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. 
<ol> <li> 稀释 EDTA 全血1:1 与1x 磷酸缓冲盐水 (PBS)。重新挂起. </li>
	<li> 淋巴细胞分离培养基上的层 (血液 PBS: 淋巴细胞分离培养基, 2:1) 在50毫升管。
	<ol> <li> 吸管30毫升血液稀释到50毫升管. </li>
		<li> 将15毫升的淋巴细胞分离培养基轻轻地放在50毫升的管子底部. </li>
	</ol> </li> <li> 757 和 #215 离心机; g 为10分钟的室温 (无刹车). </li>
	<li> 收集单个核细胞: <ol> <li> 用5毫升吸管收集单个核细胞的白色层, 无需搅拌, 并将其放入新的50毫升管中. </li>
		<li> 用20毫升 PBS 稀释收集的 PBMCs. </li>
	</ol> </li> <li> 清洗 PBMCs: <ol> <li> 在室温下以 225 x g 离心5分钟。
		<li> 通过倒置管丢弃上清。重复一次. </li>
	</ol> </li> <li> 红细胞裂解: 注意: 如果细胞颗粒是红色的, 用红细胞溶缓冲液处理细胞 (参见 材料表 )。
	<ol> <li> 将50毫升的管子放在冰上. </li>
		<li> 添加5毫升的红血球溶缓冲. </li>
		<li> 等待 3-5 分钟完成红细胞裂解。时间根据红细胞污染程度而变化. </li>
		<li> 通过添加20毫升 PBS 来停止反应. </li>
		<li> 在室温下离心 225 x g, 5 分钟。通过倒置管丢弃上清. 
		注: 可选: 如果进行红细胞裂解, 请按照下面的说明清洗 PBMCs. </li>
	</ol> </li> <li> 清洗 PBMCs: <ol> <li> 在室温下, 在 225 x g 处添加20毫升 PBS 和离心机, 5 分钟. </li>
		<li> 通过倒置管丢弃上清. </li>
	</ol> </li> <li> 在完全和 #945 中重新挂起单元格;-内存 (补充1% 谷氨酰胺 (初始浓度 100X) 和10% 胎牛血清). </li>
	<li> 用 Neubauer 室计数单元格。
	<ol> <li> 将冰乙酸中的样品1:100 稀释并使用10和 #181; 稀释的 L 以计数细胞. </li>
		<li> 计数至少两次并计算复制的平均值以获取所收集的单元格的总平均数量. </li>
	</ol> </li> <li> 板单元格: <ol> <li> 每 cm 的浓度为 75万 PBMCs 的板单元格 2 。
		<li> 在37和 #176 的恒温箱中存储镀膜细胞; C 在 5% CO 2 大气中. 
		注: 设计实验, 包括负控制, 这是处理与正常完整和 #945 的单元;-没有 MCSF 或 RANKL GFs. 记住在至少3技术复制中执行每个条件。执行至少3生物复制. </li>
	</ol> </li> <li> 更改与 GFs 一起补充的介质: <ol> <li> 在单元播种后等待约3小时, 然后移除介质 (200-1000 和 #181; L) 以丢弃碎片、未连接的细胞和红细胞. 
		注意: 更换培养基时要小心, 避免细胞脱落. </li>
		<li> 添加新的完整 #945;-MCSF (500 和 #181 的 20 ng/毫升) 补充. 
		注: 当大块与癌细胞, 种子的癌细胞 PBMC 播种后的一天. </li>
	</ol> </li> <li> 每隔 2-3 天 (& #945) 更改媒体: <ol> <li> 用 200-1000 和 #181 丢弃介质; L 吸管. </li>
		<li> 通过 200-1000 和 #181 添加新的新鲜培养基; L 吸管. </li>
	</ol> </li> <li> 在单元播种后6-7 天内, 添加 20 ng/毫升的 RANKL 以完成和 #945;-内存 + MCSF. </li>
	<li> 每2-3 天更改一次介质 (完整和 #945;-内存 + MCSF + RANKL): <ol> <li> 用 200-1000 和 #181 丢弃介质; L 吸管. </li>
		<li> 添加 200-1000 和 #181 的新介质; L 吸管。细胞播种后14天停止诱导分化. </li>
		<li> 丢弃介质并使用 PBS 清洗两次. </li>
	</ol> </li> <li> 使用甲醛修复单元格: <ol> <li> 将单元格保留为干燥. </li>
		<li> 添加4% 甲醛 (请参见 材料表 ). </li>
		<li> 室温孵育20分钟. </li>
	</ol> </li> <li> 丢弃甲醛并用500和 #181 清洗两次; PBS 的 L. 
	警告: 甲醛有毒;请戴上手套、口罩和眼镜. </li>
	<li> 允许单元格干燥. </li>
	<li> 根据制造商和 #39 的说明执行陷印着色 (请参阅 材料表 ). </li>
	<li> 在放大镜下, 在显微镜下手动计数破骨样细胞 10X;NA 0.30. 
	注: 破骨样细胞被定义为具有至少4核的陷阱 + polycarion 细胞. </li>
</ol> 2。癌细胞培养 注意: 可以使用不同类型的乳腺腺癌细胞系进行实验, 如 SCP2 17 , 这是一个 osteotropic 细胞系, 来源于三重负人乳腺癌细胞系 (MDA-MB-231) 和激素受体阳性细胞系 MCF7. 
<ol> <li> 将单元格作为单层在75厘米的 2 烧瓶中进行培养。
	<ol> <li> 种子100万的癌细胞在75厘米的 2 烧瓶中。
		<li> 将单元格存储在37和 #176; C 在完整和 #945 中, 在 5% CO 2 大气层中补充1% 谷氨酰胺和10% 胎牛血清. </li>
	</ol> </li> <li> 将单元格分离到大约 90% 70-100: <ol> <li> 丢弃介质, 用 pbs 进行洗涤, 并在 pbs 中添加 2-3 毫升的胰蛋白酶 1X. </li>
		<li> 在37和 #176 孵育细胞3-5 分钟; C. </li>
		<li> 从75厘米 2 烧瓶中收集分离的细胞由10毫升吸管, 添加8毫升的完整培养基停止酶反应. </li>
		<li> 225 x g 离心5分钟. </li>
	</ol> </li> <li> 清洗癌细胞: <ol> <li> 添加10毫升的 PBS. </li>
		<li> 600 x g 离心5分钟. </li>
	</ol> </li> <li> 用 Neubauer 室计数癌细胞。
	<ol> <li> 至少计算两次癌细胞的数量, 并算出复制的平均值, 以获取所收集的细胞总数. </li>
	</ol> </li> </ol> 3。癌细胞和单核细胞的直接共培养对破骨干细胞的分化作用 <ol> <li> 在插入物上种癌细胞 (孔径0.4 和 #181; m): <ol> <li> 种子癌细胞的浓度为4000细胞每厘米 2 。
		<li> 孵育于37和 #176; C 和 5% CO 2 . 
		注: 在0.4 和 #181 的共培养实验 PBMC 播种后的一天, 种子的癌细胞; m 插入. </li>
	</ol> </li> <li> 等待24小时以允许单元格粘附. </li>
	<li> 将种子插入区域性置于单核区域性上以开始共培养 (参见协议1.10 节) </li>
	<li> 每 2-3 天更改媒体, 并完成 #945;
	<ol> <li> 丢弃 200-1000 和 #181 的介质; L 吸管. </li>
		<li> 添加500和 #181; L 的新鲜培养基. 
		注: 共培养的主要目的是了解癌细胞的 osteoclastogenic 能力, 因此, GFs 不会添加到和 #945;在每次共培养实验中, 包括 negative 控制 (见《议定书》步骤1.10 中的说明) 和 RANKL 和 MCSF (无癌细胞) 区分的破骨细胞阳性控制。在未放置于单个核细胞的插入物中的癌细胞被用作对共培养的阴性控制. </li>
	</ol> </li> <li> 停止共培养并使用肿瘤细胞进行下游分析: <ol> <li> 在 PBMC 播种后13天和癌症细胞播种后12天, 将插入物放在新的盘子上. </li>
		<li> 停止癌症细胞培养, 并在此基础上进行下游分析 (请参阅 讨论 ). </li>
	</ol> </li> <li> 14 天, 停止破骨细胞分化并修复单元格, 如步骤1.15 所述. </li>
	<li> 立即或在 14-30 天内执行陷阱染色. 
	注: 可选染色: 为了证实陷阱 + polycarion 细胞是破骨, 研究人员可以执行额外的染色检测的中心, 一个破骨特异的标志物。F 肌动蛋白环的检测是另一种破骨细胞特异染色方法 12 , 13 , 14 。
</ol>

<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>

作者没有利益冲突透露。

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 ...

development-human-preclinical-model-osteoclastogenesis-from

Research

JoVE Journal

Cancer Research

7.0K 次观看.  IRCCS. 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。

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

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

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

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

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

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

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

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

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 cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

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

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 diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

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

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

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

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 cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

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

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in 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

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

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

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

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

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

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

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

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 ...