The project was supported by National Research Fund OTKA (K 104045).

1.细胞培养和试剂的制备<ol><li>保持在制造商推荐的培养基中的细胞培养物，补充有10％（体积/体积）胎牛血清和抗生素（称为完全培养基）。保持细胞培养烧瓶在湿润的5％CO 2气氛中培养器在37℃。按照倒置显微镜下细胞的增殖和汇合（使用10X相衬物镜）。 </li><li>当细胞达到足够汇合，取出培养基，并用2-3毫升，无菌磷酸盐缓冲盐水（PBS）洗涤培养物。移除PBS并加入0.5毫升，无菌0.25％胰蛋白酶-EDTA溶液向细胞培养，并在37℃下孵育直至细胞分离（约10分钟）。 </li><li>暂停细胞3-4毫升无菌完全培养基停止胰蛋白酶和他们转移到无菌离心管中。离心细胞，在150×g离心，在室温（RT）下4分钟。小心弃上清，并暂停在pellet在2-3毫升无菌完全培养基。 </li><li>取出100μL的细胞悬浮液，并用100微升0.4％（M / V）台盼蓝溶液混合，以染色死细胞。负载10微升该混合物成血球和确定存活细胞倒置显微镜的数量。 </li><li>稀释细胞悬浮液，在步骤1.3制得10毫升的无菌的完全培养基的所需量，含有4×10 4个细胞/毫升。 </li><li>加入每孔细胞悬液（步骤1.5准备）的250微升（10 4个细胞/孔）到七口井第一玻璃底部的8孔显微镜载玻片的。在使用促性腺激素释放激素摄入法这张幻灯片。 </li><li>添加每孔的细胞悬浮液（在步骤1.5中制备）的250微升（10 4个细胞/孔）到第二8孔显微镜载玻片的五井。这种滑动将在的GnRH-IR表达的方法中使用。 </li><li>加1，每孔的细胞悬浮液（在步骤1.5中制备）中的溶液（4×10 4个细胞/孔）成七个逢一个12孔板的LS。该板将在GnRH的量化方法中。 </li><li>让细胞附着在两个显微镜载玻片和板。孵育他们在湿润，5％的CO 2气氛中培养器在37℃下48小时。 </li><li>孵化后检查倒置显微镜下细胞（使用10X相衬物镜）。如果细胞是健康和连接，请继续下面的步骤。 </li><li>准备从每三个偶联物18的在二甲亚砜（DMSO）的10mM的GnRH-FITC储液。 （使用这些稀释浓度:16.43微克/μL[D-赖氨酸6（FITC） -的GnRH-I，16.97微克/μL[D-赖氨酸6（FITC） -的GnRH-II和16.47微克/μL[赖氨酸8（ FITC）〕 - 的GnRH-III）中保持这些储备溶液在暗处在室温下并在几周内使用它们。 </li><li>稀释每三个10毫米的GnRH-FITC股票解决方案1.7毫升完全培养基1.7微升。撼动这些解决方案。 （帖e为10μM的GnRH-FITC处理介质。）稀释每三个10μM的GnRH-FITC处理介质的150微升1.35中完全溶液中。摇这些解决方案，以及（这些是1μM的GnRH-FITC处理介质）。 注:含有处理介质的所有的GnRH-FITC应避光和几个小时内用完。 </li><li>预热的六个的GnRH-FITC处理介质（在步骤1.12的方法制备）和4毫升完全培养基中至37℃。 </li><li>稀的5mM的荧光探针溶液3微升在3毫升PBS中（在材料清单中提到核染剂）至5微米。该溶液应避光，其保持在RT，并在几个小时内使用完。 </li></ol> 2.激光共聚焦显微镜（CLSM） <ol><li> 促性腺激素释放激素摄入法 <ol><li>采取步骤准备1.6的第一个显微镜载玻片，并吸取了来自各的七的完全培养基。加入预热完整的中介的250微升微米到第一井。使用此作为阴性对照。添加预热的GnRH-FITC的250微升处理介质（从1.13）到每个未来六井（3个孔用1μM和3与10μM）。孵育在湿润，5％的CO 2气氛中培养器滑动，在37℃5小时。 </li><li>吸取出从每个孔的培养基，并用250微升PBS洗涤该细胞。加定影溶液（10％中性缓冲福尔马林）的250微升每孔，并培育在RT滑动，10分钟。 </li><li>吸取出从每个孔定影液，并用250微升PBS洗涤该细胞。添加250微升含在步骤1.14制备成​​每孔5微米的荧光探针溶液的PBS并孵育在RT滑动，10分钟。 </li><li>吸取了从每个荧光探针解决好，并用250微升PBS仔细清洗细胞两次。添加安装介质3-4滴到每口井，终于。保持室温在黑暗的幻灯片，直到成像。 </LI&gt; </ol></li><li> 促性腺激素释放激素-IR表达法 <ol><li>取在步骤1.7制得的第二8孔显微镜载玻片和吸取出从各五个井的完全培养基。 </li><li>添加预热的完全培养基的250微升到第一井，用此作为阴性对照。加入预热的完全培养基250μL进入第二也很好，并使用此GnRH的治疗前检查的GnRH-IR表达。 </li><li>加入这三个预热10μM的GnRH-FITC处理介质的250微升到下一个三口井。使用这些井检查的GnRH-IR表达之前，用预处理的GnRH-FITC结合物的细胞。孵育在湿润，5％的CO 2气氛中培养器滑动，在37℃1小时。 </li><li>吸取出从每个将被用于检查的GnRH处理后的GnRH-IR表达并用预热完全培养基250μL的洗涤这些孔中的三个孔的处理介质。加入250μL的预热完全培养基到每个三口井的。孵育在湿润，5％的CO 2气氛中培养器滑动，在37℃1小时。 </li><li>吸取出从每五个孔的培养基，并用250微升PBS洗涤该细胞。添加250微升定影溶液导入各孔中，并孵育在RT滑动，10分钟。 </li><li>吸取出从每个孔定影液并用PBS 250微升洗细胞。添加的PBS 250微升含5％牛血清白蛋白（BSA）封闭溶液导入各孔中，并孵育在RT滑动，1小时。 </li><li>稀的GnRH-IR初级抗体的10μL的1毫升PBS中（1:100的比例）。将其保持在RT，并在几个小时内使用完。 </li><li>吸取出从每个孔除外阴性对照（第一阱）的BSA封闭溶液。洗净用250微升PBS细胞（除阴性对照好）。加入250包含步骤2.2.7制备成EAC的GnRH-IR一抗的PBS微升ħ井（除了阴性对照）。孵育所述滑动在RT，1小时，在黑暗的地方。 </li><li>稀释的Alexa 546标记的第二抗体的2.5微升在1.25毫升的PBS（1:500的比例）。 注意:此溶液应该避光，其保持在RT，并在几个小时内使用完。 </li><li>吸取出从每五个井的解决方案，并用250微升PBS洗涤该细胞。添加的PBS 250微升含在步骤2.2.9制备成每个孔的AF 546标记的第二抗体。孵育所述滑动在RT，在黑暗1小时。 </li><li>吸取出从每个孔中的溶液，并用250微升PBS洗涤该细胞。添加包含在步骤1.14制备成​​每孔5微米的荧光探针溶液的PBS 250微升，孵育滑动10分钟，在RT。 </li><li>吸取出从每个孔的荧光探针溶液，并用250微升的PBS小心洗细胞两次。添加安装介质3-4滴进每口井，后来。避光幻灯片在室温下，直到显微成像。 </li></ol></li><li> 成像 <ol><li>图像的单元倒置共聚焦激光扫描显微镜（使用63X油浸物镜） <ol><li>使用下面的激发/发射波长。 GnRH的结合物:五百十四分之四百八十八纳米，Alexa的546标记的抗体:五百四十六分之四百八十八纳米（仅使用促性腺激素释放激素-IR表达法），DRAQ5荧光探针:680分之633纳米。 </li><li>设置使用阴性对照细胞中的摄像参数。使用这些参数来图像处理的细胞（ 图3）。重新调整上在分析的开始每个显微镜载玻片成像参数。微调和出口的图像与在必要时由制造商提供的软件。 </li></ol></li></ol></li></ol> 3.荧光激活细胞分选（FACS） <ol><li> GnRH的定量方法 <ol><li>采取步骤准备1.8板，并吸取了来自EA的完全培养基七口井的通道。添加1毫升预热的完全培养基到第一井。使用此作为阴性对照。加入1毫升每个预热六个处理介质（3口井1μM和3口井，10μM）进入下一个六口井的。孵育在湿润，5％的CO 2气氛中孵化该板在37℃5小时。 </li><li>吸取出从每个孔的介质。用2毫升的PBS仔细清洗细胞两次。加的胰蛋白酶-EDTA溶液500微升到每个孔中。在37℃孵育板，直至细胞分离（约10分钟）。 </li><li>加入1毫升完全培养基到每口井停止胰蛋白酶。摇板。轻轻悬浮细胞用吸管，并从各孔转移悬浮液到FACS管。离心管，在150×g离心4分钟，在4℃。 </li><li>小心倒出上清液，用一招。添加冰冷的PBS 500微升到每个FACS管中，轻轻地重新悬浮细胞。保持冰管，直到结束的实验。 </li></ol></li><li> 分析计算 <ol><li>分析通过流式细胞术细胞。激发，使用488nm的氩激光器波长和用于检测使用530nm的波长。通过运行阴性对照细胞建立细胞仪参数的流动。使用这些参数来分析处理的细胞（ 图4A）。用制造商的软件对数据进行评估，确定平均荧光强度（MFI）值，并且计算相对的MFI值。 </li></ol></li></ol>

<ol>
	<li>Gilad, Y., Firer, M., Gellerman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Innovations+in+Peptide+Based+Targeted+Drug+Delivery+to+Cancer+Cells.">Recent Innovations in Peptide Based Targeted Drug Delivery to Cancer Cells.</a> Biomedicines. 4 (2), 11 (2016).</li><li>Majumdar, S., Siahaan, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-mediated+targeted+drug+delivery.">Peptide-mediated targeted drug delivery.</a> Med Res Rev. 32 (3), 637-658 (2012).</li><li>Ramakrishnappa, N., Rajamahendran, R., Lin, Y. M., Leung, P. C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GnRH+in+non-hypothalamic+reproductive+tissues.">GnRH in non-hypothalamic reproductive tissues.</a> Anim Reprod Sci. 88 (1-2), 95-113 (2005).</li><li>Schally, A. V., 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=Hypothalamic+hormones+and+cancer.">Hypothalamic hormones and cancer.</a> Front Neuroendocrinol. 22 (4), 248-291 (2001).</li><li>Limonta, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GnRH+receptors+in+cancer:+From+cell+biology+to+novel+targeted+therapeutic+strategies.">GnRH receptors in cancer: From cell biology to novel targeted therapeutic strategies.</a> Endocr Rev. 33 (5), 784-811 (2012).</li><li>Ma, Y. T., Zhou, N., Liu, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+drug+delivery+system+based+on+gonadotropin-releasing+hormone+analogs:+Research+advances.">Targeting drug delivery system based on gonadotropin-releasing hormone analogs: Research advances.</a> J of Int Pharm Res. 41 (2), 140-148 (2014).</li><li>Manea, 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=In-vivo+antitumour+effect+of+daunorubicin-GnRH-III+derivative+conjugates+on+colon+carcinoma-bearing+mice.">In-vivo antitumour effect of daunorubicin-GnRH-III derivative conjugates on colon carcinoma-bearing mice.</a> Anti-Cancer Drugs. 23 (1), 90-97 (2012).</li><li>Argyros, O., 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=Peptide-Drug+conjugate+gnrh-sunitinib+targets+angiogenesis+selectively+at+the+site+of+action+to+inhibit+tumor+growth.">Peptide-Drug conjugate gnrh-sunitinib targets angiogenesis selectively at the site of action to inhibit tumor growth.</a> Cancer Res. 76 (5), 1181-1192 (2016).</li><li>Karampelas, 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=GnRH-Gemcitabine+conjugates+for+the+treatment+of+androgen-independent+prostate+cancer:+pharmacokinetic+enhancements+combined+with+targeted+drug+delivery.">GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.</a> Bioconjug Chem. 25 (4), 813-823 (2014).</li><li>Millar, R. P., Pawson, A. J., Morgan, K., Rissman, E. F., Lu, Z. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+actions+of+GnRHs+mediated+by+ligand-induced+selective+signaling.">Diversity of actions of GnRHs mediated by ligand-induced selective signaling.</a> Front Neuroendocrinol. 29 (1), 17-35 (2008).</li><li>Millar, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotropin-releasing+hormone+receptors.">Gonadotropin-releasing hormone receptors.</a> Endocr Rev. 25 (2), 235-275 (2004).</li><li>Olberg, D. 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=Synthesis+and+in+vitro+evaluation+of+small-molecule+[18F]+labeled+gonadotropin-releasing+hormone+(GnRH)+receptor+antagonists+as+potential+PET+imaging+agents+for+GnRH+receptor+expression.">Synthesis and in vitro evaluation of small-molecule [18F] labeled gonadotropin-releasing hormone (GnRH) receptor antagonists as potential PET imaging agents for GnRH receptor expression.</a> Bioorg Med Chem Lett. 24 (7), 1846-1850 (2014).</li><li>Zoghi, 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=Development+of+a+Ga-68+labeled+triptorelin+analog+for+GnRH+receptor+imaging.">Development of a Ga-68 labeled triptorelin analog for GnRH receptor imaging.</a> Radiochimica Acta. 104 (4), 247-255 (2016).</li><li>Guo, H., Gallazzi, F., Sklar, L. A., Miao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+indium-111-labeled+gonadotropin-releasing+hormone+peptide+for+human+prostate+cancer+imaging.">A novel indium-111-labeled gonadotropin-releasing hormone peptide for human prostate cancer imaging.</a> Bioorg Med Chem Lett. 21 (18), 5184-5187 (2011).</li><li>Nederpelt, I., 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=Characterization+of+12+GnRH+peptide+agonists+-+A+kinetic+perspective.">Characterization of 12 GnRH peptide agonists - A kinetic perspective.</a> Br J Pharmacol. 173 (1), 128-141 (2016).</li><li>Szabo, I., 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=Comparative+in+vitro+biological+evaluation+of+daunorubicin+containing+GnRH-I+and+GnRH-II+conjugates+developed+for+tumor+targeting.">Comparative in vitro biological evaluation of daunorubicin containing GnRH-I and GnRH-II conjugates developed for tumor targeting.</a> J Pept Sci. 21 (5), 426-435 (2015).</li><li>Leurs, U., 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=GnRH-III+based+multifunctional+drug+delivery+systems+containing+daunorubicin+and+methotrexate.">GnRH-III based multifunctional drug delivery systems containing daunorubicin and methotrexate.</a> Eur J Med Chem. 52, 173-183 (2012).</li><li>Mur&#225;nyi, 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=Synthesis,+characterization+and+systematic+comparison+of+FITC-labelled+GnRH-I,+-II+and+-III+analogues+on+various+tumour+cells.">Synthesis, characterization and systematic comparison of FITC-labelled GnRH-I, -II and -III analogues on various tumour cells.</a> J Pept Sci. 22 (8), 552-560 (2016).</li></ol>

The authors have nothing to disclose.

本文所述实验中使用的选择性标记GnRH类似物进行筛选粘附 细胞培养体外 。共聚焦显微镜和流式细胞仪方法适合于跟踪和量化以时间和浓度依赖的方式，这些的GnRH-FITC缀合物的细胞摄取。这些实验有以下关键步骤:1）保持无菌的，健康的细胞培养物; 2）的GnRH肽必须是高质量的; 3）合理的浓度和孵育时间必须遵循; 4）一种治疗和洗涤步骤; 5）以及调整仪器设置。 ?...

<html>基于肽靶向给药系统已成为癌症治疗中一个快速显影和希望的领域，在过去几年中1,2。人促性腺激素释放激素受体I型（GnRH的-IR）是主要位于脑垂体，但也存在于其他几个它们负责自我繁殖3的组织。的GnRH-IR也表示在许多癌组织中，相关或无关的生殖系统4,5。促性腺激素释放激素-IR的几种恶性肿瘤细胞高表达与正常组织相比，提供了有针对性的治疗5,6的机会。 许多促性腺激素释放激素（GnRH）类似物已在过去的几十年中，它可以用来作为靶向部分开发F"&gt; 7，8，9，这些肽是能够提供以高选择性抗癌剂为恶性肿瘤细胞，其过量表达的GnRH-IR 6。几种的GnRH偶联的抗肿瘤药物具有更高的选择性和更高的效率比相应的未缀合游离药物已经报道了7，8，9。 有关的GnRH肽及其受体以前的出版物报道，GnRH的红外可以假设具有不同的选择性GnRH类似物10的各种构象。高度可变的GnRH-IR已复杂和各种信号通路被赋予了对他们的自然和人工配体11个不同的活动。这些事实使得基于促性腺激素释放激素系统的调查具有挑战性。另一方面，在<html> Ÿ具有前途的治疗潜力。用放射性标记的GnRH肽几个实验以前报道12， 第13， 第 14，第 15，但实验，其中使用荧光标记的GnRH类似物仍然有限。而放射性标记具有高灵敏度，荧光标记有几个其他的优点，例如容易处理，并用不同的荧光团给染液的能力。已成功地被用于药物递送三种常见GnRH类似物是[D-赖氨酸6] -GnRH-I，D-赖氨酸6] -GnRH-II和GnRH的-Ⅲ，但这些肽的作为靶向部分的有效性是很少相比16，17。另一方面，在其中不同的癌细胞和GnRH类似物被用于从不同的实验结果是多样的。 ntent"&gt;基于这些考虑，我们集中在肿瘤靶向和药物递送的潜在这些的GnRH肽的，并且由此合成和表征的[D-赖氨酸6（FITC）〕 -的GnRH-I，D-赖氨酸6（FITC ）〕 -的GnRH-II和[赖氨酸8（FITC）〕 -的GnRH-III肽缀合物18这些类似物有选择地与FITC标记上的他们的赖氨酸或D-赖氨酸（肽-FITC比率侧链1:1每个。缀合物），其思想是，选择性荧光标记可以提供有关这些肽新颖的信息，并允许他们的良好的跟踪和可靠的定量。这些结合物具有安全处理和可靠探测，这使得它更容易比较他们的肿瘤靶向效率，并且许多类型的恶性肿瘤细胞的筛选，我们希望达至最新的实验与这些肽偶联物可能有助于新型癌症的靶向发展促性腺激素释放激素药物结合物，并有助于发现新的治疗焦油得到为好。 本手稿演示用的GnRH-FITC结合物以及一些重复性好，快速实验。的GnRH-R的细胞表面表达是关于GnRH的摄取决定性条件，因此我们同时研究了所测试的细胞系中的GnRH-1R的细胞表面水平。我们可视通过共聚焦激光扫描显微镜（CLSM）GnRH的IR和GnRH的-FITC缀合物和使用荧光激活细胞分选（FACS）定量的GnRH-FITC缀合物的细胞摄取。

<table border="0" cellpadding="0" cellspacing="0" width="496">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">EBC-1</td>
			<td style="width:79px;">JCRB Cell Bank</td>
			<td style="width:79px;">JCRB0820</td>
			<td style="width:191px;">Human lung squamous cell carcinoma</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">BxPC-3</td>
			<td style="width:79px;">ATCC</td>
			<td style="width:79px;">CRL-1687</td>
			<td style="width:191px;">Human pancreatic adenocarcinoma</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:149px;">Detroit-562</td>
			<td style="width:79px;">ATCC</td>
			<td style="width:79px;">CCL-138</td>
			<td style="width:191px;">Human pharyngeal carcinoma</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">RPMI-1640 Medium with L-glutamine</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">BE12-702F</td>
			<td style="width:191px;">Culture medium for BxPC-3 cells</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:149px;">Eagle&#39;s Minimum Essential Medium</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">&#160;BE12-611F</td>
			<td style="width:191px;">Culture medium for EBC-1 cells, and complemented with 0,1% sodium pyruvate for Detroit-562 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Fetal Bovine Serum</td>
			<td style="width:79px;">Euro Clone</td>
			<td style="width:79px;">ECS0180L</td>
			<td style="width:191px;">Complements the culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">MycoZap Plus-CL&#160;</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">VZA-2012</td>
			<td style="width:191px;">Complements the culture medium (antibiotics)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Standard Line Cell Culture Flasks</td>
			<td style="width:79px;">VWR</td>
			<td style="width:79px;">10062-872</td>
			<td style="width:191px;">Provide consistent, sterile growth environment for cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:149px;">Trypsin-EDTA Mixture&#160;</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">BE17-161E</td>
			<td style="width:191px;">Remove attached cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">BE17-516F</td>
			<td style="width:191px;">Solvent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Dimethyl sulfoxide</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:79px;">D8418-100ML</td>
			<td style="width:191px;">Solvent</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:79px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">15250061</td>
			<td style="width:191px;">Used to counting cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">[D-Lys6(FITC)]-GnRH-I</td>
			<td style="width:79px;">made by us</td>
			<td style="width:79px;">-</td>
			<td style="width:191px;">Purity &#8805;98% (HPLC)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">[D-Lys6(FITC)]-GnRH-II&#160;</td>
			<td style="width:79px;">made by us</td>
			<td style="width:79px;">-</td>
			<td style="width:191px;">Purity &#8805;98% (HPLC)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">[Lys8(FITC)]-GnRH-III</td>
			<td style="width:79px;">made by us</td>
			<td style="width:79px;">-</td>
			<td style="width:191px;">Purity &#8805;98% (HPLC)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">glass bottom 8-well microscopic slide</td>
			<td style="width:79px;">Ibidi</td>
			<td style="width:79px;">80826</td>
			<td style="width:191px;">Use in CLSM experiment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Corning Costar cell culture plate, 12 well</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:79px;">CLS3513-50EA</td>
			<td style="width:191px;">Use in FACS experiment</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Fixing solution (10% neutral buffered formalin)</td>
			<td style="width:79px;">Bio-Optica</td>
			<td style="width:79px;">01V60P</td>
			<td style="width:191px;">Fix adherent cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Bovine Serum Albumin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:79px;">A7906-10G</td>
			<td style="width:191px;">Prevent the nonspecific binding of the antibodies</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">GnRHR Antibody</td>
			<td style="width:79px;">Proteintech</td>
			<td style="width:79px;">19950-1-AP</td>
			<td style="width:191px;">Immunocytochemistry reagent, bind to the human type-I GnRH receptors</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Secondary Antibody, Alexa Fluor 546 conjugate</td>
			<td style="width:79px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">A11035</td>
			<td style="width:191px;">Immunocytochemistry reagent, bind to the primary antibody</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Fluorescent probe solution (Draq5)</td>
			<td style="width:79px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">62254</td>
			<td style="width:191px;">Counterstain nuclei</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Fluoromount Aqueous Mounting Medium</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:79px;">F4680-25ML</td>
			<td style="width:191px;">Use in CLSM experiment, preserving fluorescence</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Inverted microscope</td>
			<td style="width:79px;">Elektro-Optika Ltd.&#160;</td>
			<td style="width:79px;">Alpha XDS-2T</td>
			<td style="width:191px;">Check the phenotype and confluency of cell cultures</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Inverted confocal laser scanning microscope</td>
			<td style="width:79px;">Zeiss</td>
			<td style="width:79px;">LSM-710</td>
			<td style="width:191px;">Use in CLSM experiment</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Flow cytometer</td>
			<td style="width:79px;">BD Biosciences</td>
			<td style="width:79px;">BD FACSCalibur</td>
			<td style="width:191px;">Use in FACS experiment</td>
		</tr>
	</tbody>
</table>

in vitro imaging and quantification of the drug targeting efficiency of fluorescently labeled gnrh analogues

GnRH类似物是有效的靶向部分和能够提供抗癌剂选择性为恶性肿瘤细胞，其高表达的GnRH受体。然而，GnRH类似物&#39;细胞摄取的定量分析以及基于GnRH的药物递送系统的研究的细胞类型，目前的限制。以前介绍过，选择性荧光标记的GnRH I，-II和-III衍生产品提供了很大的可探测性，和他们有重复性的和健壮的实验合适的化学性质。我们还发现，适当的向上的最新方法与这些标记GnRH类似物能够提供有关基于促性腺激素释放激素的药物递送系统的新信息。该原稿介绍关于[D-赖氨酸6（FITC）]的细胞摄取了一些简单的和快速的实验-促性腺激素释放激素I，D-赖氨酸6（FITC）〕 -的GnRH-II和[赖氨酸8（FITC）〕 -的GnRH -III在EBC-1（肺），在中BxPC-3（胰腺）和底特律-562-（咽）恶性Ťumor细胞。在与这些的GnRH-FITC缀合物平行，促性腺激素释放激素-1受体的细胞表面水平也之前和促性腺激素释放激素治疗通过共聚焦激光扫描显微镜后检查了这些细胞系。的GnRH-FITC缀合物的细胞摄取是通过荧光激活细胞分选进行定量。在这些实验中观察到GnRH类似物和细胞类型之间的主要区别之间的微小差别。其中细胞系的显著差异有其独特的细胞表面的GnRH-I受体水平相关。引入的实验包含实用的方法来可视化，量化和在各种粘附细胞培养以时间和浓度依赖的方式进行比较的GnRH-FITC缀合物的吸收效率。这些结果可以预测在给定的细胞培养物的GnRH偶联物的药物靶向效率，并提供用于在基于促性腺激素释放激素的药物递送系统的检查的进一步实验的良好基础。

激光共聚焦扫描显微镜（CLSM）获得的图像提供有关的GnRH-FITC结合物在给定的细胞培养的时间和浓度依赖性摄取壮观信息。在与这些的GnRH-FITC缀合物的同时，促性腺激素释放激素-1R的细胞表面上的存在也由CLSM实验验证。另外，通过使用一远红DNA染色荧光探针，能够为了对细胞的细胞核以外的GnRH和GnRH的-1R。然而，虽然共焦图像不是量化的，简单的"GnRH的摄入法"可以容易地估...

Watch this Scientific Journal Video about In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues at JoVE.com

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

选择性标记的荧光的GnRH-I，-II和-III衍生物对于跟踪可靠的工具和量化它们的细胞摄取。这个手稿介绍实验来可视化，量化和比较在各种细胞系这些GnRH的缀合物的吸收效率。

体外成像和荧光标记的GnRH类似物的药物靶向性的定量分析

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

in-vitro-imaging-quantification-drug-targeting-efficiency

Research

JoVE Journal

Cancer Research

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

7.5K 次观看.  Semmelweis University. 选择性标记的荧光的GnRH-I，-II和-III衍生物对于跟踪可靠的工具和量化它们的细胞摄取。这个手稿介绍实验来可视化，量化和比较在各种细胞系这些GnRH的缀合物的吸收效率。 

视频: In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

肝大肠癌转移的高级动物模型:成像技术和转移性克隆性质

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

舌细胞外基质的制备与舌鳞癌体外重建

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

腺病毒报告系统 TGF β/Smad3 信号通路的活体细胞成像

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of both synthetic lethal interactors leads to loss of cell viability or cell death. The best studied synthetic lethal interaction is between BRCA1/2 and PARP1, with PARP1 inhibitors being used in clinical practice to treat patients with BRCA1/2 mutated tumors. Large genetic screens in model organisms but also in haploid human cell lines have led to the identification of numerous additional synthetic lethal interaction pairs, all being potential targets of interest in the development of novel tumor therapies. One approach is to therapeutically target genes with a synthetic lethal interactor that is mutated or significantly downregulated in the tumor of interest. A second approach is to formulate drug combinations addressing synthetic lethal interactions. In this article, we outline a data integration workflow to evaluate and identify drug combinations targeting synthetic lethal interactions. We make use of available datasets on synthetic lethal interaction pairs, homology mapping resources, drug-target links from dedicated databases, as well as information on drugs being investigated in clinical trials in the disease area of interest. We further highlight key findings of two recent studies of our group on drug combination assessment in the context of ovarian and breast cancer.

Large genetic screens in model organisms have led to the identification of negative genetic interactions. Here, we describe a data integration workflow using data from genetic screens in model organisms to delineate drug combinations targeting synthetic lethal interactions in cancer.

数据集成工作流，以识别针对合成致命相互作用的药物组合

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

斑马鱼中具有功能化荧光硅纳米粒子的异种移植人类癌细胞的Vivo靶向

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

利用计算机图像分析提高4T1乳腺癌模型中肺转移的定量

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...