Name: an ex vivo brain slice model to study and target breast cancer brain metastatic tumor growth
Uploaded: 2021-09-22T13:00:00
Description: Watch this Scientific Journal Video about An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth at JoVE.com

我们要感谢Julia Farnan，Kayla Green和Tiziana DeAngelis的技术援助。这项工作得到了宾夕法尼亚州联邦通用研究增强资助计划（MJR，JGJ），UO1CA244303（MJR），R01CA227479（NLS），R00CA207855（EJH）和W.W.史密斯慈善信托基金（EjH）的部分支持。

该协议已获得批准，并遵循德雷塞尔大学医学院机构动物护理和使用委员会（IACUC）的动物护理指南。本研究使用Nu / Nu无胸腺炎雌性小鼠（6-8周龄）。
1. 颅内注射肿瘤细胞
<ol><li>在高压灭菌器的干燥循环下，在灭菌袋中对所有设备（镊子，剪刀，缝合剪刀，手钻）进行灭菌长达45分钟的灭菌，包括灭菌指示剂。如果对多只动物进行手术，请使用加热的珠子灭菌器（最多3次）...

<ol>
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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=Heralding+a+new+paradigm+in+3D+tumor+modeling.">Heralding a new paradigm in 3D tumor modeling.</a> Biomaterials. 108, 197-213 (2016).</li><li>Parker, J. 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=A+human+glioblastoma+organotypic+slice+culture+model+for+study+of+tumor+cell+migration+and+patient-specific+effects+of+anti-invasive+drugs.">A human glioblastoma organotypic slice culture model for study of tumor cell migration and patient-specific effects of anti-invasive drugs.</a> Journal of Visualized Experiments: JoVE. (125), e53557 (2017).</li><li>Chuang, H. 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=Coculture+system+with+an+organotypic+brain+slice+and+3D+spheroid+of+carcinoma+cells.">Coculture system with an organotypic brain slice and 3D spheroid of carcinoma cells.</a> Journal of Visualized Experiments: JoVE. (80), e50881 (2013).</li><li>Hohensee, I., 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=PTEN+mediates+the+cross+talk+between+breast+and+glial+cells+in+brain+metastases+leading+to+rapid+disease+progression.">PTEN mediates the cross talk between breast and glial cells in brain metastases leading to rapid disease progression.</a> Oncotarget. 8 (4), 6155-6168 (2017).</li><li>van de Merbel, A. 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=An+ex+vivo+Tissue+culture+model+for+the+assessment+of+individualized+drug+responses+in+prostate+and+bladder+cancer.">An ex vivo Tissue culture model for the assessment of individualized drug responses in prostate and bladder cancer.</a> Frontiers in Oncology. 8, 400 (2018).</li><li>Martin, S. 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=Ex+vivo+tissue+slice+culture+system+to+measure+drug-response+rates+of+hepatic+metastatic+colorectal+cancer.">Ex vivo tissue slice culture system to measure drug-response rates of hepatic metastatic colorectal cancer.</a> BMC Cancer. 19 (1), 1030 (2019).</li><li>Orimo, A., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer:+a+novel+tumor-promoting+cell+type.">Stromal fibroblasts in cancer: a novel tumor-promoting cell type.</a> Cell Cycle. 5 (15), 1597-1601 (2006).</li><li>Lim, C. 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=Organotypic+slice+cultures+of+pancreatic+ductal+adenocarcinoma+preserve+the+tumor+microenvironment+and+provide+a+platform+for+drug+response.">Organotypic slice cultures of pancreatic ductal adenocarcinoma preserve the tumor microenvironment and provide a platform for drug response.</a> Pancreatology. 18 (8), 913-927 (2018).</li><li>Gerlach, M. 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=Slice+cultures+from+head+and+neck+squamous+cell+carcinoma:+a+novel+test+system+for+drug+susceptibility+and+mechanisms+of+resistance.">Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance.</a> British Journal of Cancer. 110 (2), 479-488 (2014).</li><li>Koerfer, 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=Organotypic+slice+cultures+of+human+gastric+and+esophagogastric+junction+cancer.">Organotypic slice cultures of human gastric and esophagogastric junction cancer.</a> Cancer Medicine. 5 (7), 1444-1453 (2016).</li><li>Palmieri, 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=Her-2+overexpression+increases+the+metastatic+outgrowth+of+breast+cancer+cells+in+the+brain.">Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.</a> Cancer Research. 67 (9), 4190-4198 (2007).</li><li>Kyeong, 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=Subtypes+of+breast+cancer+show+different+spatial+distributions+of+brain+metastases.">Subtypes of breast cancer show different spatial distributions of brain metastases.</a> PLoS One. 12 (11), 0188542 (2017).</li><li>Hengel, 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=Attributes+of+brain+metastases+from+breast+and+lung+cancer.">Attributes of brain metastases from breast and lung cancer.</a> International Journal of Clinical Oncology. 18 (3), 396-401 (2013).</li><li>Jackson, J. G., 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=Neuronal+activity+and+glutamate+uptake+decrease+mitochondrial+mobility+in+astrocytes+and+position+mitochondria+near+glutamate+transporters.">Neuronal activity and glutamate uptake decrease mitochondrial mobility in astrocytes and position mitochondria near glutamate transporters.</a> Journal of Neuroscience. 34 (5), 1613-1624 (2014).</li><li>Farnan, J. K., Green, K. K., Jackson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+imaging+of+mitochondrial+dynamics+and+trafficking+in+astrocytes.">Ex vivo imaging of mitochondrial dynamics and trafficking in astrocytes.</a> Current Protocols in Neuroscience. 92 (1), 94 (2020).</li><li>Simone, N. 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=Ionizing+radiation-induced+oxidative+stress+alters+miRNA+expression.">Ionizing radiation-induced oxidative stress alters miRNA expression.</a> PLoS One. 4 (7), 6377 (2009).</li><li>Couturier, C. 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=Single-cell+RNA-seq+reveals+that+glioblastoma+recapitulates+a+normal+neurodevelopmental+hierarchy.">Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy.</a> Nature Communications. 11 (1), 3406 (2020).</li><li>Candolfi, 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=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathological+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.</a> Journal of Neuro-Oncology. 85 (2), 133-148 (2007).</li><li>Fitzgerald, D. 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=Reactive+glia+are+recruited+by+highly+proliferative+brain+metastases+of+breast+cancer+and+promote+tumor+cell+colonization.">Reactive glia are recruited by highly proliferative brain metastases of breast cancer and promote tumor cell colonization.</a> Clinical &amp; Experimental Metastasis. 25 (7), 799-810 (2008).</li><li>Kondru, 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=An+Ex+Vivo+Brain+Slice+Culture+Model+of+Chronic+Wasting+Disease:+Implications+for+Disease+Pathogenesis+and+Therapeutic+Development.">An Ex Vivo Brain Slice Culture Model of Chronic Wasting Disease: Implications for Disease Pathogenesis and Therapeutic Development.</a> Scientific Reports. 10 (1), (2020).</li><li>Abu Samaan, T. 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=Paclitaxel's+mechanistic+and+clinical+effects+on+breast+cancer.">Paclitaxel's mechanistic and clinical effects on breast cancer.</a> Biomolecules. 9 (12), (2019).</li><li>Mewes, A., Franke, H., Singer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+of+adult+transgenic+P301S+mice--a+model+for+tauopathy+studies.">Organotypic brain slice cultures of adult transgenic P301S mice--a model for tauopathy studies.</a> PLoS One. 7 (9), 45017 (2012).</li><li>Valiente, 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=Brain+metastasis+cell+lines+panel:+A+public+resource+of+organotropic+cell+lines.">Brain metastasis cell lines panel: A public resource of organotropic cell lines.</a> Cancer Research. 80 (20), 4314-4323 (2020).</li></ol>

本研究建立了一种新的外植异种移植脑肿瘤的 离体 脑培养方法。我们表明，颅内注射在小鼠大脑中的BCBM细胞MDA-MB-231BR细胞可以在 离体 脑切片中存活和生长。该研究还测试了颅内注射的U87MG胶质母细胞瘤（GBM）细胞，还发现这些癌细胞在脑切片中存活和生长（数据未显示）。我们相信这个模型可以从BCBM和GBM扩展到其他容易转移到大脑的癌症，包括肺癌和黑色素瘤。选择颅内注射乳?...

当细胞从原发性乳腺肿瘤扩散到大脑时，乳腺癌脑转移瘤（BCBM）就会发展。乳腺癌是仅次于肺癌的第二大脑转移原因，转移发生在10-16%的患者中1。不幸的是，脑转移瘤仍然无法治愈，因为>80%的患者在脑转移诊断后一年内死亡，并且由于神经功能障碍，他们的生活质量受损2。迫切需要确定更有效的治疗方案。单层二维或三维培养模型是在实验室中测试治疗剂最常用的方法。然而，它们不能模仿复杂的BCBM微环境，这是肿瘤表型和生长的主要驱动因素。虽然这些模型是有用的，但它们不能捕获复杂的肿瘤 - 基质相互作用，独特的代谢需求以及肿瘤的异质性3。为了更忠实地概括肿瘤 - 基质相互作用和微环境异质性，我们的小组和其他人已经开始用患者来源的肿瘤细胞（原发性或转移性）或癌细胞系4，5，6产生有机指型脑转移“切片”培养物。与传统的 体外 系统相比，这种短期 离体 模型可能为在大型动物队列的临床前评估之前筛选新疗法提供更多相关条件。
已经构建并成功用于鉴定各种癌症的成功治疗。它们需要几天的评估，此外还可以根据患者特定的药物筛查进行定制。例如，人膀胱癌和前列腺癌的离体组织已经显示出多西紫杉醇和吉西他滨7的剂量依赖性抗肿瘤反应。开发了类似的结直肠癌离体组织来筛选化疗药物奥沙利铂，西妥昔单抗和Pembrolizumab8。该应用已被广泛用于胰腺癌，考虑到基质环境与胰腺导管腺癌的基因型和表型特征之间的基本相互作用9，10。此外，已经开发了这种有机细胞模型，用于头，颈，胃和乳腺肿瘤的类似筛查11，12。
在这里，正在产生异种移植乳腺癌脑转移性肿瘤细胞在其微环境中的 离体 脑切片模型。将小鼠颅内注射乳腺癌脑转移性脑营养MDA-MB-231BR细胞13 在脑皮层顶叶 - TNBC转移的常见部位14，15 并允许发展肿瘤。从这些异种移植动物中产生脑切片，并作为有机细胞培养物进行 体外 保存，如16，17所述。这种新颖的 离体 模型可以分析BCBM细胞在脑实质内的生长，并可用于测试治疗剂和对脑微环境中肿瘤细胞的辐射作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe, slip tip</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G1/2 Needles</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Genessee</td>
			<td>25-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated microscope and LUMAVIEW software</td>
			<td>Etaluma</td>
			<td>LS720</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 (GEM21)</td>
			<td>Gemini Bio-Products</td>
			<td>400-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker 50 mL</td>
			<td>Fisher</td>
			<td>10-210-685</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt sable paintbrush, Size #5/0</td>
			<td>Electron Microscopy Sciences</td>
			<td>66100-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Wax</td>
			<td>ModoMed</td>
			<td>DYNJBW25</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain injection Syringe</td>
			<td>Hamilton Company</td>
			<td>80430</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher Scientific</td>
			<td>BP510-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaved caspase 3 Antibody</td>
			<td>Cell Signaling</td>
			<td>14220S</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin Potassium Salt</td>
			<td>Perkin Elmer</td>
			<td>122799</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge razor blade</td>
			<td>VWR</td>
			<td>55411-060(95-0043)</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper (#1), quantitative circles, 4.25 cm</td>
			<td>Fisher</td>
			<td>09-805a (1001-042)</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine sable paintbrush #2/0</td>
			<td>Electron Microscopy Sciences</td>
			<td>66100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma-H2AX antibody</td>
			<td>Millipore</td>
			<td>05-636</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Thermo Fisher</td>
			<td>13-0300</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP antibody</td>
			<td>Santa Cruz</td>
			<td>SC-9996</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine (200 mM)</td>
			<td>Corning cellgrow</td>
			<td>25-005-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>H&#38;E and KI-67</td>
			<td>Jefferson Core Facility Pathology staining</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hand Drill Set with Micro Mini Twist Drill Bits</td>
			<td>Amazon</td>
			<td>YCQ2851920086082DJ</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, free acid</td>
			<td>Fisher Scientific</td>
			<td>BP299-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Just for mice Stereotaxic Frame</td>
			<td>Harvard Apparatus (Holliston, MA, USA).</td>
			<td>72-6049, 72-6044</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Large surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14001-18</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231BR cells</td>
			<td>Kindly provided by Dr. Patricia Steeg</td>
			<td>Ref 14</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Fisher Scientific</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice imaging device</td>
			<td>Perkin Elmer</td>
			<td>IVIS 200 system</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice imaging software</td>
			<td>Caliper Life Sciences (Waltham, MA, USA).</td>
			<td>Living Image Software</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Tecan Spark</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting solution</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS reagent</td>
			<td>Promega CellTiter 96 Aqueous One Solution</td>
			<td>(Cat:G3582)</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nu/Nu athymic mice</td>
			<td>Charles Rivers Labs (Wilmington, MA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Affymetrix</td>
			<td>19943</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Life Technologies</td>
			<td>145140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Suture</td>
			<td>Medex supply</td>
			<td>ETH-8556H</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone Iodine Swab sticks</td>
			<td>DME Supply USA</td>
			<td>Cat: 689286X</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade #11 (pk of 100)</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula/probe</td>
			<td>Fine Science Tools</td>
			<td>10090-13</td>
			<td></td>
		</tr>
		<tr>
			<td>SS Double edge uncoated razor blades (American safety razor co (95-0043))</td>
			<td>VWR</td>
			<td>55411-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Amresco</td>
			<td>57-50-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>Exelint International</td>
			<td>D29702</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Chopper</td>
			<td>Brinkman</td>
			<td>(McIlwain type)</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture inserts</td>
			<td>Millipore</td>
			<td>PICMORG50 or PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray machine</td>
			<td>Precision 250 kVp</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo brain slice model to study and target breast cancer brain metastatic tumor growth

脑转移瘤是乳腺癌对女性的严重后果，因为这些肿瘤难以治疗，并且与不良的临床结果有关。乳腺癌脑转移（BCBM）生长的临床前小鼠模型是有用的，但价格昂贵，并且很难跟踪活细胞和肿瘤细胞在脑实质内的侵袭。这里介绍的是来自异种移植小鼠的 离体 脑切片培养方案，其中包含颅内注射的乳腺癌寻脑克隆亚线。 将MDA-MB-231BR 荧光素酶标记的细胞颅内注射到Nu / Nu雌性小鼠的大脑中，肿瘤形成后，将大脑分离，切片并 离体培养。对肿瘤切片进行成像以鉴定表达荧光素酶的肿瘤细胞，并监测它们在脑实质中的增殖和侵袭长达10天。此外，该协议描述了使用延时显微镜对电离放疗或化疗后肿瘤细胞的生长和侵袭行为进行成像。肿瘤细胞对治疗的反应可以通过实时成像显微镜可视化，测量生物发光强度，并在含有BCBM细胞的脑切片上进行组织学。因此，这种 离体 切片模型可能是一个有用的平台，用于单独或与辐射联合使用新型治疗剂的快速测试，以鉴定个性化药物，以靶向个体患者的脑微环境中的乳腺癌脑转移生长。

如上所述，将MDA-MB-231BR-GFP-荧光素酶细胞颅内注射到4-6周龄Nu / Nu小鼠的右半球（图1A）并允许生长12-14天，在此期间通过生物发光成像监测肿瘤生长（图1B）。正如其他小组19所报告的那样，我们在颅内注射了100，000个癌细胞，但有可能注射低至20，000个细胞20。如上所述，在脑切片生成（图1C）?...

Watch this Scientific Journal Video about An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth at JoVE.com

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

我们介绍了一种在有机结构脑切片模型中测量乳腺癌脑转移细胞的实时药物和辐射反应的方案。这些方法提供了一种定量测定法，以研究各种治疗对脑微环境界面内乳腺癌脑转移瘤的治疗效果。

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

该协议允许脑转移性肿瘤细胞的形成和离体脑切片的产生，从而可以快速测试药物疗效和预成型肿瘤的辐射。该技术的主要优点是能够在生理相关环境中快速测试多种药物或治疗方法，从而维持脑 - 肿瘤 - 宿主相互作用。这种方法可能有助于了解脑转移细胞如何生长，存活和侵入脑实质，并了解脑肿瘤微环境在癌症生长和扩散中的作用。演示该程序的将是Lorela Ciraku和Emily Esquea，他们都是我实验室的博士候选人。首先，将麻醉的小鼠放入立体定位框架中，并使用标准耳杆固定头部。在切口前，通过反复交替施用碘拭子和70%乙醇对头部表面进行三次灭菌。使用手术刀，以对角线角度做一个一厘米的中线切口，将小鼠的大脑分成两个对称的半部分，露出头骨。用棉签擦拭任何血液后，横向偏转皮肤以暴露注射部位。使用0.73毫米毛刺钻头穿透颅骨，并使用轻微的压力和扭曲运动钻一个孔。要注射5微升稳定表达GFP荧光素酶的细胞，在大脑中插入3.5毫米深的高精度注射器。让它静置两分钟，然后将注射器向上拉约一毫米。两分钟后，慢慢注入前半个体积，再等待两分钟，然后再注射剩余的体积。三分钟后，慢慢拉动注射器。将骨蜡涂在颅骨上的注射部位，并用聚丙烯缝合线缝合组织。定期监测手术后立即小鼠的行为，以确定是否需要特殊治疗，包括盐水注射和软食，以帮助快速恢复。为了通过生物发光成像监测肿瘤生长，首先打开成像系统上的氧气和异氟醚，并允许两者被分配到成像盒外的单独腔室。然后，在打开软件并初始化仪器后，选择适当的选项来可视化和捕获整个鼠标主体。接下来，将小鼠转移到提供氧气和异氟醚的成像盒外的腔室中。一旦小鼠处于麻醉作用下，腹膜内注射200微升每毫升荧光素30毫克。将小鼠置于成像室中，胃朝下到成像室鼻唇。锁定腔室，选择两分钟时间曝光开始成像，然后完成成像。将组织切片机放入无菌层流罩中后，用70%乙醇清洁所有工具和仪器。对小鼠实施安乐死后，取出并将大脑迅速放入冰冷的蔗糖- ACSF溶液中。使用刮刀将大脑转移到60毫米盘盖上的ACSF润湿滤纸上。使用锋利的无菌剃须刀片，切除大脑中不含任何肿瘤的多余部分，包括大脑的底部，使大脑的形状变成一个不完美的立方体。将几张ACSF润湿的滤纸放在切割平台上后，将堵塞的组织放在滤纸上。要将组织切成200至250微米厚的部分，请在提供者尺上将切割尺寸设置为2或2.5单位并开始切割。使用油漆刷将脑切片转移到含有蔗糖ACSF的培养皿中，并使用27号针在光学显微镜下分离切片。在荧光显微镜下鉴定GFP阳性切片。然后，使用具有宽开口的无菌一毫升破损移液器，将鉴定的切片转移到含有两到三毫升成年切片培养基的新的60毫米培养皿中。然后，将三至五片转移到安全距离内六孔板的每个孔中的每个组织培养插入物上。从插入物表面除去多余的培养基后，在每个插入物下方加入一毫升平衡的成年小鼠切片培养基。在成像前，将组织在37摄氏度，5%二氧化碳和95%湿度下孵育24小时。将五微升每毫升30毫克荧光素溶液移液到无菌罩内插入物下方的培养基中。将带盖子的六孔板放在固定相机下方的仪器成像室内，并锁定腔室门。打开 软件 并 初始化 仪器。选择允许每张图像仅可视化和捕获一个孔的相机设置后，将要成像的孔直接放置在相机下方。使用ROI工具，将其围绕肿瘤形状和大小进行调整，测量ROI，并报告总读数以量化切片上每个肿瘤的生物发光信号。将板带回无菌层流罩后，使用镊子将插入物从孔中略微抬起。吸出培养基后，用一毫升新鲜培养基替换培养基，然后将插孔放回孔中。对于用各种化合物（例如抑制剂和代谢物）处理切片的实验，在1.5毫升管中在1.2毫升的脑切片培养基中制备适当的试剂浓度，然后将一毫升试剂转移到含有切片的孔中。将MDA-MB-231 BR-GFP荧光素酶细胞注射到裸鼠体内，让肿瘤生长，解剖，切片，离体生长，通过生物发光成像监测肿瘤生长。H&E染色和GFP阳性荧光证实了脑切片中存在肿瘤。增加的Ki-67染色证实了高度增殖性癌细胞的存在。未暴露于照射的脑切片中的肿瘤继续向外生长，而暴露于照射的肿瘤显示出停滞的生长。还通过实时成像监测含有肿瘤的脑切片，以可视化照射治疗后的肿瘤生长。与生物发光成像一致，对照癌细胞随着GFP强度的增加和细胞开始侵入脑实质而迅速生长。相比之下，用辐照处理的脑切片中的癌细胞是细胞抑制的，GFP强度得以维持。在IR处理的癌细胞中检测到大的多核癌细胞和DNA损伤标志物染色的增加。此外，检测到反应性星形胶质细胞的增加。在无肿瘤小鼠脑切片中未检测到IR治疗诱导的细胞凋亡。含有肿瘤的脑切片用不同浓度的紫杉醇（一种化疗药物）治疗。治疗在治疗的第10天后显着减小了肿瘤的大小。在紫杉醇治疗的肿瘤细胞中检测到细胞凋亡的增加。抑制剂治疗没有改变脑组织活力。在紫杉醇治疗的无肿瘤小鼠脑切片中，通过切割半胱天冬酶-3染色测量细胞凋亡没有增加。当用感兴趣的药物治疗终止时，可以通过免疫细胞化学，组学研究或实现适合流式细胞术的单细胞悬浮液来分析切片的蛋白质表达。

Research

JoVE Journal

Cancer Research

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

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 ...

科研

癌症研究

Acellular and Cellular Lung Model to Study Tumor Metastasis

脱细胞和细胞肺模型研究肿瘤转移

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

肿瘤治疗胶质母细胞瘤的临床应用

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

In Vitro Assay to Study Tumor-macrophage Interaction

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

一种研究并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型