作者要感谢“博凡吴基督教Raithel的为他介绍活细胞成像技术援助和迈克尔·丽格。由国家癌症研究所，美国国立卫生研究院RO1CA112176（PAF），美国国立癌症研究所，美国国立卫生研究院支持。 R01CA89041-10S1（PAF），，丁斯特的EV A/09/72227编号德国学术Austaush。 316（REN），国防部W81XWH-11-1-0074（REN），WCU（世界一流大学）计划，通过教育，科学和技术部（R31-10069）由韩国国家研究基金会（PAF ），美国国立卫生研究院IG20 RR025828-01（啮齿动物屏障设施设备），和，NIH：NCI 5P30CA051008（显微镜和影像和动物共享资源）。

<html> 1。总体方案<ol><li>生成基因工程乳腺的癌前病变的乳腺上皮细胞的原代培养和控制野生型小鼠。 </li><li>捕捉活细胞图像，每15分钟使用Volocity图像采集软件（版本5.3.1，珀金埃尔默，位于Waltham，MA）长达5天。 </li><li>查看时间推移图像直接评估的时间和上皮细胞的集落形成机制，细胞凋亡的发生率，并逐步形态的变化。 </li><li> Volocity产生。TIFF堆栈转换的JPG文件使用的MetaMorph的显微镜自动化图像分析软件（Molecular Devices公司，LLC，加利福尼亚州桑尼维尔市）和重命名的数字图像文件（重命名2.1.6， <a href="http://www.softpedia.com/get/System/File-Management/THE-Rename.shtml" target="_blank">http://www.softpedia.com/get /系统/文件管理/ THE Rename.shtml</a> ），使与蒂姆的跟踪工具软件（TTT的兼容性，tz-muenchen.de/en/isf/hematopoiesis/software-download/index.html“目标=”_blank“&gt; http://www.helmholtz-muenchen.de/en/isf/hematopoiesis/software-download/index。 HTML）。 </li><li>使用的TTT，产生的细胞分裂之间的时间（电池寿命）和模式（对称与不对称的细胞分裂）测定细胞的命运地图。 </li><li>分析上述步骤3和5的定量数据，以确定是否存在统计上的显着差异不同的遗传工程小鼠模型和/或野生型对照。 </li></ol> 2。原代乳腺上皮细胞培养物的生成<ol><li>准备完整的介质隔离的单乳腺上皮细胞的变化，制造商的说明（EpiCult-B鼠标中包，干细胞技术，温哥华，BC）。结合450毫升EPI-邪教-B中，50毫升EPI-邪教乙扩散补充，5％胎牛血清（FBS），50微克/ ml青霉素/链霉素（PenStr聚酯）和10 ng / ml的表皮生长因子（EGF）。 </li><li>准备解离媒体通过组合1的一部分，10倍胶原酶/透明质酸酶混合物与9份完整的媒体从2.1。 </li><li>安乐死鼠标，并立即进行尸检。把鼠标在它的后面一个发泡胶剖检平台上，并确保所有四肢，所以腹侧皮肤被拉紧。饱和腹侧的皮肤和毛发，包括覆盖在四肢，用70％的乙醇。暴露2/3（胸）和＃4/5（腹股沟）正中切口，通过皮肤（不进入腹膜），通过Y形切口延伸到每个前翼内侧的皮肤和乳腺每个后肢内侧皮肤的Ÿ切口通过一个上下颠倒的。 </li><li>使用钝性分离，分离的皮肤从underlyingperitoneum，双方的皮肤上拉回来，直到它被拉紧，并确保它的发泡胶剖检平台，利用销。从外侧开始，使用钝性分离，如果使用得当，狄氏ction剪刀分离出完整的的腹股沟及/或从底层的结缔组织和肌肉的胸乳腺。将不超过两个乳腺在10厘米无菌培养皿中，并移动到组织培养罩。 </li><li>在组织培养罩，检查小，外接结节，一个黄色的颜色被确定为乳腺淋巴结肿大的腺体。清除淋巴结周围腺体，用无菌手术刀和镊子。 3〜1毫米的立方体，用无菌手术刀，每手切碎，乳腺组织。 </li><li>地点在5ml解离媒体，混合乳腺组织绞碎轻轻移液管向上和向下的2-3倍，使用了10毫升移液管，并孵育过夜（可达〜16小时），在37℃的培养箱中，用5％的CO 2，在一个50毫升锥形管与放松帽。 </li><li>第二天早上，准备一个寒冷的1 Hanks平衡盐溶液（HBSS）含2％FBS和4份混合缓冲氯化铵溶液，以促进红色bl洪水细胞的裂解（HFAmCl）以及冷的HBSS补充含2％FBS（HF）。预暖胰蛋白酶-EDTA（0.25％），5毫克/毫升中性蛋白酶在汉克的平衡盐溶液改性，1毫克/毫升的DNA酶I，和完整的媒体。 </li><li>与乳腺组织从孵化器中取出离心管中，轻轻的脉冲涡流2-3秒2倍。管在450×g下离心5分钟（室温下或4℃），并弃去上清液。 </li><li>将沉淀重悬于10毫升HFAmCl和离心机在450×g下最低为5分钟（室温下或4℃），并弃去上清液。 </li><li>加入3ml胰蛋白酶-EDTA至沉淀中，并充分混合先用5毫升移液管，然后用P1000的微量移液器直到弦（1-3分钟）。添加10毫升HF，离心450×g的5分钟（室温或4°C），弃去上清液。 </li><li>加入2毫升5毫克/毫升Dispase酶和200微升的1毫克/毫升DNA酶I的颗粒和混合1分钟与P1000微量移液器。样品应CLO乌德，但没有粘性的。如果弦，加入100μl的DNA酶I，再次混合。 </li><li>加入10毫升冷HF，通过40微米的细胞过滤器和离心机的压力在450×g的5分钟（室温或4°C），弃去上清液。 </li><li>最终的沉淀重悬在完整的媒体。量化的细胞总数（使用血球计数器或Coulter计数器）。两个乳腺收率约1×10 6〜1×10 7个细胞。板原发性上皮细胞在平底的6孔板中，在1.5×10 5个细胞每孔的密度。 </li></ol> 3。活细胞成像<ol><li>电镀后的细胞，立即安全地放置在6孔板中，在显微镜载物台上，在5％CO 2，/饱和humidity/37°C的温度孵化室内。调整冷凝器柯勒照明和中心的相位环。对于这里提出的实验，一个倒置的尼康的Eclipse TE300/PerkinElmer纺纱影碟共焦显微镜系统在广角与10倍物镜相衬模式。 </li><li>打开图像采集软件，创建和命名时间的推移图像库，并有足够的空间大文件保存到的位置。这里介绍的实验使用Volocity图像采集软件（版本5.3.1，珀金埃尔默，位于Waltham，MA）。 </li><li>允许板在显微镜培养箱平衡为最少15分钟。降低镜片以避免阶段透镜干扰。在“舞台”的标题，选择“校准阶段。”重新获得的焦平面，集Z =零，选择“添加点”下的“舞台”标题下的x，y，z标签和标记成像点。将在6孔板的各孔中的中间点作为相位衬度成像的成像可能会扭曲塑料井的周边附近。排列在4×4个字段（650微米×700微米的字段），每一个与一小部分重叠（约5％）在一个正方形的点。保存选定的点下“雄鹿（五）“ </li><li>在“舞台”，选择“焦点图”，并按照提示设置的每一个点的重点。设置图像采集的定时拍摄4张照片每小时（每15分钟）。这取决于特定的实验的要求，可以调整。保存焦点图下的“舞台”。 </li><li>右键单击右侧工具栏中的“图像采集并保存的成像设置。按下录制按钮开始进行成像。 1小时后，检查的重点的图像，看看是否有任何需要调整。 </li><li>监控和​​持续5天，结束细胞成为合流时，实时成像。 </li></ol> 4。评估的时机和机制的初步上皮细胞集落形成，细胞凋亡的发生率，并逐步形态的变化直接观察时间的推移图片<ol><li>直接观察时间推移图像可以使用任何兼容的视频观看软件（ 如 <a href="http://www.real.com/" target="_blank">HTTP：</a>/ / www.real.com/的Realplayer或其他免费或商业软件）。在这一步后转换为JPG文件（见步骤5），可以查看图像。TIFF格式。 </li><li>要确定初始群体的形成机制，从一个单一的图像堆栈的成像板网站。在初始帧，细胞都会有所上浮。按照串行的图像，以确定当第一上皮细胞粘附到板。在此步骤中，可以识别和上皮细胞从他们更多立方形的形态的成纤维细胞的分化。按照这个单一的上皮细胞，通过序列图像，并按照它的命运在随后的24小时。记录，如果它变得包围额外的上皮细胞进行细胞分裂或细胞凋亡。如果由上皮细胞包围，集落形成的机制可以直接测定，如果周围的细胞来源于漂浮的细胞，然后聚合的初始粘附细胞（s）或通过查看从最初的贴壁细胞的分裂。在第一个24小时的细胞聚集对细胞分裂的菌落形成的记录的数目。 </li><li>最初在特定的时间内贴壁细胞发生凋亡的数量来确定的，按照序列图像的外观经典功能的发展已经贴壁的细胞凋亡（ <a href="http://www.alsa.org/research/about-als-research/cell-death-and-apoptosis.html" target="_blank">http://www.alsa.org/research/about -als-research/cell-death-and-apoptosis.html</a> ），凋亡细胞的数量确定的成像的整个持续时间期间或在特定时间范围内记录。 </li><li>随着时间的推移，上皮细胞的培养改变其形态从最初的长方体形状以与EMT更细长的外观一致。按照序列图像发生这种情况时，确定电镀后的日子/小时。 </li></ol> 5。图片文件修改<ol><l我通过该Volocity软件，导出图像文件到TIFF栈。这里的MetaMorph的显微镜自动化与图像分析软件（Molecular Devices公司，有限责任公司桑尼维尔，CA）用于打开TIFF堆栈文件，然后将它们保存在的MetaMorph单。JPG图像。 </li><li>根据要求蒂姆的跟踪工具软件，重命名图像文件夹和文件使用“重命名2.1.6（ <a href="http://www.softpedia.com/get/System/File-Management/THE-Rename.shtml" target="_blank">http://www.softpedia.com/get/System/File-Management/THE-Rename.shtml</a> ）的一个免费的程序，如重命名文件组。创建一个文件夹中的所有文件夹和文件的实验和命名的文件夹（ 例如： </eM&gt; EXPERIMENTNAME）。 </li><li>创建一个子文件夹中的每个点/位置的文化成像。命名的子文件夹：EXPERIMENTNAME_pPOSITIONNUMBER。的位置应该有3个数字。例：072511RN_p001。把所有的图像文件，子文件夹中的个别位置。 </li><li>添加时间点（5位）和信道数（因为只有一个明亮的磁场通道，使用“0”），在这种格式中的每个文件名的末尾： EXPERIMENTNAME_pPOSITIONNUMBER_tTIMEPOINT_wCHANNELNUMBER.jpgExample：072511RN_p001_t00001_w0.jpg </li><li>每个位置生成一个日志文件，先下载的的免费程序TTTlogfileconverter从TTT网站。启动TTTlogfileconverter程序和选择实验文件夹。输入图像的时间间隔（以秒计）。每15分钟拍摄的照片中，输入900秒。然后按绿色的秘密日志文件按钮。 </li></ol> 6。产生单细胞的细胞命运地图使用TTT <ol><li> Çreate一个称为TTTexport的文件夹和子文件夹称为TTTfiles TTT软件指令后，所有细胞的跟踪数据将被存储</li><li>启动的TTT程序，选择“用户名缩写，单击”继续“。 </li><li>按“设置NAS”，选择创建的用户的“TTTexport”文件夹，然后单击“确定”。在浏览器中，选择一个实验文件夹，选择一个位置文件夹，然后按“加载位置”按钮。 </li><li>眼系数设置为“10倍”。加载所需的图像（建议第一次加载的所有图像）。 </li><li>在单元格编辑器“窗口中，选择”新殖民地“下的”文件“菜单上。在电影“窗口中，按”营业细胞“或F2开始跟踪一个细胞。 </li><li>确定一个单元格中的MOVIE窗口，并用鼠标将光标在单元格。的细胞的数量的圆与应出现后F2已被点击。使用键盘的数字键盘上的“0”键的位置跟踪的细胞和提前至下一张照片。移动光标跟随的每一个细胞每一帧的位置。删除曲目并返回到上一张图像按“Del”的数字键盘上的。要前进的框架没有跟踪的单元格按“3”，并没有向后移动跟踪数字键盘上按“1”。 </li><li>一个细胞分裂标记在电影“窗口中单击”该部门“按钮。为了纪念性细胞死亡在电影“窗口中，单击”细胞死亡“按钮。一旦发生了分裂，子细胞可以在同一个细胞的命运地图上点击右键开始自动跟踪模式的单元格编辑器“窗口中指定的子细胞中的圆圈符号的跟踪。 </li><li>按F10保存的树。每棵树将保存在指定的输出倍ER（TTTExport）。要开始一个新的细胞命运的地图中，选择“新殖民地”下的“文件”，然后“打开”菜单中的“单元格编辑器”窗口中。 </li><li>制表电池的寿命数据后收集在单元格编辑器“窗口中的”单元格数据“选项卡。 </li></ol> 7。统计分析<ol><li>使用任一学生的吨-测试（如果比较两种基因型）或方差分析（如果比较&gt; 2基因型），以确定如果参数数据如初始细胞菌落，电池的寿命或小时数的差异，直到的外观EMT的不同基因型之间有统计学显著。细胞凋亡的频率可以作为数/总数镀敷用Student的t检验或方差分析的细胞或采用Mann-Whitney或Kruskal-Wallis来自作为贴壁细胞的百分比的比较。 </li><li>使用的数据从最初的实验分析，以确定有多少实验重复进行功率测试需要有足够的小号，从每个重复产生的细胞的命运地图tatistical电源使用现有的免费（ 例如 <a href="http://statpages.org/" target="_blank">http://statpages.org/</a> ）。在实验中，来自三只小鼠基因型的细胞培养足够，以确定是否有统计上的显着差异，细胞集落形成和细胞寿命时，特定的基因进行了修改。 </li></ol>

<ol>
	<li>Jones, L. 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=Activation+of+estrogen+signaling+pathways+collaborates+with+loss+of+Brca1+to+promote+development+of+ERalpha-negative+and+ERalpha-positive+mammary+preneoplasia+and+cancer.">Activation of estrogen signaling pathways collaborates with loss of Brca1 to promote development of ERalpha-negative and ERalpha-positive mammary preneoplasia and cancer.</a> Oncogene. 27, 794-802 (2008).</li><li>Burga, L. 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=Altered+proliferation+and+differentiation+properties+of+primary+mammary+epithelial+cells+from+BRCA1+mutation+carriers.">Altered proliferation and differentiation properties of primary mammary epithelial cells from BRCA1 mutation carriers.</a> Cancer Res. 69, 1273-1278 (2009).</li><li>Li, M., Wagner, K., Furth, P. A., Ip, M., Asch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+primary+mammary+epithelial+cells+and+transfection+by+viral+and+nonviral+methods.">Preparation of primary mammary epithelial cells and transfection by viral and nonviral methods.</a> Methods in Mammary Gland Biology and Breast Cancer. , (2000).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+stem-cell-driven+regeneration+in+mammals.">Imaging stem-cell-driven regeneration in mammals.</a> Nature. 453, 345-351 (2008).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+single-cell+imaging+of+mammalian+stem+cells.">Long-term single-cell imaging of mammalian stem cells.</a> Nature Methods. 8, S30-S35 (2011).</li><li>Eilken, H. M., Nishikawa, S. -. I., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+single-cell+imaging+of+blood+generation+from+haemogenic+endothelium.">Continuous single-cell imaging of blood generation from haemogenic endothelium.</a> Nature. 457, 896-900 (2009).</li><li>Kokkaliaris, K. D., Loeffler, D., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+tracking+hematopoiesis+at+the+single-cell+level.">Advances in tracking hematopoiesis at the single-cell level.</a> Current opinion in hematology. , (2012).</li><li>Rieger, M. A., Hoppe, P. S., Smejkal, B. M., Eitelhuber, A. C., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+cytokines+can+instruct+lineage+choice.">Hematopoietic cytokines can instruct lineage choice.</a> Science. 325, 217-218 (2009).</li><li>Rieger, M. A., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+of+lineage+choice+by+hematopoietic+cytokines.">Instruction of lineage choice by hematopoietic cytokines.</a> Cell Cycle. 8, 4019-4020 (2009).</li><li>Heinrich, 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=Generation+of+subtype-specific+neurons+from+postnatal+astroglia+of+the+mouse+cerebral+cortex.">Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex.</a> Nat. Protoc. 6, 214-228 (2011).</li></ol>

作者宣称，他们没有竞争的金融利益。

关键步骤重要的是要确保从小鼠同年龄控制在乳腺上皮细胞的行为与年龄有关的变化，乳腺腺体的收获。当镀敷的细胞，细胞的相同数目的应被镀在各孔中，为每一个实验。细胞应该是相对稀疏时，镀敷，使文化不成为汇合太快使得可以按照多个单独的细胞通过串行图像。至关重要的是，该板是在孵化器中平衡，因为焦点成像之前将更改后的板平衡。一次成像已经开始，应经常...

遗传工程小鼠模型的工具来研究和了解不同的遗传病变患乳腺癌的风险。例如，已经表明，遗传工程小鼠的三个因素的结合：损失的全长1，乳腺癌发病初期（BRCA1基因 ）在乳腺上皮细胞中的基因，肿瘤蛋白p53（TP53）种系单倍剂量不足，和乳腺上皮细胞靶向上调雌激素受体​​α（ERA）的表达在乳腺癌的发展，在100％的BRCA1基因两侧装接loxP（F）11/f11/Mouse乳腺肿瘤病毒（MMTV）-Cre/p53 + / -/tetracycline-operator（ TET-运）-ER/MMTV-reverse“四环素反式激活（rtTA 12个月的年龄在小鼠相比，比例较低报道的 BRCA1 f11/f11/MMTV-Cre/p53 + / -小鼠没有时代的过度表达（约50 - 60％）和BRCA1基因f11/f11/MMTV-Cre没有TP53 haploinsuf的小鼠效率（&lt;5％）。 癌前原发性乳腺上皮细胞的行为的动态时间推移成像揭示在细胞的行为差异，在静态的组织切片，不太容易理解。在细胞增殖和分化的改变主要观察乳腺细胞从人类BRCA1基因突变携带者。2创造的单细胞悬液，主要从正常乳腺上皮细胞和基因工程小鼠产生切除的乳腺组织通过酶的解离。时间的推移观看影像，以评估机制和时机的细胞集落的外观和细胞的形态学变化包括上皮 - 间质转化（EMT）和细胞凋亡的发生率。细胞命运的图的生成，促进细胞分裂（电池寿命），并且确定的细胞分裂的模式之间的时间的长度的定量通过使用单细胞的跟踪。蒂姆“跟踪工具（TTT）是公开可用的软件，用于生成单细胞的命运地图。其效用在阐明细胞命运的机制已经建立4,5检查正常造血干细胞发育6-9和神经元的产生。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> EpiCult-B的基础培养基鼠标</td><td>干细胞技术</td><td> 05610 </td><td></td></tr><tr><td> EpiCult-B扩散补充鼠标</td><td>干细胞技术</td><td> 05612 </td><td></td></tr><tr><td>重组人表皮生长因子（rhEGF） </td><td>干细胞技术</td><td> 02633 </td><td></td></tr><tr><td>胶原酶/透明质酸酶</td><td>干细胞技术</td><td> 07912 </td><td></td></tr><tr><td>一次性Scapels </td><td>羽毛</td><td> 2975第10 </td><td></td></tr><tr><td> Hanks&#39;平衡盐溶液</td><td>干细胞技术</td><td> 37150 </td><td></td></tr><tr><td>一mmonium氯化物</td><td>干细胞技术</td><td> 07800 </td><td></td></tr><tr><td> Tryspin-EDTA </td><td>干细胞技术</td><td> 07901 </td><td></td></tr><tr><td>中性蛋白酶</td><td>干细胞技术</td><td> 07913 </td><td></td></tr><tr><td> DNA酶I </td><td>干细胞技术</td><td> 07900 </td><td></td></tr><tr><td> 40微米的细胞过滤器</td><td>干细胞技术</td><td> 27305 </td><td></td></tr><tr><td> FBS </td><td>干细胞技术</td><td> 06100 </td><td></td></tr><tr><td> PenStrep </td><td> Gibco公司</td><td> 15140 </td><td></td></tr></tbody></table>

time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer

延时成像可以用来比较原代培养的癌前来自不同的遗传工程小鼠模型的乳腺癌的乳腺上皮细胞的行为。例如，之间的时间（电池寿命）的细胞分裂，凋亡细胞的数量，进化形态的变化，和集落形成机制，可以量化和比较携带特定遗传病变的细胞。原发性乳腺上皮细胞培养物产生的未扪及肿瘤的乳腺。有明确的分离从邻近的肌肉，仔细切除腺体，淋巴结被删除，和切碎的乳腺组织，然后用酶解和过滤所产生的丰富的乳腺上皮细胞的单细胞悬液。单细胞悬液，接种和内直接放置在显微镜下活细胞成像的孵化室。十六个650微米×700微米领域的一个4x4的配置每一个W埃尔的6孔板中的成像，每15分钟，连续5天。时间推移的影像检查，直接测量细胞的行为，可以包括细胞集落形成的机理和频率范围内的第一个24小时的电镀细胞（聚集与细胞增殖），细胞凋亡的发生率，并逐步形态的变化。单细胞跟踪是用来产生用于测量个别细胞寿命和调查的细胞分裂模式的细胞命运地图。定量数据进行统计分析，以评估与特定的遗传病变显着的行为差异。

上皮细胞和成纤维细胞，可以区分细胞的形态。上皮细胞有一个长方体形状（ 图1A-B）和形式的细胞集落（ 图1A）。成纤维细胞，基质细胞的类型，有一个细长的形态（ 图1C）。 进行了舍入细胞，并漂浮在成像（ 图2A-D）的发病。连接到该板后，他们变得平坦，并表现出立方形型外观（ 图2E中的H）。按4天的培养?...

Watch this Scientific Journal Video about Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer at JoVE.com

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

时间延时成像是用来评估初级癌前来自乳腺癌风险遗传工程小鼠模型，以确定是否有特定的行为参数之间的相关性和不同的遗传病变的乳腺上皮细胞的行为。

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

time-lapse-imaging-primary-preneoplastic-mammary-epithelial-cells

Research

JoVE Journal

Medicine

11.8K 次观看.  Georgetown University. 时间延时成像是用来评估初级癌前来自乳腺癌风险遗传工程小鼠模型，以确定是否有特定的行为参数之间的相关性和不同的遗传病变的乳腺上皮细胞的行为。

视频: Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

科研

医学

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

跟踪乳腺建筑特色与检测乳腺癌的磁共振扩散张量成像

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...