我们感谢J. Cappellani和J.邱的技术支持。这项工作是支持的资金由美国国家癌症研究所（U01 CA141451），斯塔尔癌症协会，苏珊科曼为治愈，长岛第2天城抗击乳腺癌，曼哈塞特妇女的抗乳腺癌联盟ME，和从国会导演的乳腺癌研究计划的博士后奖学金，，美国ESNESN是收件人的Leslie C.快速和威廉·伦道夫·赫斯特基金会奖学金从沃森生物科学学院。 HAA得到了资金从研究理事会挪威（160698/V40 151882），和东南地区卫生局（2007060）。

<ol>
	<li>Egeblad, M., Nakasone, E. S., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors+as+organs:+complex+tissues+that+interface+with+the+entire+organism.">Tumors as organs: complex tissues that interface with the entire organism.</a> Dev. Cell. 18, 884-901 (2010).</li><li>Ahn, G. -. O., Tseng, D., Liao, C. -. H., Dorie, M. J., Czechowicz, A., Brown, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Mac-1+(CD11b/CD18)+enhances+tumor+response+to+radiation+by+reducing+myeloid+cell+recruitment.">Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 8363-8368 (2010).</li><li>Tlsty, T. D., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+stroma+and+regulation+of+cancer+development.">Tumor stroma and regulation of cancer development.</a> Annu. Rev. Pathol. 1, 119-150 (2006).</li><li>DeNardo, D. G., Brennan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+complexity+predicts+breast+cancer+survival+and+functionally+regulates+response+to+chemotherapy.">Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy.</a> Cancer Discov. 1, 54-67 (2011).</li><li>Nakasone, E. S., Askautrud, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer Cell. 21, 488-503 (2012).</li><li>Kim, J. B., Stein, R., O'Hare, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+tissue+culture+models+of+breast+cancer--+a+review.">Three-dimensional in vitro tissue culture models of breast cancer-- a review.</a> Breast Cancer Res. Treat. 85, 281-291 (2004).</li><li>Gilbert, L. A., Hemann, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage-mediated+induction+of+a+chemoresistant+niche.">DNA damage-mediated induction of a chemoresistant niche.</a> Cell. 143, 355-366 (2010).</li><li>Pittet, M. J., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging.">Intravital imaging.</a> Cell. 147, 983-991 (2011).</li><li>Lohela, M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+stromal+cell+dynamics+in+tumors.">Intravital imaging of stromal cell dynamics in tumors.</a> Curr. Opin. Genet. Dev. 20, 72-78 (2010).</li><li>Boissonnas, A., Fetler, L., Zeelenberg, I. S., Hugues, S., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+cytotoxic+T+cell+infiltration+and+elimination+of+a+solid+tumor.">In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor.</a> J. Exp. Med. 204, 345-356 (2007).</li><li>Breart, B., Lema&#238;tre, F., Celli, S., Bousso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+intratumoral+CD8++T+cell+cytotoxic+activity+during+adoptive+T+cell+therapy+in+mice.">Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice.</a> J. Clin. Invest. 118, 1390-1397 (2008).</li><li>Engelhardt, J. J., Boldajipour, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marginating+dendritic+cells+of+the+tumor+microenvironment+cross-present+tumor+antigens+and+stably+engage+tumor-specific+T+cells.">Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells.</a> Cancer Cell. 21, 402-417 (2012).</li><li>Brown, E., McKee, T., 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=Dynamic+imaging+of+collagen+and+its+modulation+in+tumors+in+vivo+using+second-harmonic+generation.">Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation.</a> Nat. Med. 9, 796-800 (2003).</li><li>Wyckoff, J. B., Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+macrophage-assisted+tumor+cell+intravasation+in+mammary+tumors.">Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.</a> Cancer Res. 67, 2649-2656 (2007).</li><li>Yuan, F., Salehi, H. A., Boucher, Y., Vasthare, U. S., Tuma, R. F., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+and+microcirculation+of+gliomas+and+mammary+carcinomas+transplanted+in+rat+and+mouse+cranial+windows.">Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows.</a> Cancer Res. 54, 4564-4568 (1994).</li><li>Jain, R. K., Munn, L. L., Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+tumour+pathophysiology+using+intravital+microscopy.">Dissecting tumour pathophysiology using intravital microscopy.</a> Nat. Rev. Cancer. 2, 266-276 (2002).</li><li>Egeblad, M., Ewald, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+stromal+cell+dynamics+in+different+tumor+microenvironments+by+spinning+disk+confocal+microscopy.">Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.</a> Dis Model Mech. 1, 155-167 (2008).</li><li>Cheng, N., Lambert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+Transplantation+of+Stromal+Cells+and+Carcinoma+Cells+in+C57BL/6J.">Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J.</a> Mice. J. Vis. Exp. (54), ee2716 (2011).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+vital+signs+for+long-term+survival+of+mice+under+anesthesia.">Monitoring of vital signs for long-term survival of mice under anesthesia.</a> Cold Spring Harb. Protoc. 2011, pdb.prot5563 (2011).</li><li>Sounni, N. E., Dehne, 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=Stromal+regulation+of+vessel+stability+by+MMP14+and+TGFbeta.">Stromal regulation of vessel stability by MMP14 and TGFbeta.</a> Dis. Model Mech. 3, 317-332 (2010).</li><li>Magness, S. T., Bataller, R., Yang, L., Brenner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+reporter+gene+transgenic+mouse+demonstrates+heterogeneity+in+hepatic+fibrogenic+cell+populations.">A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations.</a> Hepatology. 40, 1151-1159 (2004).</li><li>Motoike, T., Loughna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+GFP+reporter+for+the+study+of+vascular+development.">Universal GFP reporter for the study of vascular development.</a> Genesis. 28, 75-81 (2000).</li><li>Fukumura, D., Xavier, R., 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=Tumor+induction+of+VEGF+promoter+activity+in+stromal+cells.">Tumor induction of VEGF promoter activity in stromal cells.</a> Cell. 94, 715-725 (1998).</li><li>Sugimoto, H., Mundel, T. M., Kieran, M. W., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+fibroblast+heterogeneity+in+the+tumor+microenvironment.">Identification of fibroblast heterogeneity in the tumor microenvironment.</a> Cancer Biol. Ther. 5, 1640-1646 (2006).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic,+long-term+in+vivo+imaging+of+tumor-stroma+interactions+in+mouse+models+of+breast+cancer+using+spinning-disk+confocal+microscopy.">Dynamic, long-term in vivo imaging of tumor-stroma interactions in mouse models of breast cancer using spinning-disk confocal microscopy.</a> Cold Spring Harb. Protoc. 2011, pdb.top97 (2011).</li></ol>

作者有没有利益冲突的披露。按照IACUC批准的协议，所有的动物进行了实验。

肿瘤在体内的全身治疗的反应，可以显着不同的，因为在体外癌细胞的微环境影响的急性反应和复发5。诉说在体内的耐药途径的最大障碍之一是肿瘤细胞和微环境之间的相互作用的复杂性。特别是，因为已经开发固体肿瘤癌症和基质细胞，扰动的一种成分，如抗肿瘤治疗的过程中发生的细胞死亡之间的平衡，可以导致对组织的组织的戏剧性的效果。 活体成像技术提供了允许直接在现场，麻醉小鼠的肿瘤的肿瘤细胞 - 基质相互作用的可视化与抗癌药物在治疗过程中。我们使用旋转盘共聚焦显微镜，它有一个相对有限的穿透深度（见17 </sup&gt;的），但我们的外科手术和标记技术可用于与其它类型的显微镜。从这些实验中获得的影可以用来跟踪过程，包括的荧光强度的变化（ 例如，由于标记人口或诱导细胞死亡的渗透），细胞运动，注射剂的分布（ 例如，跟踪血管渗漏），和合作本地化的荧光信号5,12,17,20。 我们经常在图像小鼠超过6小时，超过18小时，并进行成像。这需要保持不断在麻醉状态下的小鼠，这些长的时间周期。通过适当的麻醉，超过90％的动物生存6个小时，约80％存活超过18小时。有关如何进行长期的麻醉先前已公布的19。在我们的经验中，有五个因素是至关重要的:避免低温，避免脱水，保持的生理血液中氧饱和度水平，U唱加湿，异氟醚的载气，并保持动物在麻醉的最低水平，在它们不显示疼痛的迹象。为了保持体温，我们养老鼠在加热毯（38°C）。为了避免脱水，在整个成像过程中，我们注入少量的生理盐水IP。保持生理血液的氧饱和度水平，我们开始与载气中的（而不是常用的100％）的21％的氧气。这使我们吸入的氧含量增加，如果鼠标 - 小时为一个实验 - 显示血氧饱和度下降。根据我们的经验，增加吸入氧气水平的时间（如30-40％）几乎总是稳定的动物，让几个小时的额外的影像。麻醉的最佳水平，为大多数小鼠与1-1.5％异氟烷在60-65/min和脉冲率400-450/min的呼吸率和结果取得。在我们的成像实验中，我们主要使用转基因小鼠模型（MMTV-PyMT和C3 [1]-标签），其中肿瘤是多焦点，允许在不同的阶段的肿瘤进展的单个成像会话期间在多个的x，y位置的平行的多个病灶成像。这减小关切鼠标鼠标相对于实验旨在识别阶段依赖性的治疗反应的变化，并减少所需的成像的动物的数目。 通过渐进的病理组织学阶段，可以在活体成像识别的MMTV-PyMT模型的（和其他自发肿瘤模型）去，虽然可以做的活体成像过程中的肿瘤性病变的病理分期是不同的，不太详细，比组织切片5,17。相比之下，移植的模型往往反映晚期肿瘤最好的，因为晚期肿瘤的癌细胞通常是孤立的。因此，这种模型更适合于研究肿瘤间质晚期肿瘤的相互作用在不同阶段之间的相互作用大于分歧。 我们的例子形象核结构所发生的变化后诱导细胞死亡的治疗。在抗癌治疗的情况下，该标记策略，而不是可能可用于在原位的图像细胞分裂，阐明，例如，细胞的增殖和休眠如何的影响的微环境。在未来，线粒体成分的荧光标记可能有可能图像线粒体细胞凋亡的细胞死亡过程中所发生的变化。 在这个协议中，我们还如何图像治疗后癌细胞的免疫细胞间的相互作用，但其他微环境的组成部分，也可以是可视化和成像的例子。现有的转基因记者基质成分包括那些为成纤维细胞（ 如 ，FSP1-EGFP 17，21αSMA-RFP，COL1A1-EGFP 21）或细胞的脉管（ 如 Tie2的-GFP 22; VEGF-GFP的23）。 活体成像允许发生在体内以下化疗给药的相互作用和过程的实时可视化。目前的技术，我们已经取得了重大的进展，在了解髓细胞如何影响治疗的反应;然而，由于肿瘤微环境是如此复杂，重大的进步仍然需要开始钻研机械地环境介导的耐药性的过程。仍然需要的最重要的进步之一是更好的标记物的识别，特定细胞群。更好的标记物的重要性，是很好的说明有两个最突出的乳房肿瘤微环境，成纤维细胞和免疫细胞的基质细胞组件。 α-平滑肌肌动蛋白（αSMA）是经常被用来识别成纤维细胞，但该蛋白也表达的血管平滑肌细胞和肌上皮细胞24。因此，尽管存在αSMA-GFP记者小鼠，在活体成像实验确定特定的细胞类型，以下是具有挑战性的。同样，一个单一的荧光标记物，如EGFP表达的c-FMS子下，往往会识别多种免疫细胞亚群的12,17。因此，虽然理想情况下，人会喜欢使用一个单独的标记来识别特定的细胞类型使用这种方法的基本生物学的复杂性构成了重大挑战。 对这个问题的部分解决方案之一是使用注射型标签，以进一步区别细胞亚群的表达相同的荧光报道。例如，荧光葡聚糖由巨噬细胞，并可以用来标记所标记的c-fms的-EGFP和FSP1-EGFP转基因报告小鼠的巨噬细胞的亚群<sup&gt; 17。抗体共轭荧光探针也被用来识别特定亚群（ 例如 GR1-阳性人口的骨髓细胞标记的C-FMS-EGFP记者17）。 不同的游标卡尺测量，它提供的小机械信息如何或为何肿瘤给药治疗，或组织学系列在不同的时间点，需要大样本的动物组织中收获来自肿瘤生成的响应曲线，我们的技术提供了直接的，可量化的信息上的动态的细胞死亡，并在小同伙的基质细胞与癌细胞上的相互作用。因此，这里报告的使用过程的优点是它提供了对药物的反应的上下文中，一个完整的肿瘤微环境中的实时的动态信息。这些信息可以到底层的进程，推动对治疗的反应导致了重要的见解&lt;/ SUP&gt;。

实体肿瘤包括两大隔间:癌细胞和基质成分（蜂窝和非蜂窝）协助维持一个适当的环境为肿瘤的生长1。这些间质的的的选民中发挥着重要的的角色，，在的开发，"的进展，和"转移中的许多类型的癌症，其中包括那些的的乳房2-5的的的中 。的的间质的环境也地影响对治疗的反应2,4,5。因此，确定癌细胞如何响应常规和新疗法的原封未动的微环境的上下文内，重要的是我们进一步认识癌症生物学和改善目前的治疗策略。此外的是，对于理解的生物学的肿瘤复发的，定义了如何癌症的细胞-间质的相互作用的会，随着投药的治疗而改变是至关重要的的的的。 器官型的的合作培养系统已被有用的，用于研究肿瘤 - 基质的的相互作用的<s最多&gt; 6，但它是明显的的，，目前可用的的方法不能成功地扼要重述的完好无损的的肿瘤微环境在体外培养，置于特别是相对于到的免疫细胞的血管功能和征聘。事实上，癌症细胞的反应，顷在体外观察到的的治疗，的客人也常常显着不同的从那些在体内 ，在那里的微环境是完整的5的中观察到的。因此，， 在了上的的治疗反应正研究肿瘤-基质的的的的相互作用和他们的的的的影响的体内实验模型为中，可能会更好地反映临床相关的的机制，7。 研究到在体内的抗肿瘤治疗的响应的主要手段，已被通过来自处理过的动物或患者，的的派生的组织的的肿瘤的大小和的组织学评估的测量的。 ，则即使，在显微镜和荧光记者，中的的进步不会明显缩小超过的过去的的两个几十年来已经启用了的的间-和细胞内的的流程的可视化，在高分辨率下在活的的，麻醉后的的的动物（的活体成像）8 JOURNAL。这些的活体显微镜技术已被证明到是尤其是宝贵的的用于在空间上上和时间上地解剖所述在体内动力学的肿瘤-基质的蜂窝式的和子-蜂窝式的的的决议的的相互作用在8,9的 。 由的活体成像的揭开了的被的生产工艺进行过包括的的抗-肿瘤的T细胞的的细胞毒作用10,11，T细胞的系和髓系的的服务小区12的之间的相互作用，的动态变化的"在胶原蛋白的的组成和"组织以下食疗的治疗13日 ，巨噬细胞-而引发相关癌症的细胞的的血管内的和转移"的变化14日 ，和血管通透性的15。 有是三个主要的的的的为的活体成像的要求。这些措施包括的组织中的适当的准备和曝光为成像，的的的组织的组件的的兴趣时在荧光灯的标签中，和一个已配对的显微术-摄影相机S变体系能够采集图像16。的用于，以解决这些的要求的的最常见的的战略包括:1）异位准备工作（ 例如 ，。，的耳朵或眼睛简直轨道的的接种的），永久的的成像的窗户，的或形象化的的准备工作（ 例如 ，。，背鳍的皮肤瓣）; 2）转基因荧光记者，的，并荧光血管注射剂到组织成分贴上标签;，和3）与的光电倍增管或押记偶联型的移动设备（CCD）相机，分别为9配对的多光子或共聚焦显微镜系统。 ，我们使用一个外科手术准备的的的皮肤的皮瓣模型，以暴露的腹股沟的的乳腺腺为表达的编码荧光记者，的转殖基因的的的麻醉动物的，的成像技术在。的画像顷得到的过了一段中的12小时到第40小时，使用一个增强型CCD（ICCD）的摄像头与一个四-激光纺丝磁盘共聚焦显微镜观察系统17配对。在以这种方式中的影像学已经允许我们，以研究这样的的的的进程，，作为在的体内的药物分布，阶段-依赖的的的CH中emotherapeutic的的反应，的化疗-诱导的细胞死亡，系和髓系的细胞的行为5开始的类型。 我们提供了一个的的活体的成像的癌症细胞的反应，的，以的的抗-癌的疗法和在乳房癌症的小鼠模型中的中的肿瘤 - 基质的的相互作用施治，的的协议，用于。此协议可以用于来跟踪的两种癌症细胞和间质组件的行为和死亡的与内容广泛且具有的各种各样的转基因的和可注射的的的荧光标记在转基因和器官移植两种模型中为期限最长可达到第40小时在一个单一的的成像届会议的。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> MMTV-PyMT小鼠</td><td>杰克逊实验室</td><td> 2374 </td><td></td></tr><tr><td> C3（1）标记的小鼠</td><td>杰克逊实验室</td><td> 13591 </td><td></td></tr><tr><td> ACTB ECFP小鼠</td><td>杰克逊实验室</td><td> 3773 </td><td></td></tr><tr><td> ACTB-H2B-EGFP小鼠</td><td>杰克逊实验室</td><td> 5418 </td><td></td></tr><tr><td> 1×PBS </td><td>内部</td><td></td><td></td></tr><tr><td> 2 MD葡聚糖，荧光素共轭的</td><td> Invitrogen公司</td><td> D-7137 </td><td>重组2 O（4毫克/毫升，储存在-20°C）蒸馏水稀释至1 mg / ml的1×PBS </td></tr><tr><td> 10 kD的葡聚糖的Alexa Fluor 647-共轭</td><td&gt; Invitrogen公司</td><td> D-22914 </td><td>重组2 O（4毫克/毫升，储存在-20°C）蒸馏水稀释至1 mg / ml的1×PBS </td></tr><tr><td>盐酸阿霉素</td><td> Sigma-Aldrich公司</td><td> 44583 </td><td>重组在卫生署2 O和存储在4°C，在1×PBS稀释</td></tr><tr><td> Isothesia（异氟醚） </td><td>巴特勒动物保健供应</td><td> 029450 </td><td> 250毫升</td></tr><tr><td>碘化丙啶</td><td> Invitrogen公司</td><td> P3566 </td><td> 1毫克/毫升，稀1:15 1×PBS中</td></tr><tr><td>氮</td><td></td><td></td><td></td></tr><tr><td>氧</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td> 18G×1½"定期锥针</td><td> BD </td><td> 305196 </td><td></td></tr><tr><td> μManager </td><td>淡水河谷实验室，加州大学旧金山分校</td><td> <a href="www.micro-manager.org" target="_blank">www.micro-manager.org</a> </td><td>开放源码软件</td></tr><tr><td>酒精棉签</td><td> BD </td><td> 326895 </td><td> 70％的异丙醇拭子</td></tr><tr><td>麻醉系统</td><td>分子影像产品，公司</td><td></td><td></td></tr><tr><td>盖玻片</td><td>康宁</td><td> 2940-245 </td><td>第1½，24x50毫米; 70％的异丙醇擦拭消毒</td></tr><tr><td> Curity纱布（无菌） </td><td>肯德尔</td><td> 6939 </td><td></td></tr><tr><td>显微镜幻灯片</td><td>康宁</td><td> 2948-75x25 </td><td>预清洗，如果没有预先清洗，消毒用70％的异丙醇湿巾</td></tr><tr><td>硬化精剪</td><td>精细科学工具</td><td> 14090-11 </td><td>不锈钢，锋利的尖，直头，26毫米的切削刃，11厘米长2对; </td></tr><tr><td>电热毯</td><td> Gaymar产业</td><td></td><td></td></tr><tr><td>热珠灭菌器</td><td>精细科学工具</td><td> 18000-45 </td><td>在使用前打开约30分钟;消毒工具，在&gt; 200℃下进行30秒</td></tr><tr><td> Imaris </td><td>位面</td><td> <a href="www.bitplane.com" target="_blank">www.bitplane.com</a> </td><td></td></tr><tr><td> KRAZY胶</td><td> Elmer的产品</td><td> KG484 </td><td></td></tr><tr><td>实验室带（½"） </td><td></td><td></td><td></td></tr><tr><td>实验室磁带（1"） </td><td></td><td></td><td></td></tr><tr><td>盖保丽龙航运冷却器</td><td></td><td></td><td>这将被用作手术平台</td></tr><tr><td>微解剖钳</td><td> Roboz </td><td> RS-5153 </td><td> 1x2齿，轻微的曲线，0.8毫米刀头宽，4"长度</td></tr><tr><td>微解剖钳</td><td> Roboz </td><td> RS-5135 </td><td>锯齿状的，轻微的曲线，0.8毫米的刀尖宽度4"长度</td></tr><tr><td>显微镜</td><td></td><td></td><td>纺纱盘共聚焦，XYZ压电的阶段，epifluourescence capablility 17,25; </td></tr><tr><td>显微镜载物台插入</td><td>应用科学仪器</td><td></td><td>自定义制作，有两个圆形成像端口</td></tr><tr><td> MouseOx血氧饱和度，软件和传感器</td><td> STARR生命科学</td><td> <a href="www.starrlifesciences.com" target="_blank">www.starrlifescience</a>s.com </td><td></td></tr><tr><td>干的Nalgene超级砂劳越实验室soakers </td><td> Thermo Scientific的</td><td> 74218-00 </td><td>排队手术的平台，切成四分</td></tr><tr><td>喷雾器</td><td>索尔特实验室</td><td> 8901 </td><td>加湿气体，防止刺激肺组织</td></tr><tr><td>滑尖一次性结核菌素注射器（1ml）中</td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> Surflo翅输液器</td><td>泰尔茂</td><td> SV-23BLK </td><td> 23G x¾"超薄针，12"管</td></tr><tr><td>聚维酮碘喷雾</td><td> Purdue Pharma公司</td><td> BASP3H </td><td></td></tr></tbody></table>

live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer

肿瘤微环境中起着举足轻重的作用，在肿瘤的发生，发展，转移和抗癌疗法的反应。三维共培养系统经常被用来阐明肿瘤 - 基质的相互作用，包括它们的作用，药物的反应。然而，许多完整的微环境中发生在体内的相互作用不能被完全复制在这些体外设置。因此，直接可视化这些进程在实时了解肿瘤反应疗法和识别肿瘤细胞之间的相互作用和基质，可以影响这些反应已成为一个重要的工具。在这里，我们提供了一种方法，使用旋转盘的活，麻醉小鼠的共聚焦显微镜直接观察药物分布，癌症细胞的反应和在给药后的全身性治疗乳腺癌模型中的肿瘤 - 基质相互作用的变化。我们为labeli描述程序ng的不同肿瘤组件，用于观察治疗反应治疗的动物，和用于暴露长达40小时的用于成像的肿瘤组织的外科手术。此协议从所获得的结果的时间推移的电影，在其中可以使用图像分析软件评价药物浸润，肿瘤细胞死亡和基质细胞迁移等过程。

Watch this Scientific Journal Video about Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer at JoVE.com

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

我们描述了一种方法，成像响应于抗肿瘤治疗的<em在体内</em在单细胞分辨率。

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 ...

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24.8K 次观看.  Watson School of Biological Sciences. 我们描述了一种方法，成像响应于抗肿瘤治疗的

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

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

科研

医学

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 ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

癌症研究

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

使用生物发光监测小鼠乳腺癌生长和转移集落形成

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

生物学

<meta charset="utf8"></head><body>靶向致癌 miRNA-10b 治疗转移性乳腺癌的纳米药物

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

<meta charset="utf8"></head><body>监测小鼠乳腺癌转移灶中纳米药物的积累

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

监测小鼠乳腺癌转移灶中纳米药物的积累

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

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

摘要

探索更多视频

此视频中的章节

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

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

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

使用生物发光监测小鼠乳腺癌生长和转移集落形成

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

活体成像和治疗卵巢癌的原位小鼠模型

监测小鼠乳腺癌转移灶中纳米药物的积累

监测小鼠乳腺癌转移灶中纳米药物的积累

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

摘要

探索更多视频

此视频中的章节

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

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

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

使用生物发光监测小鼠乳腺癌生长和转移集落形成

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

活体成像和治疗卵巢癌的原位小鼠模型

监测小鼠乳腺癌转移灶中纳米药物的积累

监测小鼠乳腺癌转移灶中纳米药物的积累

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移