This work was conducted with funds to M.B. from the Canadian Institutes of Health Research (CIHR) grant MOP 107972. M.B. is a recipient of a CIHR New Investigator Salary Award. D.C. is a recipient of studentships from the Translational Breast Cancer Research Unit and the CIHR Strategic Training Program in Cancer Research and Technology Transfer, London Regional Cancer Program. C.G. is recipient of studentships from the Translational Breast Cancer Research Unit, London Regional Cancer Program and from the CIHR- Strategic Training Program in Cancer Research and Technology Transfer. SGD.

1，三维基底膜基质乳腺癌细胞培养（埋置技术） <ol><li>处理基底膜基底膜基质:在4℃下在冰上解冻过夜基底膜基质是液体在低温下固化，但在室温下。保持基底膜基质冰（ 图1A-B）上。 </li><li>覆盖共聚焦一号玻璃底培养皿用50μl基底膜基质的利用P-200的Pipetman的前端在螺旋形图案（ 图1C）扩频矩阵的装置。传播基底膜基质时避免气泡的形成谨慎使用。同样，避免传播矩阵到接近玻璃底菜的边界，以防止半月板形成。如果没有经验的操作基底膜基质，然后预冷的菜，P-200的Pipetman移液管，并能在冰上（可替换地，在4℃放置过夜，在冰箱）。这一步报价额外的时间凝固之前扩散基底膜基质。放置在培养皿（ES）在细胞培养孵育箱（37℃，5％CO 2）为至少30分钟，以使基底膜基质固化（ 图1D）。 </li><li>而基质正在发生凝固，胰蛋白酶消化细胞的70-80％汇合100毫米的钢板。一旦细胞已开始剥离所述板，它们悬浮在10ml含10％（V / V）胎牛血清（FBS）的RPMI 1640培养基中，以灭活胰蛋白酶（ 图1E）。然后将悬浮的细胞转移到15毫升锥形管中（ 图1F）。 </li><li>在一个专门的细胞培养物离心分离（ 图1G-H）旋转的细胞（存在于锥形管中），在100×g离心3分钟。 </li><li>虽然在细胞被离心下来，等份加入50μl基质成1毫升微量离心管（注意:各玻底培养皿被分配一个微量离心管中，因此，如果实验要求3的菜肴，那么它遵循3微量离心管是必要的也一样），然后将管置于冰上（ 图1B）。 </li><li>从锥形管从步骤1.4吸出培养基，同时保留原状（ 图1I）的粒料。重悬细胞（已降速）1毫升FBS的RPMI培养基（ 图1J）的。 </li><li>一旦细胞被使用血细胞计数器（ 图1K），或细胞颗粒计数器等分试样2.5×10 4细胞放入离心管中计数，并用适当的介质，以获得50微升（ 图1L），总体积为顶它关闭。 </li><li>从步骤1.7（25,000个细胞在50μl）与来自步骤1.5以1:1的比例的含有基质的离心管中的细胞混合;最终体积为100微升（ 图1M）。 </li><li>从步骤1.8细胞混合到S:轻轻板100微升的矩阵olidified基底膜基质涂层菜从步骤1.2（ 图1N）。这使得细胞将要被嵌入在基底膜基质。 </li><li>传送盘（ES）到细胞培养孵化器中（37℃，5％CO 2），并允许矩阵:细胞混合物以固化至少30分钟。 </li><li>一旦矩阵:细胞混合物凝固后，加入2ml FBS的RPMI媒体的菜（ 图1-10），并采取了菜回到原来的位置将被保存为实验（ 图1P）的其余部分的孵化器。 </li><li>每一天，为期5天更换介质（或按照客户要求进行化验，化验的MDA-MB-231细胞的持续时间为5天的）。 </li><li>使用光学显微镜，采取微分干涉对比的MDA-MB-231的菌落悬浮在基底膜基质（ 图3A）条 （DIC）的图像。每天一次图像20代表地区在10倍物镜5天，测定菌落形态（ 图3C）。 </li><li> 盲目地分析图像，以确定细胞的集落形成星状（ 图3B）。一个群体被认为是星状，如果从细胞的球状体一个或多个突起被感知到。以确定的侵入星状菌落的百分比，由每所获取的图像的细胞集落的总数除以星状的菌落数，然后平均20个图像的每一天的星状菌落的百分比。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig1highres.jpg" width="500" /> 图1:MDA-MB-231乳腺癌细胞的基底膜基质（在嵌入技术）的三维培养。 AB）的实验装置（在通风橱中进行）的玻底培养皿涂层的。 三）良好的示意图用50μl基底膜基质的D）美食放置在细胞培养孵育箱（37℃，5％CO 2），以使矩阵以固化至少30分钟。E）的细胞用胰蛋白酶处理。F ）将细胞重悬于15毫升锥形管中。GH）细胞（存在于锥形管）是在组织培养离心机离心沉降，在100×g离心3分钟。Ⅰ）细胞沉淀J）细胞（已停止旋转）重新悬浮于1ml FBS的RPMI培养基中的K）细胞用血细胞计数器:L计数）2.5×10 4个细胞等分至微量离心管，并配上关闭使用适当的介质，以便得到50μl的总体积:M从步骤1.7（25,000个细胞在50μl）与来自步骤1.5以1:1的比例的含有基质的微量离心管）混合细胞;最终体积为100μ从第8步细胞混合物至步骤1.2固化基质涂层菜O）一旦矩阵:，L N）轻轻板100微升基质细胞混合物凝固后，加入2ml FBS的RPMI媒体来。菜，P）将菜在那里将被储存在余下的实验孵化器。 的三维培养与免疫荧光形态发生特点2。考试<ol><li>取出培养皿（ES）之前（ 图2A），80％甲醇（固定溶液）和3％牛血清白蛋白（封闭液BSA）:设置在冰托盘，冷磷酸盐缓冲溶液（PBS）中，20％丙酮从培养箱中。 </li><li>将菜（ES）上的冰盘和抽吸介质，然后用2毫升冷的PBS（ 图2B）洗3次。 </li><li>一旦最后的PBS冲洗吸出，加2 mL 20％的丙酮:80％甲醇溶液放入盘中（ES）（ 图2C </st荣&gt;）以固定细胞20分钟，在4℃下（无论是在冰上或在冰箱中）（ 图2D）。 </li><li>一旦固定的20分钟结束时，使菜回到室温，吸出固定液，并随后用PBS洗涤3次。一旦最后的PBS洗涤被吸入，加入2ml的3％BSA中的菜（ES）在室温下（ 图2E），以阻断至少30分钟。 </li><li>期间的抑制期间准备的主（层粘连蛋白V 1:100）和二级抗体（在适当的稀释度）溶解在3％牛血清白蛋白（BSA）封闭缓冲液中稀释。请注意:400-500微升的"BSA +初级抗体的解决方案应直接加入到基质:细胞混合物。 </li><li>一旦阻塞的30分钟已经过期，添加一级抗体并孵育至少1小时，在室温下（ 图2F）。请注意，没有PBS洗涤应遵循30分钟blockin进行克期。 </li><li>根据步骤2.6完成后，除去&#39;BSA +初级抗体"溶液，并用PBS洗涤3次。 </li><li>加溶解在3％BSA的二级抗体（在适当的稀释度），直接在基质:细胞混合物，盖菜和孵育1小时，在室温下（ 图2G）。 </li><li>删除&#39;的BSA +二次抗体"溶液，并用PBS洗涤3次。 </li><li>加入2ml的Hoechst 33258（1:10000）溶解在PBS的核染色，孵育铝箔下5分钟（或覆盖不透明的容器，以限制光漂白）（ 图2H）。 </li><li>一旦5分钟孵化用Hoechst33258吨已过期，用PBS洗碟（ES）5倍。 </li><li>添加封固剂直接喷在矩阵:在共聚焦培养皿细胞混合物，并用盖玻片覆盖小心，以防止诱发任何中断在基底膜马特里殖民地的完整性X:细胞混合物（ 图2I）。 </li><li>让培养皿（ES），以在室温下（ 图2I）干燥过夜（或24小时）。一旦干燥，菜（ES）可以储存在-20°C，但它强烈建议他们应该尽可能快地成像。 </li><li>用荧光显微镜使用（ 图3D-E）抗体的合适的激光波长采集图像。 </li></ol><img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig2highres.jpg" width="500" /> 图2。考试3D培养免疫荧光的。 A）实验装置的示意图B）美食置于冰盘和媒体吸气;随后洗涤细胞三次，用2ml冷的PBS C）2毫升20％丙酮:80％的甲醇溶液加入到培养皿（ES）D）固定细胞。20分钟，在4℃下（无论是在冰上或在冰箱中）。E）2×3％BSA的添加到培养皿（ES）在室温下阻断了至少30分钟。F）一旦的30分钟阻挡已过期，添加一级抗体并孵育至少1小时，在室温下G）溶解在3％BSA（在适当的稀释）次级抗体进行直接添加到基底膜基质:细胞混合物; 。随后菜肴覆盖并温育1小时，在室温下高）2毫升Hoechst公司（1:10,000;溶于PBS中）加入到dishe（ES）细胞和培养下铝箔5分钟I）的安装介质直接添加。到矩阵:在共聚焦菜和菜细胞混合物覆盖着玻璃盖玻片。菜（ES）在室温下干燥过夜（或24小时）。

<ol>
	<li>Chambers, A. F., Groom, A. C., MacDonald, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Dissemination and growth of cancer cells in metastatic sites.</a> Nat Rev Cancer. 2, 563-572 (2002).</li><li>Kramer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration+and+invasion+assays.">In vitro cell migration and invasion assays.</a> Mutat Res. 752, 10-24 (2013).</li><li>Shaw, K. R., Wrobel, C. N., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+three-dimensional+basement+membrane+cultures+to+model+oncogene-induced+changes+in+mammary+epithelial+morphogenesis.">Use of three-dimensional basement membrane cultures to model oncogene-induced changes in mammary epithelial morphogenesis.</a> J Mammary Gland Biol Neoplasia. 9, 297-310 (2004).</li><li>Eccles, S. A., Welch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+recent+discoveries+and+novel+treatment+strategies.">Metastasis: recent discoveries and novel treatment strategies.</a> Lancet. 369, 1742-1757 (2007).</li><li>Poincloux, R., Lizarraga, F., Chavrier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+invasion+by+tumour+cells:+a+focus+on+MT1-MMP+trafficking+to+invadopodia.">Matrix invasion by tumour cells: a focus on MT1-MMP trafficking to invadopodia.</a> Journal of cell science. 122, 3015-3024 (2009).</li><li>Bissell, M. J., Labarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context,+tissue+plasticity,+and+cancer:+are+tumor+stem+cells+also+regulated+by+the+microenvironment.">Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment.</a> Cancer Cell. 7, 17-23 (2005).</li><li>Burgstaller, G., Oehrle, B., Koch, I., Lindner, M., Eickelberg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Profiling+of+Cellular+Invasion+in+3D+Cell+Culture+Models.">Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models.</a> PLoS One. 8, (2013).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30, 256-268 (2003).</li><li>Mroue, R., Bissell, 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+cultures+of+mouse+mammary+epithelial+cells.">Three-dimensional cultures of mouse mammary epithelial cells.</a> Methods Mol Biol. 945, 221-250 (2013).</li><li>Vidi, P. A., Bissell, M. J., Lelievre, S. A. <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+culture+of+human+breast+epithelial+cells:+the+how+and+the+why.">Three-dimensional culture of human breast epithelial cells: the how and the why.</a> Methods Mol Biol. 945, 193-219 (2013).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li><li>Debnath, 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=The+role+of+apoptosis+in+creating+and+maintaining+luminal+space+within+normal+and+oncogene-expressing+mammary+acini.">The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini.</a> Cell. 111, 29-40 (2002).</li><li>Weaver, V. M., Bissell, 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=Functional+culture+models+to+study+mechanisms+governing+apoptosis+in+normal+and+malignant+mammary+epithelial+cells.">Functional culture models to study mechanisms governing apoptosis in normal and malignant mammary epithelial cells.</a> J Mammary Gland Biol Neoplasia. 4, 193-201 (1999).</li><li>Weaver, V. M., Howlett, A. R., Langton-Webster, B., Petersen, O. W., Bissell, 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=The+development+of+a+functionally+relevant+cell+culture+model+of+progressive+human+breast+cancer.">The development of a functionally relevant cell culture model of progressive human breast cancer.</a> Semin Cancer Biol. 6, 175-184 (1995).</li><li>Kenny, H. A., Krausz, T., Yamada, S. D., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+novel+3D+culture+model+to+elucidate+the+role+of+mesothelial+cells,+fibroblasts+and+extra-cellular+matrices+on+adhesion+and+invasion+of+ovarian+cancer+cells+to+the+omentum.">Use of a novel 3D culture model to elucidate the role of mesothelial cells, fibroblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells to the omentum.</a> Int J Cancer. 121, 1463-1472 (2007).</li><li>Cougoule, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+leukocytes+and+macrophages+of+various+phenotypes+have+distinct+abilities+to+form+podosomes+and+to+migrate+in+3D+environments.">Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments.</a> European journal of cell biology. 91, 938-949 (2012).</li><li>Sung, K. E., 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=Understanding+the+Impact+of+2D+and+3D+Fibroblast+Cultures+on+In+Vitro+Breast+Cancer+Models.">Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models.</a> PLoS One. , (2013).</li><li>Li, T. 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=Beta-arrestin/Ral+signaling+regulates+lysophosphatidic+acid-mediated+migration+and+invasion+of+human+breast+tumor+cells.">Beta-arrestin/Ral signaling regulates lysophosphatidic acid-mediated migration and invasion of human breast tumor cells.</a> Mol Cancer Res. 7, 1064-1077 (2009).</li><li>Zajac, 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=GPR54+(KISS1R)+transactivates+EGFR+to+promote+breast+cancer+cell+invasiveness.">GPR54 (KISS1R) transactivates EGFR to promote breast cancer cell invasiveness.</a> PLoS One. 6, (2011).</li><li>Underwood, J. 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=The+ultrastructure+of+MCF-10A+acini.">The ultrastructure of MCF-10A acini.</a> J Cell Physiol. 208, 141-148 (2006).</li><li>Lelievre, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+phenotype+depends+on+reciprocal+interactions+between+the+extracellular+matrix+and+the+structural+organization+of+the+nucleus.">Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus.</a> Proc Natl Acad Sci U S A. 95, 14711-14716 (1998).</li><li>Petersen, O. W., Ronnov-Jessen, L., Howlett, A. R., Bissell, 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=Interaction+with+basement+membrane+serves+to+rapidly+distinguish+growth+and+differentiation+pattern+of+normal+and+malignant+human+breast+epithelial+cells.">Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.</a> Proc Natl Acad Sci U S A. 89, 9064-9068 (1992).</li><li>Weaver, V. 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=beta4+integrin-dependent+formation+of+polarized+three-dimensional+architecture+confers+resistance+to+apoptosis+in+normal+and+malignant+mammary+epithelium.">beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium.</a> Cancer Cell. 2, 205-216 (2002).</li><li>Cvetkovic, D., 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=KISS1R+Induces+Invasiveness+of+Estrogen+Receptor-Negative+Human+Mammary+Epithelial+and+Breast+Cancer+Cells.">KISS1R Induces Invasiveness of Estrogen Receptor-Negative Human Mammary Epithelial and Breast Cancer Cells.</a> Endocrinology. , 1999-2014 (2013).</li><li>Alemayehu, 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=beta-Arrestin2+regulates+lysophosphatidic+acid-induced+human+breast+tumor+cell+migration+and+invasion+via+Rap1+and+IQGAP1.">beta-Arrestin2 regulates lysophosphatidic acid-induced human breast tumor cell migration and invasion via Rap1 and IQGAP1.</a> PLoS One. , (2013).</li></ol>

我们什么都没有透露。

三维细胞培养技术的发展使得研究人员研究乳腺上皮细胞的转化，使我们能够想象的巨大的形态学变化。除了分析细胞的侵袭，单或多细胞的乳腺上皮细胞球体可用于评估改变细胞的粘附，增殖，大小和基底根尖极性。相反，以前报道的方法，其中所述细胞覆盖有ECM 8，我们的方法嵌入了细胞在细胞外基质18,19,24，这允许多方向侵入到被量化。这些创新的三维培养模型使研究人员?...

迁移和侵袭能力的个人或集体的细胞是癌症的两大特色，以及所需癌细胞1-4的转移扩散。癌症细胞的启动转移的能力取决于它们的能力来迁移和侵入使用的invadopodia以降解细胞基底膜邻近组织。 invadopodia的是动态的肌动蛋白富含基质降解的突起，通过基质降解蛋白酶5的释放使胞外基质的降解。癌细胞侵袭涉及矩阵接着癌症细胞迁移的降解，这是伴随着所述三维（3D）矩阵环境2的重组。因此，为了穿过基质，细胞必须改变其形状和与细胞外基质（ECM）2交互。 乳腺组织完整性的维持依赖于紧密微机控制三维组织体系结构，因为细胞-细胞外基质和细胞-细胞粘附路口影响基因表达和上皮极性的破坏可导致癌症6-10的发作。然而，大多数体外迁移和侵袭测定法如Transwell小室测定法或伤划痕测定是2维（2D），因此这些忽视的细胞和它们的相邻环境3,6,8,11-14之间的错综复杂的相互作用。相当大的形态和功能的多样性包括不同的细胞形态，细胞分化，细胞-基质粘连和基因表达模式已经由培养细胞在三维培养那些在一般2D缺乏实验2,6,8,11检测。因此，采用了3D试验是显著有益的扼要体内的状况更符合生理，导致基础研究到临床6-10更好的翻译突破性的发现。然而，应该注意的尽管获得与使用三维培养的诸多优点，这种模式不能捕获所有的体内肿瘤微环境，包括各种细胞类型的复杂性。然而，有可能把基质细胞分化成的三维模型（例如，成纤维细胞，白细胞，巨噬细胞），研究了对癌症的细胞粘附和侵入15-17肿瘤-基质的相互作用的影响。 乳腺上皮细胞的培养生长最有效的，当细胞外基质蛋白，如层粘连蛋白和胶原蛋白都存在。与该已知的，市售的基质混合物是来自于Engelbreth-的Holm-群（EHS）小鼠肿瘤和被称为基质胶基底膜基质2,8。已经建立了多种技术来增加上皮细胞的3D殖民地基底膜基质2,8。三维基底膜基质的模型是有效的建立既恶性及非恶性乳腺细胞生长，类似什么是发生在体内环境18,19。 MCF10A细胞非恶性乳腺上皮细胞。当在基底膜基质生长，这些细胞表现出在正常乳腺细胞的体内特性，并进行控制，细胞增殖，细胞分化，凋亡和建立的内腔空间8,12,20。此外，MCF10A细胞的形成在三维培养腺泡细胞的细胞核的外观更接近于乳腺上皮细胞在组织中比在单层培养21。通过研究比塞尔和同事们首次揭示了恶性乳腺细胞可以从非恶性乳腺细胞相鉴别时，层粘连蛋白丰富的环境中成长，因为恶性细胞显示高度混乱的表型，增加增殖，降低细胞对细胞粘附，间质标记物的表达增加，并增加了侵入结构的数量形成3,6,22。 在细胞环境中的异常可影响肿瘤的形成20。在三维培养方法可用于有效地学习，肿瘤细胞和其周围环境之间发生的通信，并确定如何蛋白表达的影响，例如通信14,20,21,23。本文提供了详细的方法生长的MDA-MB-231乳腺癌细胞在三维培养分析侵袭，并研究上皮形态的利用上皮标志物层粘连蛋白，细胞基底膜18,19,24的一个组成部分的损失， 25。详细的程序提供准确和可重复地量化星状（侵入型）结构形成由任何侵入性肿瘤细胞，而不是限定的共同的乳腺癌细胞系（如MDA-MB-231，HS578T，MCF-7，T47D或能力）。因此，该测定可以作为评价或在细胞如何表达治疗与平台亲或反侵入性化合物调节细胞外基质的降解，由单个或多个细胞。

<table border="0" cellpadding="0" cellspacing="0" width="1062">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Material/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL tubes</td>
			<td>VWR</td>
			<td>CA10011-700</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100-mm culture dish BD353003</td>
			<td>VWR</td>
			<td>CABD353003</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Falcon tube</td>
			<td>VWR</td>
			<td>CA21008-918</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL filtered tips</td>
			<td>VWR</td>
			<td>10011-350</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-016</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-008</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35-mm glass-bottomed Confocal No.1 culture dishes&#160;</td>
			<td style="width:265px;">MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Precooled before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>BioShop</td>
			<td>ALB003.100</td>
			<td>Used at 3% for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Life Technologies</td>
			<td>&#160;A11029</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>&#160;A11011</td>
			<td>1:1200 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-Beta-Catenin&#160;</td>
			<td>BD Transduction Laboratories</td>
			<td>610153</td>
			<td>Mouse-monoclonal; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>Used at 10% (v/v)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg&#8260;mL Solution in Water</td>
			<td>Life Technologies</td>
			<td>H3569</td>
			<td>Used at 0.1% (1:10000 dilution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InVivo Analyzer Suite&#160;</td>
			<td style="width:265px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kisspeptin-10 (KP-10)</td>
			<td style="width:265px;">EZ Biolabs</td>
			<td style="width:119px;">PT0512100601</td>
			<td>Used at 100nM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Laminin&#160;</td>
			<td>Cedarlane</td>
			<td>AB19012(CH)</td>
			<td>Rabbit-polyclonal full length human; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM-510 META laser scanning microscope&#160;</td>
			<td style="width:265px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Used at 63X objective; oil immersion lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel phenol red free (BD356237)</td>
			<td style="width:265px;">VWR</td>
			<td>CACB356237</td>
			<td>&#160;Lot No.2180819; 10.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus IX-81 microscope&#160;</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Antibiotic (added to media; used at 0.01%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipet&#160;</td>
			<td>VWR</td>
			<td>CA53300-523</td>
			<td>Sterile, disposeable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium with Glutamine</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>Used for culturing of MDA-MB-231 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>25200-072</td>
			<td>Used to trypsinize MDA-MB-231 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)</td>
			<td style="width:265px;">Lonza</td>
			<td style="width:119px;">CC-3150</td>
			<td>Used for culturing of MCF10A cells</td>
		</tr>
	</tbody>
</table>

quantification of breast cancer cell invasiveness using a three-dimensional (3d) model

现在是众所周知的细胞和组织微环境是影响肿瘤起始和进展的关键调节因子。此外，细胞外基质（ECM）已被证明是在培养和稳态体内细胞行为的关键调节剂。在二维（2D）培养细胞的当前方法中，塑料表面的结果的细胞和它们的微环境之间复杂的相互作用的干扰和丢失。通过使用三维（3D）培养物测定，用于细胞的微环境相互作用的条件成立类似的体内微环境。本文提供了详细的方法生长的乳腺癌细胞在三维基底膜蛋白基质，体现三维培养的细胞侵袭的评估潜在进入周围环境。此外，我们将讨论这些3D分析如何有潜力，研究信号molec的损失ULES，通过免疫组化方法调节上皮细胞的形态。这些研究有助于识别重要的机械细节成调节入侵的方法中，需要对乳腺癌的扩散。

在MDA-MB-231细胞侵入在三维矩阵的一个例子示于图3C。将细胞包埋在基质（第1天），并开始由第3天形成侵入性（星状）结构，以及完全侵入到基质由5天（ 图3C）。形成星状的菌落数进行计数，并表示为每培养皿的菌落（侵入性和非侵入性）的总数的百分比。另外，由于测量是为五天每日进行，入侵的速率也可以进行评价。 建立形态发生事件的时间线?...

Watch this Scientific Journal Video about Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model at JoVE.com

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

本文提供的详细方法为使用三维（3D）测定法来量化乳腺癌细胞的侵袭。具体来说，我们将讨论建立这样的检测要求的程序，量化和数据分析，以及方法来探讨及细胞膜的完整性，当细胞侵入发生的损失。

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

quantification-breast-cancer-cell-invasiveness-using-three

Research

JoVE Journal

Medicine

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

15.7K 次观看.  University of Western Ontario. 本文提供的详细方法为使用三维（3D）测定法来量化乳腺癌细胞的侵袭。具体来说，我们将讨论建立这样的检测要求的程序，量化和数据分析，以及方法来探讨及细胞膜的完整性，当细胞侵入发生的损失。 

视频: Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

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 ...

科研

医学

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

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 ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三维文化:一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

癌细胞球体检测，以评估入侵的3D设置

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

乳腺癌细胞侵袭定量使用三维（3D）模型

摘要

探索更多视频

此视频中的章节