这项研究是由美国国防部支持的授予TSA 140010到MK和BC 111038到MMM由作者本人观点和意见，推荐并不会反映这些美国陆军和国防部的。

所有动物研究已经批准了机构动物护理和德州理工大学健康科学中心使用委员会，并遵循"指南实验动物的护理和使用"由美国国立卫生研究院发表在列出的各项规​​定。用八到十二周龄的雌性BALB / c小鼠是市售的。注射1×10 5个 4T1或1×10 5个 4T1细胞表达GFP，可从不同的供应商处购买，到乳腺脂肪垫。 1. 4T1文化和4T1-GFP的细胞和肿瘤细胞悬浮用于注射14准备<ol><li> 4T1和4T1-GFP细胞培养 注:除非另有规定执行在层流（LAF）生物安全柜使用无菌溶液的所有步骤。 <ol><li>保持4T1和4T1-GFP细胞中的RPMI培养基中补充有10％热灭活胎牛血清和100U / ml青霉素G和100微克/ ml硫酸链霉素。 注:由细胞的每一个胰蛋白酶和电镀计算确定的通道数。使用的细胞具有相同的通道编号，以所有小鼠实验中是重要的，因为通道的数目影响细胞的致瘤性和转移潜能。 </li><li>在此之前细胞注射去除T75厘米2瓶，含有肿瘤细胞在80％汇合，从孵化器和吸出网上平台。加入10毫升的PBS并轻轻旋转烧瓶洗出剩余的培养基中，然后吸PBS中。 </li><li>用EDTA加2ml的0.25％胰蛋白酶的烧瓶中并倾斜它均匀地在表面上，在37℃，5％CO 2的分发溶液孵育在湿润的培养箱中3分钟。 </li><li>点击烧瓶，以促进细胞的分离和加入8毫升新鲜培养基。移液器上下数次破坏团块，并获得单细胞悬浮液。 </li><li>离心细胞5分钟。在室温500 XG。吸出上清液并洗涤细胞在10ml PBS中。 </li><li>移液器上下数次破坏团块，并获得单细胞悬浮液。取100微升细胞悬浮液，并使用细胞活力分析仪按照制造商的说明计数细胞。 </li><li>计算公式使用所有注射所需的细胞数量:每个实验中使用的小鼠的鼠X＃10 5个细胞（总是准备好备用电池是在安全方面）。 </li><li>离心机的细胞在1.1.5。除去通过在新鲜的PBS的量抽吸和悬浮细胞上清液需取得的1×10 6个细胞/ ml的最终细胞浓度。保持冰细胞悬液，直到准备注射。 </li></ol></li><li> 4T1或4T1-GFP细胞注射鼠程序 <ol><li>肿瘤细胞注射前一天，麻醉小鼠，用3％异氟醚的感应腔室。一旦老鼠是睡着了，经常呼吸，地方Ť他鼠标上的手术垫与鼻锥内鼻，并把它连接到异氟烷蒸发器。保持用2.5％异氟烷麻醉。捏住鼠标脚趾以确保该鼠标被麻醉。 </li><li>用棉签将注射部位（右胸部乳房脂肪垫）应用脱毛膏，等待2分钟。清洁用的湿纸巾的网站，将鼠标放回笼子里，直到它重新获得足够的意识，以保持胸骨斜卧监视动物。 </li><li>上注射当天，麻醉小鼠为在1.2.2和放置在手术垫。 </li><li>涡含有肿瘤细胞以获得均匀的细胞悬浮液的管中。抽吸100微升细胞悬浮液，含有1×10 5个肿瘤细胞，成0.5 ml胰岛素注射器（29克，12.7mmol毫米针长度）。 </li><li>解除用拇指和手指指数靠近第2 次和第3 次乳头的皮肤，将注射器的针头插入mammarŸ脂肪垫只是手指之间的皮肤下的第三个乳头下方，并注入细胞慢慢形成泡沫（皮下注射）。 </li><li>将鼠标放回注射后的笼子和监测在1.2.2。 </li></ol></li><li> 监测肿瘤生长 <ol><li> 卡尺测量14 注:注入4T1或4T1-GFP细胞形成积极的乳腺肿瘤。细胞接种后5 - 通常情况下，可触及的肿瘤在第4天出现〜。 4T1-GFP的细胞形成生长速度慢，后来给转移瘤。测量肿瘤，每周二至三次。如果动物的临床状况恶化时，动物损失超过10％的体重，肿瘤超过体重的10％或变得溃烂，立即安乐死的小鼠。 <ol><li>麻醉小鼠如在1.2.1。，称重，放置在手术垫和湿用70％乙醇的区域。 </li><li> （ - 5天后细胞接种4）在注射部位的触诊肿瘤。 </li> &lt;利&gt;测量的最大直径（D）和更小的直径（d1）的带厚度。 </li><li>使用式体积=（DX D1点¯xD1）/ 2 mm 3的计算肿瘤体积。监控麻醉小鼠恢复为1.2.2。 </li></ol></li><li> 成像（仅适用于4T1 GFP的细胞） <ol><li>麻醉小鼠为1.2.1。将鼠标在成像仪器内部的可动阶段。保持通过鼻锥麻醉。 </li><li>打开显像仪和光源按照制造商的说明。 </li><li>在标签"收购"到"照明"，并切换到白光空位置（没有过滤器）。确保光引擎为ON，并在光表中选择"跨"。 </li><li>在标签"显微镜"，选择"清除"排放过滤器和0.5X放大倍率。在"摄像头"选项卡上，确保分档捕获为1×1"和预览"4×4"。 </l我&gt; <li>点击预览来定位白光肿瘤，着力得到清晰的图像，然后单击捕获。 </li><li>进入"采集"选项卡并更改灯光过滤1（过滤器GFP按照制造商的说明）。在"显微镜"选项卡中，发射滤光片变更为"广发二十零分之五百三十○"。预览和捕捉图像中的绿色通道。调整曝光时间来获得合适的亮度。 </li><li>应用伪彩色的图像，并保存在相应的文件夹（ 图1）。 </li></ol></li></ol></li></ol> 2.脂质体的10鼻腔给药注:除非另有规定执行在层流（LAF）生物安全柜使用无菌溶液的所有步骤。 <ol><li>在肿瘤细胞注射后6天麻醉小鼠为1.2.1。 </li><li> VORTEX从制造商获得的氯膦酸盐或控制（PBS）脂质体悬浮液。取60微升脂质体小号uspension使用无菌枪头。 </li><li>缓慢释放的脂质体溶液（每次5微升）鼻孔允许鼠标在溶液中呼吸附近，重复直到60微升整个剂量给药。 </li><li>让鼠标把它放回笼子前恢复知觉。 </li><li>重复脂质体给药，每3天，直到处死小鼠。不要牺牲的日子管理脂质体。 </li></ol> 3.鼠标牺牲和组织收集注:使用高压灭菌和鼠标清扫消毒器械。安乐死而下，通过重要器官的放血和去除小鼠麻醉。 <ol><li>在第22天 （为4T1细胞）或第26天（对于4T1-GFP细胞）的肿瘤细胞注射后麻醉小鼠如在1.2.1。和放置在手术垫与连接到汽化器鼻锥，维持麻醉如在1.2.1。捏脚趾之一，以确保第m乌斯是无意识的。 </li><li>针用针给的夹层​​板的脚趾。喷雾小鼠皮肤上乙醇。 </li><li>使用镊子提起皮肤做一个切口用手术剪。慢慢地露出腹膜，并继续通过胸部切皮，直到脖子。 </li><li>用手术剪使腹膜切口，并用棉技巧避免对血管的损害仔细暴露器官。 </li><li>移动器官一边，露出了下腔静脉。穿刺静脉采血与29克的胰岛素注射器。放置收集血液到管中，并在冰上进一步处理离开。 </li><li>切割膜片露出肋骨，肺和心脏。慢慢地切断两侧使用骨刀两肋骨和解除胸腔的顶部。抓住用钳子心脏和削减结缔组织心脏和肺连接到胸腔。 </li><li>放置在PBS少量肺部在60毫米冰皮氏培养皿，直到准备成像和进一步加工成冰冻切片的免疫荧光或常规组织学[包括伊红（H＆E）染色。 </li></ol> 4.肺转移瘤的评价<ol><li> 计数和评分表面转移 <ol><li>放置培养皿用解剖显微镜下肺部。重点以获得肺表面的一个明确的说法。 </li><li>使用解剖显微镜观察肺表面。指望两肺的前部和后部侧转移。 注:正常肺是白色或粉红色和多孔，具有结构类似海绵。 4T1转移是固体和无孔，有时会出现为类似于液体（ 图2A）的下降。 </li></ol></li><li> 龙成像 <ol><li>放置培养皿在成像仪器内部的可移动台肺部。 </li><li>打开仪器和biolite多光谱源。打开软件。在该选项卡，"收购"，进入"照明"，并切换到白光空位置（没有过滤器）。光引擎 - &gt; ON，轻表 - &gt;外延。在标签"显微镜"选择"清除"的发射滤波器和1.66X的放大倍率。在"摄像头"选项卡上，确保分档捕获为1×1"和预览"4×4"。 </li><li>点击预览和重点得到肺部白光清晰的画面，然后单击捕获。 </li><li>进入"采集"选项卡，并更改过滤器1（过滤器GFP按照制造商的说明）。在"显微镜"选项卡中，发射滤光片变更为"广发二十零分之五百三十○"。预览和捕捉图像中的绿色通道。调整曝光时间来获得合适的亮度。 </li><li>适用伪的图像并保存在相应的文件夹。 </li><li>翻转肺部得到对方的图像以类似的方式。这PROCedure生成可使用适当的软件（ 图2B）14被进一步定量GFP阳性转移图像。 </li></ol></li><li> 免疫荧光 <ol><li>固定在4％低聚甲醛的肺，在4℃在黑暗4小时。 </li><li>用10毫升的PBS洗两次组织了5分钟后，完全去除固定液。转移到18％蔗糖的PBS中并在4℃下孵育O / N。 </li><li>从蔗糖中取出组织和轻拍多余。 </li><li>以一个cryomold，覆盖华侨城介质底层。放置OCT介质上的组织。填写与华侨城中等模具完全覆盖干冰组织和地点。 </li><li>在-80°C，直到切片存储块。 </li><li>准备5微米与带电幻灯片低温恒温器和地点厚的部分。 </li><li>在-80°C，直到进一步使用存放不用的幻灯片。 </li><li>空气干燥的幻灯片7分钟。并用PBS洗涤以除去倍频程WIPe为薄纸或纸巾滑动以除去过量的水，装入盖玻片带安装含有DAPI培养基并用显微镜下的GFP滤波器可视化。 </li><li>量化的GFP转移（ 图3A）与使用适当的软件14。 </li></ol></li><li> 组织学和数字病理学 <ol><li>点击手动加载按钮的开始选项卡下，等待滑动保持架从扫描器弹出。 </li><li>拿H＆E染色的组织切片，用纸巾擦拭，除去灰尘，并安全地将其放置在滑动保持架。 </li><li>请在弹出的窗口中幻灯片描述。 </li><li>选择SS成像控制台窗口中的扫描区域选项卡并调整绿化广场来定义感兴趣区域（ROI）的区域进行扫描。 </li><li>蓝色校准金刚石拖至幻灯片，其中，组织为不存在，但是，在ROI内的一个透明部分。 </li><li>接下来，选择焦点Points选项卡，并单击在ROI组织自动选择按钮来获得几个焦点（黄色加号）。 </li><li>如果有必要，手动将额外的焦点由ROI内双击。 </li><li>继续执行校准选项卡并选择校准按钮蓝色钻石校准点开始幻灯片校准。校准完成后，将显示从校准点的图像。 </li><li>检查这一形象，以确保它是明确的，无污垢或文物。 </li><li>继续扫描选项卡，选择开始扫描开始ROI的扫描。扫描组织切片被转换为数字滑动（ 图3B和图4A） </li><li>如前所述（ 图4B）14量化转移性负担。 </li></ol></li></ol> 5.流式细胞仪（FACS）肺泡巨噬细胞在肺内分析<ol><li> 单细胞Suspensi的制备上<ol><li>成像和转移的数量后，洗肺用无菌PBS和放置在培养皿中。使1 - 2毫米使用手术剪刀和镊子肺部件。 </li><li>转移肺件15毫升锥形管用3ml含有1mg / ml胶原酶P，为0.04mg / ml的DNA酶I消化缓冲液中，10微克/ ml胰蛋白酶抑制剂溶于RPMI培养基（无菌）。 </li><li>孵育在孵化器的旋转摇动器的锥形管在37℃下40分钟。 </li><li>从培养箱中取出管锥和吸取的溶液上下离解组织。 </li><li>通过40微米的过滤器，以避免团块，并获得单细胞悬浮液的溶液。如果需要的话，崩解更好的消化的组织按组织针对与针筒柱塞的帮助下细胞过滤。 </li><li>当一个单细胞悬浮液已经实现了，通过离心收集细胞并裂解红血细胞与加入2mL ACK缓冲液中，在室温10分钟。然后，洗颗粒两次于8ml RPMI中。 </li><li>在3ml的完全RPMI培养基和悬浮细胞进行细胞使用细胞活力分析仪按照制造商的说明计数。 </li></ol></li><li> 肺细胞染色表面标志 注:在4℃执行所有孵化。在500 XG和4℃离心平板。 <ol><li>吸管1×10 6个细胞在100μlFACS缓冲液（PBS中的1％FBS）中，以V型底96孔板的各个孔中的。使用V型底96孔板便于多个样品的装卸。 </li><li>预孵育用1μg小鼠Fc块的细胞悬浮液纯化的抗小鼠CD16 / CD32在100μlFACS缓冲液中15分钟。离心V型底96孔板以沉淀细胞并通过翻转板并让溶液漏极去除上清。 </li><li>用于表面染色，在预先稀释的抗体murin在一个管（预混）电子抗原:BV605 CD45（30-F11），PE的CD11b（M1 / 70），PE / Cy7的F4 / 80（BM8），APC / Cy7的表面CD11c（N418），PerCPCy5.5 I A / I E（MHCII）（M5 / 114.152），PE CD80（16-10A1），AF 647 CD86（GL-1）至在FACS缓冲液含有10微克/毫升的Fc块CD16的2.5微克/ ml的终浓度/ CD32抗体。 注意:此组的抗体是用于识别和肺泡巨噬细胞和树突状细胞的功能状态的评估是有用的。 </li><li>加入100μl稀释抗体（母液）与V型底96孔板的孔中并在4℃温育30分钟。 </li><li>离心板沉淀细胞并去除上清5.2.2。洗涤细胞用200μlFACS缓冲液中，离心以500g 5分钟。除去上清液为5.2.2。和悬浮细胞在200微升的PBS。 </li><li>离心板以沉淀细胞，并添加在PBS中（1:1000）稀释活力染料。孵育在4℃20分钟。&lt;/ LI&gt; <li>离心板沉淀细胞，去除上清为5.2.2。用200微升PBS洗，并再次离心板。 </li><li>悬浮细胞在4℃下加入100μl的1％多聚甲醛和存储的，直到获得通过FACS仪器。此前收购转移细胞悬液的聚丙烯流式细胞仪管。 </li></ol></li></ol> 6. FACS数据分析:门控策略及细胞亚群的鉴定<ol><li>如先前报道10进行FACS分析。门收购了基于前向（FSC）（SSC）细胞/事件和侧散射;然后门生活和CD45 +细胞。对于肺泡巨噬细胞识别，门的CD11b阴性，然后CD11c的+ F4 / 80 +细胞。 （ 图5A）。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages.">Tissue-resident macrophages.</a> Nat Immunol. 14, 986-995 (2013).</li><li>Holt, P. G., Strickland, D. H., Wikstrom, M. E., Jahnsen, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+immunological+homeostasis+in+the+respiratory+tract.">Regulation of immunological homeostasis in the respiratory tract.</a> Nat Rev Immunol. 8, 142-152 (2008).</li><li>Sharma, S. 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=Pulmonary+alveolar+macrophages+contribute+to+the+premetastatic+niche+by+suppressing+antitumor+T+cell+responses+in+the+lungs.">Pulmonary alveolar macrophages contribute to the premetastatic niche by suppressing antitumor T cell responses in the lungs.</a> J. Immunol. 194, 5529-5538 (2015).</li><li>Pulaski, B. A., Ostrand-Rosenberg, S., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+4T1+breast+tumor+model.">Mouse 4T1 breast tumor model.</a> Current protocols in immunology. , (2001).</li><li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+building+a+framework.">Cancer metastasis: building a framework.</a> Cell. 127, 679-695 (2006).</li><li>Buiting, A. M., Van Rooijen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+mediated+depletion+of+macrophages:+an+approach+for+fundamental+studies.">Liposome mediated depletion of macrophages: an approach for fundamental studies.</a> J. Drug Target. 2, 357-362 (1994).</li><li>Vadrevu, S. 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=Complement+c5a+receptor+facilitates+cancer+metastasis+by+altering+T-cell+responses+in+the+metastatic+niche.">Complement c5a receptor facilitates cancer metastasis by altering T-cell responses in the metastatic niche.</a> Cancer Res. 74, 3454-3465 (2014).</li><li>Walrath, J. C., Hawes, J. J., Van Dyke, T., Reilly, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+mouse+models+in+cancer+research.">Genetically engineered mouse models in cancer research.</a> Adv. Cancer Res. 106, 113-164 (2010).</li><li>Bosiljcic, 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=Myeloid+suppressor+cells+regulate+the+lung+environment--letter.">Myeloid suppressor cells regulate the lung environment--letter.</a> Cancer Res. 71, 5050-5051 (2011).</li><li>Yan, H. 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=Myeloid+Suppressor+Cells+Regulate+the+Lung+Environment-Response.">Myeloid Suppressor Cells Regulate the Lung Environment-Response.</a> Cancer Res. 71, 5052-5053 (2011).</li><li>Van Rooijen, N., Sanders, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+mediated+depletion+of+macrophages:+mechanism+of+action,+preparation+of+liposomes+and+applications.">Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications.</a> J. Immunol. Methods. 174, 83-93 (1994).</li></ol>

作者宣称，他们没有竞争的经济利益。

The recent insights into cancer biology and causative factors involved in carcinogenesis and tumor progression lead to development of genetically engineered mouse (GEM) models of cancer, in which tumors grow spontaneously, usually over a period of several months15. Although these tumor models appear to reflect better the natural history of human malignancies than xenografts or syngeneic models, much time required for tumor development and various degrees of malignant phenotype penetrance limit the use of these...

The premetastatic niche is an important process in cancer metastasis defined as a set of alterations that occur in the organs that are targets for metastases prior to arrival of tumor cells1,2. Therefore, therapeutic targeting of this step of cancer progression may prevent metastases to the vital organs that cause approximately 90% of cancer-associated deaths. Although the concept of the premetastatic niche, also known as the "seed and soil" theory, was introduced more than a century ago, no experimental proof has been provided until recently, when the bone marrow-derived cells were demonstrated to contribute to the premetastatic soil1,3-7. Despite these developments, the premetastatic niche remains a largely understudied aspect of cancer pathophysiology and further research to identify cellular players and mechanisms involved is needed. 
Here we report the in vivo approaches to study the role of alveolar macrophages in breast cancer metastases and the lung premetastatic niche. The alveolar macrophages arrive to the lungs early during the embryonic development and self-renew there during adulthood8. They also have important immunomodulatory and homeostatic functions including the protection of this organ from undesired inflammatory responses to the environmental innocuous antigens9. Therefore, we hypothesize that tumors exploit this physiological immunosuppression, imposed by alveolar macrophages, and, consequently, alveolar macrophages contribute to the lung premetastatic niche by suppressing antitumor immunity. This hypothesis is supported by our recent report demonstrating that the specific depletion of these cells reduces lung metastases and enhances antitumor T cell responses10. 
For these studies we apply a well-established syngeneic model of breast cancer (4T1), which mimics stage IV metastatic breast cancer11; and has been previously reported in studies of the premetastatic niche6. To track metastasizing tumor cells in vivo we use 4T1 cells expressing GFP (4T1-GFP) in conjunction with animal imaging and confocal microscopy. We focus on the lung premetastatic niche, since this organ is one of the most common targets of hematogenous metastases of human malignancies12. To investigate functions of alveolar macrophages in the premetastatic niche, we use clodronate liposomes to deplete these cells13; and evaluate impact of this depletion on lung metastases. Of note, this method specifically depletes alveolar macrophages but no other phagocytic cells in the lungs or in circulation10.

<table border="0" cellpadding="0" cellspacing="0" width="1155">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">4T1 cell line&#160;</td>
			<td style="width:417px;">American Type Culture Collection, Manassas, VA, USA</td>
			<td style="width:139px;">CRL 2539</td>
			<td style="width:287px;">Tumor cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4T1-GFP cell line</td>
			<td>Caliper life sciences/ Perkin Elmer, Waltham, MA, USA</td>
			<td>BW128090</td>
			<td>Tumor cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Corning, Corning, NY, USA</td>
			<td>10-040-CM</td>
			<td>Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat inactivated FBS</td>
			<td>Gibco (Thermo Scientific), USA</td>
			<td>10082147</td>
			<td>Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin Streptomycin</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>MT-300-02-CI</td>
			<td>Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>BP399-20</td>
			<td>Dilute with distilled water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin 0.25% with EDTA</td>
			<td>Hyclone, Logan, Utah, USA</td>
			<td>SH30042.02</td>
			<td>Tissue culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T75 cm2 flask</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>12-565-32</td>
			<td>Tissue culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15ml conical tube</td>
			<td>BD falcon, Franklin Lakes, NJ, USA</td>
			<td>352096</td>
			<td>Tissue culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50ml conical tube</td>
			<td>BD falcon, Franklin Lakes, NJ, USA</td>
			<td>352098</td>
			<td>Tissue culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm2 Petri dish</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>AS4052</td>
			<td>For lung imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane (Isothesia)</td>
			<td>Butler Schein Animal health, Dublin, OH, USA</td>
			<td>NDC 11695-6776-2</td>
			<td>Mouse anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clodronate liposomes</td>
			<td>Formumax Scientific Inc, Palo Alto, CA, USA</td>
			<td>F70101C-N</td>
			<td>Macrophages depletion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Control liposomes</td>
			<td>Formumax Scientific Inc, Palo Alto, CA, USA</td>
			<td>F70101-N</td>
			<td>Control PBS-liposomes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">29 gauge insulin syringes (12.7 mm and 0.5 ml capacity)- Reli-On</td>
			<td>Walmart, Bentonville, AR, USA</td>
			<td></td>
			<td>For tumor cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair removal cream (Nair)</td>
			<td>Walmart, Bentonville, AR, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde solution (4%)</td>
			<td>Affymetrix, Santa Clara, CA, USA</td>
			<td>19943-I Lt</td>
			<td>Dilute to 4% or 1% using 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT compound</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>230-730-571</td>
			<td>For freezing tissue in cryomolds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoro-Gel-II with DAPI</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>17985-51</td>
			<td>Mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma, St. Louis, MO, USA</td>
			<td>S-9378</td>
			<td style="width:287px;">Cryopreservation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase P</td>
			<td>Roche, Basel, Switzerland</td>
			<td>11249002001</td>
			<td>Components of tissue digestion buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnase I</td>
			<td>Roche, Basel, Switzerland</td>
			<td>10104159001</td>
			<td>Components of tissue digestion buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor</td>
			<td>Sigma, St. Louis, MO, USA</td>
			<td>T9253</td>
			<td>Components of tissue digestion buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 micron cell strainers</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>22-363-547</td>
			<td>Used in tissue digestion to remove clumps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trustain FcX-Fc Block (CD16/CD32)</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>101320</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BV605 CD45</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>103139</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE CD11b</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>101207</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Cy7 F4/80&#160;</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>123113</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC/Cy7 CD11c</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>117323</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCPcy5.5 IA/IE (MHCII)&#160;</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>107625</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE CD80</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>104707</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AF647 CD86</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>105019</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixable viability Dye eflour 506</td>
			<td>eBioscience, San Diego, CA,USA</td>
			<td>65-0866</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica Biosystems, Buffalo Grove, IL, USA</td>
			<td>CM1850</td>
			<td>Cryosectioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UVP iBox Explorer</td>
			<td>UVP Inc, Upland, CA, USA</td>
			<td></td>
			<td>Mouse and lung fluorescent imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aperio Scanscope CS</td>
			<td>Leica Biosystems, Buffalo Grove, IL, USA</td>
			<td></td>
			<td>Digital pathology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD LSRFortessa&#160;</td>
			<td>BD Biosciences, Franklin Lakes, NJ, USA</td>
			<td></td>
			<td>Flow cytometry/data acquisition</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Nikon A1 confocal TE2000 microscope</td>
			<td>Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A.</td>
			<td></td>
			<td style="width:287px;">Imaging and quantifying GFP fluorescence in lung cryosections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">UVP visionworks software (Version 7.1RC3.38)</td>
			<td style="width:417px;">UVP Inc, Upland, CA, USA</td>
			<td style="width:139px;"></td>
			<td>Imaging software for iBOX</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Aperio Imagescope software (v12.1.0.5029)&#160;</td>
			<td>Leica Biosystems, Buffalo Grove, IL, USA</td>
			<td></td>
			<td style="width:287px;">Imaging software for analysis of digital slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow JO software (version 9.8.1)</td>
			<td>Flow JO LLC, Ashland, OR, USA</td>
			<td></td>
			<td>Analysis of flow cytometric data</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">NIS Elements AR (version 4.20.01) 64 Bit</td>
			<td>Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A.</td>
			<td style="width:139px;"></td>
			<td style="width:287px;">Acquisition and analysis of lung cryosections for GFP&#160;</td>
		</tr>
	</tbody>
</table>

studying the role of alveolar macrophages in breast cancer metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

4T1-GFP肿瘤细胞注射到乳腺脂肪垫导致了概括人乳腺癌的转移扩散小鼠肿瘤（ 图1A）的形成，如转移灶在肺中（ 图2），肝脏，骨骼迅速地形成和老鼠11大脑。 4T1细胞用GFP稳定转染促进肿瘤生长的监测（ 图1B），跟踪转移性肿瘤细胞和定量转移负担（ 图2B，3B）。另外，肿瘤的成像提供关于几种病理特征如肿瘤细胞...

Watch this Scientific Journal Video about Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis at JoVE.com

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

研究肺泡巨噬细胞的作用乳腺癌转移

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

studying-the-role-of-alveolar-macrophages-in-breast-cancer-metastasis

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Medicine

9.6K 次观看.  Texas Tech University Health Science Center. Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used. 

视频: Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

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

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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 ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型:转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

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 ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...