We would like to thank NIH, LACaTS and Ochsner clinic foundation for the financial support.

所有的小鼠实验均按照批准的医学LSU学校的机构动物护理和使用委员会（IACUC）协议完成。所有仪器之前必须清扫高压灭菌。 从小鼠乳腺肿瘤1.准备原代细胞<ol><li>使用已遗传工程与小鼠乳腺肿瘤病毒多瘤中的T抗原（MMTV-PyMT转基因小鼠），其自发地产生肿瘤如前所述6小鼠模型。 </li><li>牺牲小鼠用异氟醚（99.9％）和喷75％的乙醇，以确保一无菌环境（ 图1A）。 </li><li>打开从尿道口与剪刀颈部的区域中的皮肤。引脚上的皮朝下，以方便获取器官（ 图1B）。 </li><li>隔离乳腺乳房肿瘤，并用冷的无菌磷酸盐缓冲盐水（PBS）（pH 7.4）中（ 图1C）。 </li><li>转移的肿瘤无菌10厘米组织培养板和用无菌刀片和剪刀砍它到约1mm 3件。随后的步骤应在无菌层流罩下进行。 </li><li>孵育切碎的肿瘤组织与胶原酶溶解在无血清DMEM培养基以1mg / ml的终浓度上的摇杆2小时，在37℃。离心肿瘤组织的悬浮液3分钟，在200×g下。 注：胶原酶孵育对于组织酶解必不可少。 </li><li>弃上清，用PBS（pH7.4）中离心3分钟，在200×g下洗涤沉淀物3次。最后一次洗涤后，倒出上清液。 </li><li>悬浮用2ml胰蛋白酶的EDTA 5分钟的组织，在37℃，同时章动。离心该悬浮液3分钟，在200×g离心并重复洗涤。 </li><li>重悬组织沉淀在正常生长培养基（DMEM，10％FBS，2X青霉素/链霉素）到10cm的板。变化每天在接下来的3天的媒体。培养细胞直到所需的细胞数量被获得。 注：约合1×10 7细胞会从这个过程中分离出来。其他小鼠乳腺肿瘤模型，如MMTV-NEU，相比MMTV-PyMT给细胞的产量较低。当使用产生较低产量模型，它是有益的开始与一个较大的肿瘤和培养细胞更长。 </li></ol> 2.小​​鼠肺提取形象化肺转移<ol><li>之前肺提取，取出肝脏和隔膜。去除肋，而不会干扰肺部。 </li><li>用细剪刀，打开更靠近颈静脉气管的上部。 </li><li>使用导管，经气管注射的试剂如1毫升Z-fix或印度油墨的填补肺（ 图2A）。嵌入的石蜡Z-修复肺部。 </li><li>德在染色菲克特的解决方案注入墨汁肺（100毫升70％的乙醇，将10毫升的10％缓冲的福尔马林和5ml冰醋酸）中5分钟。限定肿瘤结节在辅助站点与≥直径0.5mm，确定结节为转移。 注：由于支气管和细支气管阻塞的肿瘤，肿瘤结节不能吸收15％印油墨，因而正常肺组织将是黑白的，而肿瘤结节会以白色为主色调7。这将允许个体肿瘤结节（ 图2B）的可视化。 </li></ol> 3.从小鼠胚胎准备小鼠胚胎成纤维细胞（MEF中） <ol><li>牺牲孕鼠用异氟烷（99.9％）和喷75％的乙醇，以确保一无菌环境。 </li><li>打开从尿道口与剪刀颈部的区域中的皮肤和分离从子宫的胚胎。 </li><li>要小心，不要打开皮肤的时候打扰胚胎。 </li><li>取出子宫角和冲洗他们在寒冷的STE激怒的PBS（pH 7.4）中。 </li><li>在含有冰冷的PBS（pH7.4）中10厘米板分离其胎盘非常仔细地和地方的胚胎的胚胎。 </li><li>除去头，肝脏和肠从胚胎，并将其余部分在含有PBS（pH7.4）中在12孔组织培养板。 </li><li>胚体转移到另一12孔板中含有胰蛋白酶EDTA和切成使用无菌刀片和剪刀小块（约1 立方毫米）。孵化用5ml胰蛋白酶的EDTA切碎材料5分钟，在37℃。 </li><li>加入1ml胎牛血清（FBS），以短切胚胎灭活胰蛋白酶。离心悬浮液，在200×g离心速度为10分钟，小心取出上清液，重悬沉淀用1毫升温的DMEM培养基和所有的内容传送到10cm的培养皿含有10ml的DMEM（20％FBS和2X青霉素/链霉素）。 </li><li>每一天两天的改变介质。培养细胞，直到所需的细胞号为止。 </li></ol>

<ol>
	<li>Minn, A. 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=Genes+that+mediate+breast+cancer+metastasis+to+lung.">Genes that mediate breast cancer metastasis to lung.</a> Natur. 436 (7050), 518-524 (2005).</li><li>Poste, G., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+cancer+metastasis.">The pathogenesis of cancer metastasis.</a> Natur. 283 (5743), 139-146 (1980).</li><li>Wan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Metastasis+Moving+New+Biological+Insights+into+the+Clinic.">Tumor Metastasis Moving New Biological Insights into the Clinic.</a> Nat Med. 19 (11), 1450-1464 (2013).</li><li>Bergers, G., Benjamin, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenesis+and+the+angiogenic+switch.">Tumorigenesis and the angiogenic switch.</a> Nat Rev Cancer. 3 (6), 401-410 (2003).</li><li>Frese, K. K., Tuveson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nat Rev Cancer. 7 (9), 654-658 (2007).</li><li>Guy, C. T., Cardiff, R. D., Muller, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+tumors+by+expression+of+polyomavirus+middle+T+oncogene:+a+transgenic+mouse+model+for+metastatic+disease.">Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.</a> Mol Cell Biol. 12 (3), 954-961 (1992).</li><li>Wexler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+Identification+of+Experimental+Pulmonary+Metastases.">Accurate Identification of Experimental Pulmonary Metastases.</a> J Natl Cancer Inst. 36 (4), 641-645 (1966).</li><li>Boczkowski, D., Nair, S. K., Nam, J. H., Lyerly, H. K., Gilboa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+tumor+immunity+and+cytotoxic+T+lymphocyte+responses+using+dendritic+cells+transfected+with+messenger+RNA+amplified+from+tumor+cells.">Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells.</a> Cancer Res. 60 (4), 1028-1034 (2000).</li><li>Vargo-Gogola, T., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer:+one+size+does+not+fit+all.">Modelling breast cancer: one size does not fit all.</a> Nat Rev Cancer. 7 (9), 659-672 (2007).</li><li>Watts, A. M., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+Tumor+Foci+in+an+Experimental+Murine+Tumor+Model+Using+Computer-Assisted+Video+Imaging.">Quantitation of Tumor Foci in an Experimental Murine Tumor Model Using Computer-Assisted Video Imaging.</a> Anal Bioche. 256 (2), 217-219 (1998).</li><li>Rosenberg, S. 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=Regression+of+Established+Pulmonary+Metastases+and+Subcutaneous+Tumor+Mediated+by+the+Systemic+Administration+of+High-Dose+Recombinant+Interleukin+2.">Regression of Established Pulmonary Metastases and Subcutaneous Tumor Mediated by the Systemic Administration of High-Dose Recombinant Interleukin 2.</a> J Exp Me. 161 (5), 1169-1188 (1985).</li><li>Fantozzi, A., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+breast+cancer+metastasis.">Mouse models of breast cancer metastasis.</a> Breast Cancer Re. 8 (4), 212 (2006).</li><li>Veer, 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=Gene+expression+profiling+predicts+clinical+outcome+of+breast+cancer.">Gene expression profiling predicts clinical outcome of breast cancer.</a> Nature. 415 (6871), 530-536 (2002).</li></ol>

作者没有披露。

这个协议是用于从新鲜小鼠肿瘤中分离的原代细胞。从这个方法制备的裂解物是蛋白质，DNA和RNA分析是有用的。从原发性乳腺肿瘤的癌细胞往往转移到脑，肺和骨骼1。包括的是一种技术，以提取肺检测转移。染色的肺用墨汁染色将使肿瘤结节的可视化， 如图2B所示。在固定Z-修复解决方案肺部将使我们能够使肺部切片，染色并用恰当的研究转移机制的抗体。还包括的是一个协...

Metastasis is the final step in the progression of breast cancer 1. It involves a series of steps in which malignant cells are released from the primary tumor and disseminated to other organs 2. The cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system 3. Each step in tumorigenesis is tightly controlled by cells through various genetic and epigenetic changes 4. To study the mechanisms of breast cancer tumorigenesis, mouse model systems have been utilized due to their short lifespan, small size and fast breeding time. Some major advantages of using mouse models in cancer research are the molecular and physiological similarities they share with humans 5. 
Established mouse and human cell lines have been used extensively in breast cancer research. Although we can obtain some information about the steps in metastasis using these cells, genetically manipulated animal models with tumor suppressors/oncogenes will give far more information about tumor development and metastasis starting from the embryonic stage. Thus, with these mouse models, we can obtain genetic and developmental regulation of tumor growth. Furthermore, the tumor microenvironment also plays an important role in tumor cell gene expression and it can be difficult to mimic the same scenario with studies using established cell lines. Primary human mammary tumors can also be used to study mouse mammary tumorigenesis. The main disadvantage of using isolated breast tumors is that this newly implanted tumor does not have the mouse&#8217;s vasculature and lymphatic tissue. Whereas the genetic mouse model systems for studying mammary tumorigenesis will allow us to monitor the steps of metastasis, analyze the tumor microenvironment, and access to the entire body 5.
The overall goal of this protocol is to study breast tumorigenesis in mice. It is known that breast cancers typically metastasize to the brain, lung and bone. This protocol allows for the isolation of primary mammary tumor cells, as well as, studying breast cancer metastasis to the lung. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. Together, these techniques are useful in studying mouse mammary tumorigenesis.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life&#160; technologies</td>
			<td>25300-062</td>
			<td>Store at &#8211; 20 &#176;C</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td>Store at &#8211; 20 &#176;C</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H3506</td>
			<td>Store at &#8211; 20 &#176;C</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Life&#160; technologies</td>
			<td>15140-122</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gemini Bio-Products</td>
			<td>100-106</td>
			<td>Store at &#8211; 80 &#176;C</td>
		</tr>
		<tr>
			<td>Z-fix</td>
			<td>ANATECH</td>
			<td>#174</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>India Ink</td>
			<td>Yasutomo</td>
			<td></td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>AERRANE (Isoflurane)</td>
			<td>Baxter</td>
			<td>NDC 10019-773-60</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMD</td>
			<td>AX0441-3</td>
			<td>Store at room temperature</td>
		</tr>
	</tbody>
</table>

primary tumor and mef cell isolation to study lung metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

肿瘤进展的阶段，在该牺牲动物取决于特定研究和IACUC准则。在图1A中，100日龄C57BL / 6小鼠用小鼠乳腺肿瘤病毒多瘤中的T抗原（MMTV-PyMT）被处死三个月。皮肤从尿道口的区域中打开与剪刀的颈部和皮肤被牵制，以允许如图1B容易获得器官。这使得容易获得图1C中得到的肿瘤。从肿瘤中获得的原代细胞可视化在图1D。因为许多基因介导的乳腺癌转移到...

Watch this Scientific Journal Video about Primary Tumor and MEF Cell Isolation to Study Lung Metastasis at JoVE.com

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

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15.7K 次观看.  Louisiana State University Health Sciences Center. The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.