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>

<ol><li>Sceneay, J., Smyth, M. J., Moller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pre-metastatic+niche%3A+finding+common+ground%5BTitle%5D)+AND+%22Cancer+Metastasis+Rev%22%5BJournal%5D)">The pre-metastatic niche: finding common ground.</a> Cancer Metastasis Rev. , (2013).</li>
<li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pathogenesis+of+cancer+metastasis%3A+the+%27seed+and+soil%27+hypothesis+revisited%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited.</a> Nat Rev Cancer. 3, 453-458 (2003).</li>
<li>Kaplan, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VEGFR1-positive+haematopoietic+bone+marrow+progenitors+initiate+the+pre-metastatic+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.</a> Nature. 438, 820-827 (2005).</li>
<li>Hiratsuka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MMP9+induction+by+vascular+endothelial+growth+factor+receptor-1+is+involved+in+lung-specific+metastasis%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis.</a> Cancer Cell. 2, 289-300 (2002).</li>
<li>Hiratsuka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+S100A8-serum+amyloid+A3-TLR4+paracrine+cascade+establishes+a+pre-metastatic+phase%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase.</a> Nat Cell Biol. 10, 1349-1355 (2008).</li>
<li>Yan, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gr-1%2BCD11b%2B+myeloid+cells+tip+the+balance+of+immune+protection+to+tumor+promotion+in+the+premetastatic+lung%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Gr-1+CD11b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung.</a> Cancer Res. 70, 6139-6149 (2010).</li>
<li>Gao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myeloid+progenitor+cells+in+the+premetastatic+lung+promote+metastases+by+inducing+mesenchymal+to+epithelial+transition%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Myeloid progenitor cells in the premetastatic lung promote metastases by inducing mesenchymal to epithelial transition.</a> Cancer Res. 72, 1384-1394 (2012).</li>
<li>Davies, L. C., Jenkins, S. J., Allen, J. E., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-resident+macrophages%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((Regulation+of+immunological+homeostasis+in+the+respiratory+tract%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">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/pubmed?term=((Pulmonary+alveolar+macrophages+contribute+to+the+premetastatic+niche+by+suppressing+antitumor+T+cell+responses+in+the+lungs%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">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., et al. Mouse 4T1 breast tumor model. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPulaski%2C+BA+%22Current+protocols+in+immunology%22">Current protocols in immunology</a>. Coligan, J. E., et al. , Chapter 20, Unit 20 22 (2001).</li>
<li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+metastasis%3A+building+a+framework%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Liposome+mediated+depletion+of+macrophages%3A+an+approach+for+fundamental+studies%5BTitle%5D)+AND+%22J.+Drug+Target%22%5BJournal%5D)">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/pubmed?term=((Complement+c5a+receptor+facilitates+cancer+metastasis+by+altering+T-cell+responses+in+the+metastatic+niche%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Genetically+engineered+mouse+models+in+cancer+research%5BTitle%5D)+AND+%22Adv.+Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Myeloid+suppressor+cells+regulate+the+lung+environment--letter%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Myeloid suppressor cells regulate the lung environment--letter.</a> Cancer Res. 71, author reply 5052-5053 5050-5051 (2011).</li>
<li>Yan, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myeloid+Suppressor+Cells+Regulate+the+Lung+Environment-Response%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Liposome+mediated+depletion+of+macrophages%3A+mechanism+of+action%2C+preparation+of+liposomes+and+applications%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">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><tbody><tr><td>4T1 cell line&amp;nbsp;</td><td>American Type Culture Collection, Manassas, VA, USA</td><td>CRL 2539</td><td>Tumor cells</td></tr><tr><td>4T1-GFP cell line</td><td>Caliper life sciences/ Perkin Elmer, Waltham, MA, USA</td><td>BW128090</td><td>Tumor cells</td></tr><tr><td>RPMI</td><td>Corning, Corning, NY, USA</td><td>10-040-CM</td><td>Media</td></tr><tr><td>Heat inactivated FBS</td><td>Gibco (Thermo Scientific), USA</td><td>10082147</td><td>Media</td></tr><tr><td>Penicillin Streptomycin</td><td>Fisher Scientific, Waltham, MA, USA</td><td>MT-300-02-CI</td><td>Media</td></tr><tr><td>PBS</td><td>Fisher Scientific, Waltham, MA, USA</td><td>BP399-20</td><td>Dilute with distilled water</td></tr><tr><td>Trypsin 0.25% with EDTA</td><td>Hyclone, Logan, Utah, USA</td><td>SH30042.02</td><td>Tissue culture supplies</td></tr><tr><td>T75 cm&lt;sup&gt;2&lt;/sup&gt; flask</td><td>Fisher Scientific, Waltham, MA, USA</td><td>12-565-32</td><td>Tissue culture supplies</td></tr><tr><td>15 ml conical tube</td><td>BD falcon, Franklin Lakes, NJ, USA</td><td>352096</td><td>Tissue culture supplies</td></tr><tr><td>50 ml conical tube</td><td>BD falcon, Franklin Lakes, NJ, USA</td><td>352098</td><td>Tissue culture supplies</td></tr><tr><td>60 mm2 Petri dish</td><td>Fisher Scientific, Waltham, MA, USA</td><td>AS4052</td><td>For lung imaging&amp;nbsp;</td></tr><tr><td>Isoflurane (Isothesia)</td><td>Butler Schein Animal health, Dublin, OH, USA</td><td>NDC 11695-6776-2</td><td>Mouse anesthesia</td></tr><tr><td>Clodronate liposomes</td><td>Formumax Scientific Inc, Palo Alto, CA, USA</td><td>F70101C-N</td><td>Macrophages depletion</td></tr><tr><td>Control liposomes</td><td>Formumax Scientific Inc, Palo Alto, CA, USA</td><td>F70101-N</td><td>Control PBS-liposomes</td></tr><tr><td>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><td>Hair removal cream (Nair)</td><td>Walmart, Bentonville, AR, USA</td><td></td><td></td></tr><tr><td>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><td>OCT compound</td><td>Fisher Scientific, Waltham, MA, USA</td><td>230-730-571</td><td>For freezing tissue in cryomolds</td></tr><tr><td>Fluoro-Gel-II with DAPI</td><td>Electron Microscopy Sciences, Hatfield, PA, USA</td><td>17985-51</td><td>Mounting medium</td></tr><tr><td>Sucrose</td><td>Sigma, St. Louis, MO, USA</td><td>S-9378</td><td>Cryopreservation</td></tr><tr><td>Collagenase P</td><td>Roche, Basel, Switzerland</td><td>11249002001</td><td>Components of tissue digestion buffer</td></tr><tr><td>Dnase I</td><td>Roche, Basel, Switzerland</td><td>10104159001</td><td>Components of tissue digestion buffer</td></tr><tr><td>Trypsin inhibitor</td><td>Sigma, St. Louis, MO, USA</td><td>T9253</td><td>Components of tissue digestion buffer</td></tr><tr><td>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><td>Trustain FcX-Fc Block (CD16/CD32)</td><td>Biolegend, San Diego, CA, USA</td><td>101320</td><td>Antibodies for flow cytometry</td></tr><tr><td>BV605 CD45</td><td>Biolegend, San Diego, CA, USA</td><td>103139</td><td>Antibodies for flow cytometry</td></tr><tr><td>PE CD11b</td><td>Biolegend, San Diego, CA, USA</td><td>101207</td><td>Antibodies for flow cytometry</td></tr><tr><td>PE Cy7 F4/80&amp;nbsp;</td><td>Biolegend, San Diego, CA, USA</td><td>123113</td><td>Antibodies for flow cytometry</td></tr><tr><td>APC/Cy7 CD11c</td><td>Biolegend, San Diego, CA, USA</td><td>117323</td><td>Antibodies for flow cytometry</td></tr><tr><td>PerCPcy5.5 IA/IE (MHCII)&amp;nbsp;</td><td>Biolegend, San Diego, CA, USA</td><td>107625</td><td>Antibodies for flow cytometry</td></tr><tr><td>PE CD80</td><td>Biolegend, San Diego, CA, USA</td><td>104707</td><td>Antibodies for flow cytometry</td></tr><tr><td>AF647 CD86</td><td>Biolegend, San Diego, CA, USA</td><td>105019</td><td>Antibodies for flow cytometry</td></tr><tr><td>Fixable viability Dye eflour 506</td><td>eBioscience, San Diego, CA,USA</td><td>65-0866</td><td>Antibodies for flow cytometry</td></tr><tr><td>Cryostat</td><td>Leica Biosystems, Buffalo Grove, IL, USA</td><td>CM1850</td><td>Cryosectioning</td></tr><tr><td>UVP iBox Explorer</td><td>UVP Inc, Upland, CA, USA</td><td></td><td>Mouse and lung fluorescent imaging</td></tr><tr><td>Aperio Scanscope CS</td><td>Leica Biosystems, Buffalo Grove, IL, USA</td><td></td><td>Digital pathology</td></tr><tr><td>BD LSRFortessa&amp;nbsp;</td><td>BD Biosciences, Franklin Lakes, NJ, USA</td><td></td><td>Flow cytometry/data acquisition</td></tr><tr><td>Nikon A1 confocal TE2000 microscope</td><td>Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A.</td><td></td><td>Imaging and quantifying GFP fluorescence in lung cryosections</td></tr><tr><td>UVP visionworks software (Version 7.1RC3.38)</td><td>UVP Inc, Upland, CA, USA</td><td></td><td>Imaging software for iBOX</td></tr><tr><td>Aperio Imagescope software (v12.1.0.5029)&amp;nbsp;</td><td>Leica Biosystems, Buffalo Grove, IL, USA</td><td></td><td>Imaging software for analysis of digital slides</td></tr><tr><td>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><td>NIS Elements AR (version 4.20.01) 64 Bit</td><td>Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A.</td><td></td><td>Acquisition and analysis of lung cryosections for GFP&amp;nbsp;</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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.7K 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

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

内髂动脉注射显微骨转移的效率和选择性建模

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

科研

癌症研究

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

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

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

基于细胞外基质提取物 (ECME) 的三维系统中单核细胞与原代乳癌细胞的通讯分析

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

免疫与感染

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

医学

<meta charset="utf8"></head><body>检查 3D 胶原基质设置中的巨噬细胞-肿瘤球状体相互作用

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

<meta charset="utf8"></head><body>巨噬细胞-肿瘤球状体共侵袭测定

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

巨噬细胞-肿瘤球状体共侵袭测定

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

研究肺泡巨噬细胞的作用乳腺癌转移

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评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

巨噬细胞-肿瘤球状体共侵袭测定

巨噬细胞-肿瘤球状体共侵袭测定

巨噬细胞-肿瘤球状体共侵袭测定