, 这与其受体在 pre-osteoclast 表面的排列有关, 诱导 pre-osteoclast 融合, 这是分化为成熟破骨的必要过程。诱导破增加骨吸收。大量的体内研究显著提高了我们对骨转移形成的认识9,10,11。乳腺癌细胞从原发肿瘤和骨微环境扰乱骨内稳态, 促进破和骨吸收8。在这种情况下, 所有的分子相互作用发生在癌细胞和破骨细胞之间是至关重要的。正如已经提到的, 骨转移形成的机制已在体内小鼠模型中阐明。然而, 除了需要批准的所有在体内动物实验的伦理委员会, 有几个其他缺点, 以执行在体内实验, 包括高成本和耗时的方法。一些作者将临床前的在体内和体外模型的破使用小鼠线的 pre-osteoclasts 称为 RAW246.79,10,11。这个模型的缺点是由于细胞已经致力于成为 pre-osteoclasts, 而不是人类的起源。由于这些原因, 转化研究可以大大受益于体外充分的人前临床模型研究骨肿瘤细胞的相互作用。
我们优化了破体外的方法, 从人外周血样本开始,12,13。破骨细胞来源于单核细胞, 虽然在很小程度上存在于外周血标本中。通过聚密度梯度将单个核细胞从红细胞和粒中分离出来;他们然后选择, 由于他们的能力坚持塑料基板, 不同于淋巴细胞。播种后, 细胞培养14天。MCSF 和 RANKL 是单核细胞所需的 GFs, 以先分化成巨噬, 然后再进入破骨14,15。MCSF 是需要的整个期间的化验, 而 RANKL 是用来诱导分化过程的后期阶段的破。在分化的早期阶段, MCSF 帮助单核细胞增殖并存活14,15。在破的第二部分, 细胞融合并成熟为破骨, 显示了肌动蛋白 F 在环中的特征分布, 并表达了抗酒石酸酸性磷酸酶 (TRAP) 和降钙素受体等特异标记物中心)14,15. 我们的方法包括在实验的前7天将 MCSF 添加到单核细胞培养中, 并结合 MCSF 和 RANKL 从7天到14。在实验结束时, 破通过计数的分化细胞进行分析, 详见下文。
由 GFs 诱导分化的单核细胞培养是我们临床前模型的基础。我们优化了一个没有 GFs 的共培养系统, 以更好地了解乳腺癌细胞的 osteoclastogenic 能力。我们首先开发了一个间接 co-cultures 的模型, 添加了一个培养基 (80% α-最低基本培养基 (α-记忆) 和20% 条件培养基, 从培养的乳腺癌细胞, 约90% 融合到细胞进行分化12.条件培养基 (未在血清剥夺状态下收集) 在24小时后收集, 并与新鲜培养基混合, 比例为1:4。条件培养基诱导的破骨细胞分化与阴性对照有关。然而, 当使用间接 co-cultures 时, 由于癌细胞与骨细胞相互作用的信息丢失, 我们通过直接 co-cultures 来改善我们的系统。我们在0.4 µM 的插入物中植入癌细胞, 并将它们放置在单核细胞被电镀的井中。使用这种方法, 细胞共享相同的培养基和交换分泌的蛋白质。因此, 我们创造了一个完整的人类临床前模型的破诱导肿瘤细胞13。
这个系统是非常多才多艺的, 可以用于不同的研究目的,例如, 在药理研究中的作用, 调查药物在骨转移。我们的模型使得有可能研究骨靶向治疗和/或抗肿瘤药物在肿瘤细胞存在的作用和机制13。用正确的控制方法设计实验,即, 单独培养的癌细胞和破骨细胞, 使人们更容易理解共育对药物活动的影响。当研究的药物靶向癌细胞和破骨细胞时, 这种方法变得更加有趣,例如, 司16。这个模型也可以用来识别癌细胞和骨细胞之间相互作用的新途径。

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

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

骨微环境中肿瘤细胞与骨细胞之间的串扰是了解骨转移形成机制的关键。我们开发了一个体外完全人前模型的乳腺癌细胞共培养和单核化的分化为破骨病。我们优化了一个模型的破开始从一个样本的外周血从健康的捐助者收集。外周血单个核细胞 (PBMCs) 首先通过密度梯度离心分离, 在高密度下播种, 并通过增加两种生长因子诱导分化 (GFs): 核因子-nf-配体 (RANKL) 和巨噬细胞的受体激活剂集激发因子 (MCSF). 细胞被留在培养14天, 然后通过下游分析固定和分析。在骨骨转移, 肿瘤细胞到达骨的影响之一是诱导破。因此, 我们挑战我们的模型与 co-cultures 乳腺癌细胞, 以研究的分化能力的癌细胞与 GFs 有关。一个直接的方法研究癌细胞-破骨细胞相互作用是执行间接 co-cultures 的基础上, 利用条件培养基收集的乳腺癌细胞培养和混合新鲜培养基。这种混合物用于诱导破骨细胞分化。我们还优化了直接共培养的方法, 其中癌细胞和单核细胞的分化分享媒介和交换分泌的因素。这是一个显著的改进, 在原来的间接共培养方法, 因为研究人员可以观察两个细胞类型的相互作用, 并执行下游分析的癌细胞和破骨细胞。这种方法使我们能够研究药物对转移性骨微环境的影响, 以及除乳腺癌以外的种子细胞株。该模型还可用于研究其他疾病, 如骨质疏松症或其他骨骼条件。

优化了一种从人外周血单核细胞中易分化破骨的方法。单核细胞培养与癌细胞, 证实 (如文献中所描述的18), 癌细胞能够维持破骨转移。在图 1中显示了与癌细胞和 GFs 区分的破骨细胞。破骨样细胞是具有4或更多核的细胞, 对诱捕染色 (紫色细胞) 是阳性的。用于定义破骨细胞的细胞核数量的被接受的截止值是 318, 但?...

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

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

该协议描述了一个体外的发展, 从外周血单核细胞中培养的乳癌细胞系破人前模型, 模拟癌细胞-破骨细胞的相互作用。该模型可用于进一步了解骨转移形成和改善治疗方案。

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

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

Research

JoVE Journal

Cancer Research

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

开发和维护的临床前患者来源的肿瘤异种移植模型为新的抗癌疗法的调查

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

科研

癌症研究

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

临床前小鼠模型的外科手术和方法<em&gt; De Novo</em&gt;乳腺癌转移

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

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

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

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

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

建立和表征三个抗阿法替尼的肺腺癌PC-9细胞系与增加剂量的阿法替尼开发

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

用于转化免疫肿瘤学(I-O)研究的人类外周血单核细胞(PBMC)的人工化异种移植模型

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...