这项工作得到了国家卫生和医学研究理事会 (澳洲) 给 H-爵士的赠款的支持。TMBW 是澳大利亚研究生奖的接受者澳大利亚政府和 Ann 亨德森的顶部奖学金从澳大利亚扶轮健康与扶轮 Templestowe 和用餐的治疗。

所有动物实验都是由墨尔本大学动物伦理委员会批准的。
注: 腺病毒载体的序列, 构造和生成协议 pad-巨细胞病毒-Td-汤姆, pad cmv-GFP和 pad-CAGA12-卢克/Td-汤姆以前被描述了11,12, 13。所有的向量都是商用的。
1. 使用50% 组织培养感染剂量的病毒效价测定 (TCID50)
<ol><li>准备 1 x 106 HEK293A 细胞在10毫升的 DMEM + 10% 的血清介质。</li>
	<li>种子100µL 细胞悬浮在一个96井平底细胞培养板的每一个井。</li>
	<li>在完全培养基中制备1毫升1:100 稀释的病毒储存。将稀释病毒的11µL 添加到1列的每个井中。</li>
	<li>通过将稀释病毒的11µL 与100µL 细胞悬浮液混合, 在96井板中直接进行10倍稀释, 直到达到 1 x1012的最终稀释。虽然细胞在这个阶段是不黏附的, 在井之间转移的细胞的数量保持恒定。</li>
	<li>吸管11µL 每稀释入每列从最高的稀释 104开始。用每个稀释液取代吸管尖, 以限制病毒微粒的交叉。</li>
	<li>将11µL 无病毒的完整介质添加到12列作为负控制。</li>
	<li>孵育板材为 7-10 天在37°c 与 10% CO2。</li>
	<li>使用显微镜, 在每列的水井数中显示病变或细胞毒性效应的迹象。如果有任何感染迹象, 请考虑良好的阳性反应。</li>
	<li>用芦苇和明奇 (1938) 统计方法14计算每个稀释的正井的分数。</li>
	<li>计算比例距离 (PD) 使用: (50)/(a B), 其中 A 是百分比响应以上或等于50% 和 B 是百分比响应低于50%。</li>
	<li>计算 TCID50使用:10x PD, 其中 x 是稀释给予响应大于50%。</li>
	<li>使用以下方法计算病毒滴度: 0.69/(0.011 x TCID50) PFU/毫升</li>
</ol>2. 腺病毒的语言测定
<ol><li>种子 1.0-1.5 x 105 MDA-MB-231 细胞与 500 DMEM 培养基的 ul 在5% 个井板 (12 个细胞融合, 35% 个井一共) 的每井含6个血清。</li>
	<li>使用以下公式计算0、250、500、2500和5000语言所需的Ad 巨细胞病毒--Td-Tom的数量: 教学语言 = (容积 [ul] x PFU/ul)/细胞数。对于Ad 巨细胞病毒-Td-汤姆以 1 x 1010 (PFU/毫升) 的效价, 所需的感染量 mois 》杂志分别为0、3.75、7.5、37.5 和 75 ul。</li>
	<li>在2.2 节中, 通过直接将病毒吹打到水井中, 通过彻底混合, 感染经计算的Ad-CMV-Td-汤姆的细胞。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>从水井中取出介质, 并立即用 500 ul 10% 福尔马林的电池固定细胞, 5 分钟。</li>
	<li>用 500 ul 的 PBS 从水井中吸出福尔马林, 一次洗井。</li>
	<li>用 500 ul 赫斯特溶液染色细胞核, 5 分钟。</li>
	<li>取出赫斯特溶液, 用 PBS 500 ul 3 次冲洗水井。</li>
	<li>在荧光显微镜上使用200X 放大图像的 Td-番茄蛋白和细胞核信号。激发 Td-番茄蛋白在554毫微米和赫斯特染料在352毫微米。</li>
	<li>使用 ImageJ 分析图像, 确定100% 感染 Td-番茄15所需的工作语言。</li>
</ol>3. 活单细胞中的双腺病毒转导
<ol><li>种子 1.0-1.5 x 105 MDA-MB-231 细胞与 500 DMEM 培养基的 ul 在5% 个井板 (12 个细胞融合, 35% 个井一共) 的每井含2个血清。</li>
	<li>感染细胞与 75 ul 的ad CMV-GFP (效价 = 5 x 109 PFU/毫升) 和 7.5 ul 的ad CAGA12-Td-汤姆(效价 = 5 x 1010 PFU/毫升) 获得2500语言, 可以达到100% 感染阳性。</li>
	<li>治疗细胞与或没有 0.25 ul 的 TGF β每井 (10 毫克/毫升的库存, 5 ng/ul 作为最终浓度) 后立即腺病毒感染。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>图像活细胞与相对比荧光显微镜。激发 GFP 在470毫微米和 Td-蕃茄蛋白质在554毫微米。</li>
	<li>固定细胞和染色细胞核, 如步骤 1.6-1.9 所述。</li>
	<li>用荧光显微镜对 Td-番茄蛋白和赫斯特染色进行图像处理。激发赫斯特染料在352毫微米。</li>
	<li>使用 ImageJ, 计算 GFP 和 Td-番茄阳性细胞的百分比15。</li>
</ol>4. 活体单细胞信号在创面愈合试验中的作用
<ol><li>种子 4-5 x 105 MDA-MB-231 细胞与1毫升的 DMEM 培养基包含5% 个血清在6个井板 (50% 个细胞融合, 2 井总共) 的每井。</li>
	<li>感染细胞与 22.5 ul 的Ad CAGA12-Td-汤姆(效价 = 5 x 1010 PFU/毫升, 语言 = 2500)。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>用 P200 吸管尖端在每个井的细胞层上划两条交叉线。</li>
	<li>从水井中取出旧介质, 并用2毫升新鲜5% 的 TGF, 用或不含 0.5 ul 的每口井 (10 毫克/毫升的库存, 5 ng/ul 作为最终浓度)。</li>
	<li>将板材放在孵化器的37°c 与 10% CO2 5 分钟, 并采取在相对比显微镜上使用100X 放大的选择伤口区域的图像。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>在同一个区域拍摄伤口闭合图像。</li>
	<li>修复细胞, 染色细胞核, 如步骤 1.6-1.9 所述, 并可视化的 Td-番茄信号在伤口区域和非伤口区域使用200X 放大的荧光显微镜。</li>
	<li>使用 ImageJ 软件15, 量化伤口和非创面的 Td-番茄阳性细胞的百分比。</li>
</ol>5.体外荧光素酶测定
<ol><li>在1毫升的 DMEM 培养基中制备 3-5 x 104 MDA-MB-231 细胞悬浮液, 含5% 个血清。</li>
	<li>感染细胞悬浮与 2.5 ul 的Ad CAGA12-卢克(效价 = 5 x 1010 PFU/毫升, 语言 = 2500)。</li>
	<li>将受感染的细胞种子放入96井板, 在 3000 cells/100 µL/井。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>从水井中取出旧介质, 并用100µL 新鲜5% 的 TGF, 用或不含 0.1 ul (含1毫克/毫升的库存, 1 ng/ul 为最终浓度), 并在存在或没有 0.17 ul 的 TGF β抑制剂每井 (7 毫克/毫升的库存, 12 ng/ul 为最终浓度) (一种新的 TGF β配体诱捕蛋白, 陈等, 未发表的工作)。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>在双蒸馏水中解冻荧光素酶检测系统基质, 并制备1x 细胞培养裂解试剂 (DDW)。</li>
	<li>从96口井和溶解井中取出介质, 用 50 ul 1x 细胞培养裂解缓冲冰, 使用一台带有温和搅拌的眼眶, 30 分钟。</li>
	<li>将 30 ul 的裂解液从每一个井转移到一个不透明的荧光素片中, 让盘子温暖室温。</li>
	<li>在紫外线软件上, 创建一个新的协议。选择1注射器的测量前后注射。在测量和积分时间的延迟前, 将测量值设置为1秒。在注射测量后, 将注入量设置为40µL, 在注射后1秒和 5 s积分时间内进行延迟测量。通过选择"确定"确认设置。</li>
	<li>将喷油器管放入 DDW 中, 然后单击最佳设置下的喷油器 1按钮, 以在使用前清洗喷油器管。下一步从 DDW 中取出喷油器管, 然后在空气中放置两个进一步的引射器 1, 以冲洗管子中的所有溶液。将喷油器管放入萤火虫基体中, 再用两次质数准备基体。</li>
	<li>将不透明的荧光素酶板与样品放到紫外线中, 然后选择软件上的选项。突出显示感兴趣的水井, 然后单击 "应用"。按开始测量荧光素酶信号。</li>
	<li>测量完成后, 用清水除去并冲洗盘子。将管子插入空气中, 然后将其注入1回萤火虫衬底管, 将剩余的基板恢复到喷油器管中。将喷油器管放入 DDW, 再执行两个素数, 以清洁任何剩余基体的喷油器管。</li>
</ol>6. 活体信号成像的细胞制备
<ol><li>48小时前肿瘤植入, 种子 2 3x106 MDA-MB-231 细胞成175厘米2瓶 (总共10瓶)。培养细胞在20毫升的 DMEM 培养基中含有5% 的血清在37摄氏度, 10% CO2。</li>
	<li>24小时后, 添加 300 ul 的Ad CAGA12-卢克(效价 = 5 x 1010 PFU/毫升, 语言 = 2500) 到每个烧瓶。保持细胞在文化中的24小时。</li>
	<li>在植入日, 取出旧的培养基, 用20毫升的 PBS, 7.4 的 pH 值清洗腺病毒感染的细胞。在烧瓶中加入2毫升0.05% 胰蛋白酶-EDTA, 然后稍微摇动烧瓶, 让胰蛋白酶覆盖所有的烧瓶表面。放置烧瓶到孵化器37°c, 10% CO2为3分钟。</li>
	<li>将8毫升的含 DMEM 培养基加入烧瓶中, 使胰蛋白酶淬火。将所有细胞悬浮液转移到玻璃试剂瓶中。</li>
	<li>使用 hemocytometer 计数细胞数, 并在两个50ml 离心管中传输 4.8 x 107细胞。</li>
	<li>离心细胞在 400 x g 5 分钟的 RT, 以去除胰蛋白酶和并用重悬细胞在720毫升的新鲜的 DMEM 培养基。</li>
	<li>将细胞悬浮液转移到不育的5毫升管中, 加入 160 ul 胶 (10% 总胶悬浮液)。</li>
	<li>把细胞放在冰上直到植入。</li>
</ol>7. 原位植入术
<ol><li>重12只免疫缺陷小鼠, 随机将小鼠分配到控制和治疗组 (6 只小鼠/组)。</li>
	<li>在无菌的生物箱中进行植入, 以保持不育的环境。麻醉鼠标使用 2.5-4.5% 异氟醚, 氧流速为1升/分. 将鼠标放在热垫上, 并将一滴润滑眼膏放在眼睛上, 以避免角膜损伤。将鼠标固定在仰卧位的热垫上, 同时通过将鼻口放入与异氟醚相连的鼻锥中保持麻醉。</li>
	<li>通过对脚趾捏缺乏反应来确认麻醉的成功。减少异氟醚的维持剂量为 1.5-2.5% 与1升/分钟氧气提醒植入。</li>
	<li>用棉签蘸上80% 乙醇, 从两侧的第四奶嘴中清除皮肤。</li>
	<li>通过触诊找到4乳房脂肪垫, 挤压脂肪垫进一步暴露组织。</li>
	<li>通过吹打上下混合, 使细胞悬浮良好。轻轻地将50µL 的细胞混合物吸入27G 胰岛素注射器中, 并注入鼠标两侧的乳房脂肪垫。</li>
	<li>确认成功注射的脂肪垫肿胀的感觉。轻轻松开脂肪垫, 把鼠标放回笼子里。</li>
	<li>把笼子放在热垫上至少30分钟, 这样老鼠才能获得知觉, 然后把笼子放回拘留室3天。</li>
</ol>8. IVIS 成像
<ol><li>打开活生生的图像软件。</li>
	<li>通过单击 "控制面板" 上的 "初始化 IVIS 系统" 按钮初始化 IVIS 系统。等到温度标志变绿。</li>
	<li>在 "控制面板" 中选择发光成像模式。</li>
	<li>将异氟醚麻醉转向腔室和 IVIS 系统。</li>
	<li>用 2.5-4.5% 异氟醚与1升/分氧麻醉小鼠。一旦麻醉, 将异氟醚设置为 1.5-2.5% 以维护。用腹腔 (ip) 注射剂将 d-荧光素的150毫克/千克体重注入小鼠体内。</li>
	<li>立即将小鼠转移到 IVIS 系统中, 把它们放到温度控制的成像平台上, 把鼻子放到鼻锥中。</li>
	<li>单击 "控制面板" 上的 "序列设置" 按钮, 然后选择 "介质 Binning"。</li>
	<li>设置成像曝光时间如下: 十年代、二十年代、三十年代、六十年代和 120s. 在荧光素注射后开始成像大约3分钟, 并继续图像直到信号开始下降 (通常这将需要大约20分钟)。</li>
	<li>从 IVIS 中去除小鼠, 同时保持异氟醚麻醉通过鼻锥和治疗小鼠与50µL PBS 或50µL TGF β信号抑制剂 (50 微克/肿瘤) 通过肿瘤内注射。</li>
	<li>把老鼠放回笼子里, 放在热垫上至少30分钟。然后把笼子放回拘留室。</li>
	<li>重复 IVIS 成像程序, 如上文所述治疗后的前一天。</li>
</ol>9. 数据分析
<ol><li>在活体图像软件中打开保存的文件。</li>
	<li>使用视图查找图像信息 |图像信息。</li>
	<li>选择在同一时间点拍摄的照片, 同时曝光时间相同。将照片作为组加载。</li>
	<li>取消选中图像中的单个框以使强度正常化。</li>
	<li>选择光子模式来分析强度。通过在ROI 工具中选择 "感兴趣区域" 按钮来量化信号的强度。</li>
	<li>将 ROI 圆圈拖动到包含发光信号的区域, 然后单击 "测量" 按钮以获取信号强度。</li>
</ol>

<ol>
	<li>Massague, J., Like, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+receptors+for+type+beta+transforming+growth+factor.+Ligand+binding+and+affinity+labeling+in+human+and+rodent+cell+lines.">Cellular receptors for type beta transforming growth factor. Ligand binding and affinity labeling in human and rodent cell lines.</a> The Journal of biological chemistry. 260, 2636-2645 (1985).</li><li>Xu, L., Chen, Y. G., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+import+function+of+Smad2+is+masked+by+SARA+and+unmasked+by+TGFbeta-dependent+phosphorylation.">The nuclear import function of Smad2 is masked by SARA and unmasked by TGFbeta-dependent phosphorylation.</a> Nature Cell Biology. 2, 559-562 (2000).</li><li>Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+signaling+in+context.">TGFbeta signaling in context.</a> Nature reviews. Molecular cell biology. 13, 616-630 (2012).</li><li>Massague, J., Seoane, J., Wotton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smad+transcription+factors.">Smad transcription factors.</a> Genes Development. 19, 2783-2810 (2005).</li><li>Blobe, G. C., Schiemann, W. P., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transforming+growth+factor+beta+in+human+disease.">Role of transforming growth factor beta in human disease.</a> The New England journal of medicine. , 1350-1358 (2000).</li><li>Zhu, H. J., Burgess, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+transforming+growth+factor-beta+signaling.">Regulation of transforming growth factor-beta signaling.</a> Molecular cell biology research communications : MCBRC. 4, 321-330 (2001).</li><li>Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;+in+Cancer.">TGF&#946; in Cancer.</a> Cell. 134, 215-230 (2008).</li><li>Luwor, R. B., 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=Single+live+cell+TGF-beta+signaling+imaging:+breast+cancer+cell+motility+and+migration+is+driven+by+sub-populations+of+cells+with+dynamic+TGF-beta-Smad3+activity.">Single live cell TGF-beta signaling imaging: breast cancer cell motility and migration is driven by sub-populations of cells with dynamic TGF-beta-Smad3 activity.</a> Molecular cancer. 14, 50 (2015).</li><li>Clarke, D. C., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+quantitative+nature+of+TGF-beta/Smad+signaling.">Decoding the quantitative nature of TGF-beta/Smad signaling.</a> Trends in Cell Biol.ogy. 18, 430-442 (2008).</li><li>Zhao, R., Li, N., Xu, J., Li, W., Fang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+single-molecule+study+of+TGF-beta/Smad+signaling.">Quantitative single-molecule study of TGF-beta/Smad signaling.</a> Acta Biochimica et Biophysica Sinica (Shanghai). 50, 51-59 (2017).</li><li>Luwor, R. B., 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=New+reagents+for+improved+in+vitro+and+in+vivo+examination+of+TGF-beta+signalling.">New reagents for improved in vitro and in vivo examination of TGF-beta signalling.</a> Growth factors. 29, 211-218 (2011).</li><li>Dennler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+of+Smad3+and+Smad4+to+critical+TGF+beta-inducible+elements+in+the+promoter+of+human+plasminogen+activator+inhibitor-type+1+gene.">Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.</a> EMBO J. 17, 3091-3100 (1998).</li><li>Luwor, R. B., 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=Targeting+Stat3+and+Smad7+to+restore+TGF-beta+cytostatic+regulation+of+tumor+cells+in+vitro+and+in+vivo.">Targeting Stat3 and Smad7 to restore TGF-beta cytostatic regulation of tumor cells in vitro and in vivo.</a> Oncogene. 32, 2433-2441 (2013).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Method+of+Estimating+Fifty+Per+Cent+Endpoints.">A Simple Method of Estimating Fifty Per Cent Endpoints.</a> American Journal of Epidemiology. 27, 493-497 (1938).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature methods. 9, 671-675 (2012).</li><li>Dennler, S., Prunier, C., Ferrand, N., Gauthier, J. M., Atfi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Jun+inhibits+transforming+growth+factor+beta-mediated+transcription+by+repressing+Smad3+transcriptional+activity.">c-Jun inhibits transforming growth factor beta-mediated transcription by repressing Smad3 transcriptional activity.</a> The Journal of biological chemistry. 275, 28858-28865 (2000).</li><li>Fink, S. P., Mikkola, D., Willson, J. K., Markowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta-induced+nuclear+localization+of+Smad2+and+Smad3+in+Smad4+null+cancer+cell+lines.">TGF-beta-induced nuclear localization of Smad2 and Smad3 in Smad4 null cancer cell lines.</a> Oncogene. 22, 1317-1323 (2003).</li><li>Zinn, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+bioluminescence+imaging+in+small+animals.">Noninvasive bioluminescence imaging in small animals.</a> ILAR J. 49, 103-115 (2008).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and Bioanalytical Chemistry. 397, 3173-3178 (2010).</li><li>Russell, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+adenovirus+and+its+vectors.">Update on adenovirus and its vectors.</a> Journal of General Virology. 81, 2573-2604 (2000).</li><li>Lee, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Mediated+Gene+Delivery:+Potential+Applications+for+Gene+and+Cell-Based+Therapies+in+the+New+Era+of+Personalized+Medicine.">Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine.</a> Genes &amp; Diseases. 4, 43-63 (2017).</li><li>Wang, I. I., Huang, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+technology+for+gene+manipulation+and+functional+studies.">Adenovirus technology for gene manipulation and functional studies.</a> Drug Discovery Today. 5, 10-16 (2000).</li><li>Kochanek, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+adenoviral+vector:+Replacement+of+all+viral+coding+sequences+with+28+kb+of+DNA+independently+expressing+both+full-length+dystrophin+and+beta-galactosidase.">A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 5731-5736 (1996).</li><li>Wang, Q., Finer, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-generation+adenovirus+vectors.">Second-generation adenovirus vectors.</a> Nature Medicine. 2, 714-716 (1996).</li><li>Durham, H. 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=Toxicity+of+replication-defective+adenoviral+recombinants+in+dissociated+cultures+of+nervous+tissue.">Toxicity of replication-defective adenoviral recombinants in dissociated cultures of nervous tissue.</a> Experimental Neurology. 140, 14-20 (1996).</li><li>MacKenzie, K. L., Hackett, N. R., Crystal, R. G., Moore, 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=Adenoviral+vector-mediated+gene+transfer+to+primitive+human+hematopoietic+progenitor+cells:+assessment+of+transduction+and+toxicity+in+long-term+culture.">Adenoviral vector-mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture.</a> Blood. 96, 100-108 (2000).</li><li>Zhang, W. W., Koch, P. E., Roth, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+wild-type+contamination+in+a+recombinant+adenoviral+preparation+by+PCR.">Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.</a> Biotechniques. 18, 444-447 (1995).</li><li>Choi, Y., Chang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+vaccine+applications.">Viral vectors for vaccine applications.</a> Clinical and Experimental Vaccine Research. 2, 97-105 (2013).</li><li>de Cassan, S. 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=The+requirement+for+potent+adjuvants+to+enhance+the+immunogenicity+and+protective+efficacy+of+protein+vaccines+can+be+overcome+by+prior+immunization+with+a+recombinant+adenovirus.">The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus.</a> Journal of immunology. 187, 2602-2616 (2011).</li><li>Krasnykh, V. N., Mikheeva, G. V., Douglas, J. T., Curiel, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+recombinant+adenovirus+vectors+with+modified+fibers+for+altering+viral+tropism.">Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.</a> Journal of Virology. 70, 6839-6846 (1996).</li><li>Wickham, T. 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=Increased+in+vitro+and+in+vivo+gene+transfer+by+adenovirus+vectors+containing+chimeric+fiber+proteins.">Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.</a> Journal of Virology. 71, 8221-8229 (1997).</li><li>Smith, F., Jacoby, D., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+vectors+for+gene+delivery+to+the+nervous+system.">Virus vectors for gene delivery to the nervous system.</a> Restorative neurology and neuroscience. 8, 21-34 (1995).</li><li>Zhou, F., 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=Nuclear+receptor+NR4A1+promotes+breast+cancer+invasion+and+metastasis+by+activating+TGF-beta+signalling.">Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-beta signalling.</a> Nature communications. 5, 3388 (2014).</li></ol>

作者报告没有利益冲突。

我们开发了一种技术, 允许实时成像的 TGF-β/Smad3 信号在单个活细胞。利用这一新方法, 我们以前已经确定了一个子群体的细胞与动态 TGF-β/Smad3 转录活动, 与增强入侵和迁移8。该方法改进了传统的 TGF β信号检测, 如 Smad3 磷酸化和 TGF β靶向基因表达的西方印迹, 通过捕获肿瘤人群中 TGF-β/Smad3 信号的异质性, 突出单细胞生物学的重要性。此外, 单细胞成像的替代方法, 如核易位可以是费时和昂贵的, 如果在活细胞执行, 可能不会总是转化为基因转录16,17。虽然这些方法对检测信号转导的上游通路很重要, 但腺病毒报告系统提供了一个独特的视角, TGF-β/Smad3 信号在个别细胞与生物学功能。
在肿瘤进展和治疗过程中, 我们的方法为实时检测 TGF-β/Smad3 信号通路提供了一种灵敏、重现性的方法。与传统稳定的荧光素酶在活体成像中相似, 在小鼠体内检测 TGF-β/Smad3 信号依赖于细胞数量和启动子活动水平18。虽然皮下组织对分析的干扰不大, 但增加细胞深度会降低信号检测的灵敏度。有可能扩大的方法, 用于其他器官, 如肺和骨骼, 以及其他癌症;然而, 建议进行信号优化或体外成像, 以达到对癌症感兴趣的模型所需的敏感性。
使用腺病毒报告系统的另一个好处是, 它的应用可能扩展到在活细胞中同时分析多个信号通路。在图 2A中, 我们证明了 MDA-MB-231 细胞可以转基因2个单独的腺病毒载体, CMV-GFP 和 CAGA-汤姆。这种双重感染系统可以进一步扩大, 以报告两个不同的信号通路通过荧光记者蛋白和荧光素酶记者 (使用萤火虫和 Guassia 荧光素酶报告蛋白)。我们的实验室已经实现了 BMP/Smad1 和 TGF-β/Smad3 信号通路的同步信号, 在一些活的癌细胞线 (数据没有显示)。
腺病毒报告系统为 TGF β/Smad 信号通路的活体细胞成像提供了一种简便、简便的方法 . 该系统比其他常用的报告系统具有明显的优越性。首先, 报告腺病毒载体具有最高的病毒传导效率, 使其成为一个最理想的细胞系, 是难以传感器19,20,21,22。此外, 随着病毒技术的最新进展, "懦夫" 腺病毒载体可以通过去除大多数的病毒基因组, 允许增加外源 DNA 容量23。与非病毒传递系统相比, 腺病毒载体是一种成本效益高、快速、易于应用的细胞传导机制, 其传递效率大大提高19、20、21。 ,22。
该协议使用第二代腺病毒表达系统, 这是复制无能的哺乳动物细胞, 不表达 E1a 和 E1b 蛋白, 最小化生物安全风险24。然而, 尽管这些增强的生物安全功能, 腺病毒粒子仍然可以传感器的主要人类细胞的高效能25,26。在处理和处置腺病毒污染设备时, 建议美国生物安全等级2和腺病毒载体的遏制。HEK293A 细胞中腺病毒的大规模扩增, 可导致罕见的世代复制能力的 "野生型" 腺病毒27。应进行 PCR 筛选, 以确定野生型污染27。
复制不称职的腺病毒载体是短暂的表达, 不与宿主 DNA20,21集成。建议实验者在使用腺病毒报告系统时, 确定其报告蛋白表达的稳定性, 以证实长期实验的有效性。腺病毒载体在细胞内的表达时间与细胞分裂时间和病毒传播量成正比。为了确保可重现的结果, 关键是要准确地确定每种病毒批次的病毒滴度。此外, 过多的腺病毒可能导致细胞毒性或病变的影响, 重要的是, 一个最佳的教学方法是经验确定每一个细胞排列, 以确保最佳效果。此外,在体内使用腺病毒载体可以诱发免疫应答;在免疫应答不是实验的主要观察, 免疫受损的动物被推荐20,21。当使用腺病毒载体来靶向免疫应答时, 需要额外的质量控制, 尽管使用的是疫苗治疗28,29的佐剂。
腺病毒报告系统可用于广泛的主要和培养细胞线的分裂和非分裂自然20,22。使用腺病毒报告系统, 我们已经达到100% 感染率在一些癌细胞系, 包括脑, 乳房, 结肠直肠以及原代小鼠胚胎成纤维细胞 (MEF) 和犬肾上皮 (MDCK) 细胞。此外, 腺病毒载体可以进一步修改, 以增加细胞类型特异受体的亲和性, 为转导30,31提供改进的器官靶向和功效。
这种方法的应用可以扩展到其他信号通路和细胞宿主。腺病毒报告系统的主要优点包括低价、高传导效率、快速实验设置和缺乏与宿主 DNA21、32的集成。这种方法可应用于许多目前的化验和体内模型, 包括评估新的靶向疗法33。我们希望使用这一技术将增强我们对信号通路和生物过程之间的关系的理解, 导致新的生物发现和新疗法的发展。

转化生长因子β (TGF β) 是人类发展中的一项重要的细胞因子, 它通过由 II 型和 I 型受体1组成的 heterodimeric 复合物发出信号。对 II. 型受体的结合导致 I 型受体的招募和磷酸化, 进而 phosphorylates 下游 Smad2/3 蛋白2,3。这些活化 Smad2/3 蛋白绑定到 Smad4, 形成一个复杂的 translocates 进入细胞核和调控基因转录4。在恒定的情况下 TGF β/Smad 信号被严密地调控;然而, 在许多疾病中, 信号通路被解除管制, 并且经常抗原导致疾病的进展5,6,7。最近的研究表明, 细胞对 TGF β的反应是异质的, TGF β/Smad 活性细胞的亚群是以时间依赖性的方式8,9负责生物功能。TGF β/Smad 信号的常见细胞分析涉及使用固定端点化验, 只提供细胞活动的快照, 而且通常定量平均 TGF β/Smad 效应10。然而, 这些方法可能不能准确地反映 TGF-β/Smad 信号在疾病进展过程中生理状态下的分子行为。基于图像的活细胞分析捕捉细胞和生物过程的动态, 既具有空间又有时间的理解。
我们的目标是开发一种灵敏的高通量方法用于活细胞成像的 TGF β/Smad 信号使用腺病毒试剂。在这里, 我们感染了人类乳腺癌细胞系 MDA-MB-231 与腺病毒表达 Smad3 CAGA 的主题结合序列和荧光素酶 (卢克) 或 td-番茄 (td-汤姆) 报告基因。腺病毒报告系统为质粒的引入提供了一种快速、廉价的方法, 可导致癌细胞株感染率100%。腺病毒报告系统也已成功地应用于细胞系难以染与常规质粒11。在本协议中, 我们将描述一个可重现和无创的过程, 以实现 TGFβ/Smad 信号通路的活体细胞成像在体内和体外。

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>1881024</td>
			<td>Warm in 37 &#176;C waterbath before use</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Scientifix Life</td>
			<td>FBS500-S</td>
			<td>Heat inactivated before use</td>
		</tr>
		<tr>
			<td>Recombinant Human TGF-&#946;1</td>
			<td>PEPROTECH</td>
			<td>100-21</td>
			<td>Aliquot in DDW to make final concentration at 10 mg/mL</td>
		</tr>
		<tr>
			<td>Hoechst-33258</td>
			<td>Tocris Bioscience</td>
			<td>5117</td>
			<td>Dilute in to PBS to make final concentration at 1 &#956;L/mL</td>
		</tr>
		<tr>
			<td>Luciferase Reporter Assay Kit</td>
			<td>Promega</td>
			<td>197897</td>
			<td>Dilute 5x in PBS before use</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>9100-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast fluorescence microscopy</td>
			<td>OLYMPUS</td>
			<td>IX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoGl Luciferin</td>
			<td>Promega</td>
			<td>P1041</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina III In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>CLS136334</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA (10x)</td>
			<td>ThermoFisher</td>
			<td>15400-054</td>
			<td>Diltue to 0.05% (1x) in PBS</td>
		</tr>
		<tr>
			<td>Cell Culture Lysis 5x Reagent</td>
			<td>Promega</td>
			<td>E153A</td>
			<td>Dilute to 1x in DDW</td>
		</tr>
		<tr>
			<td>10% Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>F5554-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293A</td>
			<td>ThermoFisher</td>
			<td>R70507</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>CRM-HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>PRKDC-SCID</td>
			<td>Animal Resources Centre</td>
			<td>SCIDF6</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Chem-supply</td>
			<td>EA043-10L-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Refresh Night Time</td>
			<td>Allergan</td>
			<td>1750D</td>
			<td>Lubricating Eye Ointment</td>
		</tr>
		<tr>
			<td>Solution</td>
			<td></td>
			<td></td>
			<td>Composition</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td>NaH2PO4.2H2O (4 mM); NaHPO4 (16 mM); NaCl (0.12M)</td>
		</tr>
		<tr>
			<td>FBS-DMEM&#160;</td>
			<td></td>
			<td></td>
			<td>5% heat inactivated FBS; 10 &#956;g/mL penicillin; 100 &#956;g/mL streptomycin</td>
		</tr>
	</tbody>
</table>

live cell imaging of the tgf-	&beta;/smad3 signaling pathway in vitro and in vivo using an adenovirus reporter system

转化生长因子β (TGF) 信号调节细胞稳态所需的许多重要功能, 在许多疾病, 包括癌症中普遍发现抗原。TGF β在晚期癌症进展中与转移有强烈牵连, 激活了迁移和侵袭性肿瘤细胞的一个子集。目前的信号通路分析方法侧重于端点模型, 通常试图测量生物事件后的信号, 不反映疾病的渐进性质。在这里, 我们展示了一个新的腺病毒记者系统特定的 TGF-β/Smad3 信号通路, 可以检测转录激活的活细胞。利用Ad CAGA12-Td-汤姆记者, 我们可以在24 小时内达到 100% MDA-MB-231 细胞感染率。使用荧光记者可以实时地对活单细胞进行成像, 直接识别转录活性细胞。用 TGF β刺激感染细胞, 只显示转录活性和参与特定生物学功能的细胞子集。这种方法允许在单个细胞水平高特异性和敏感性, 以增强对与 TGF β信号的生物学功能的认识。Smad3 转录活动也可以在体内实时报告通过应用一个Ad CAGA12-卢克记者。CAGA12-卢克可以用与传统稳定转染的荧光素酶细胞系相同的方式来测量。Smad3在体内植入细胞的转录活性可以通过常规的 IVIS 影像分析, 并在肿瘤进展过程中进行监测, 为 TGF β信号通路的动力学提供独特的洞察力。我们的协议描述了一个有利的报告传递系统, 允许快速高通量成像的活细胞信号通路的体外和体内。这种方法可以扩展到一系列基于图像的检测, 并呈现为基础生物学和治疗发展的敏感和可重复的方法。

活体单细胞成像的体外研究
为了准确评估 TGF-β/Smad 信号在单细胞中的活化作用, 使用腺病毒试剂, 首先确定每一个细胞系的最佳感染多样性 (语言) 是很重要的。当100% 的细胞对腺病毒感染有阳性反应时, 不存在病变或细胞毒性作用, 就确定了最佳的教学语言。为了确定这一点, 我们使用了一个组成性活跃的Ad-巨细胞病毒-Td-汤姆的记者, 作为一个标志的积极感染。腺病毒感染细胞的比例从0到5000的语言依赖方式增加。在2500语言中, 我们观察到了 100% MDA-MB-231 细胞中的 Td-Tom 表达式 (图 1A, 1B)。像素强度/细胞也增加与语言, 提供增强灵敏度和检测转录输出 (图 1C)。因此, 2500 的工作室被用来进行双重感染, 其中细胞感染了Ad-巨细胞病毒-GFP (作为阳性控制, 以积极的方法感染) 和Ad CAGA12-Td-汤姆同时。单细胞呈现不同程度的 Td-汤姆表达式, 表明异种活化的 TGF-β/Smad3 转录在细胞之间的活和固定细胞。(图 2A, 2B)。100% 的 MDA-MB-231 细胞呈现 GFP 表达, 而只有约26% 的细胞显示可检测 TGF-β/Smad3 转录活动24小时后 TGF β刺激, 相比0% 阳性没有 TGF β治疗 (图 2C)。用腺病毒试剂研究生物过程中的 TGF-β/Smad3 转录活性, MDA-MB-231 细胞感染了Ad CAGA12-Td-汤姆在2500语言和受伤口愈合试验。感染的 MDA-MB-231 细胞表现正常的迁移行为和细胞治疗 5 ng/毫升 TGF β显示加强伤口闭合相比, 非 TGF β治疗细胞 (图 3A)。值得注意的是, 在伤口区约62% 个细胞呈现阳性 TGF-β/Smad3 转录活性, 这比 TGF 治疗后的非创伤区 (32%) 明显高于观察 (图3B)。因此, 以腺病毒为基础的试剂验证了它们在活体单细胞体外TGF β信号通路成像的应用。
活体 TGF-β信号成像在体内的实验
CAGA12-卢克记者的敏感性首先是在体外测试, 以响应 TGF β刺激和存在的 TGF β抑制剂。当受感染的 MDA-MB-231 细胞被刺激与 1 ng/毫升 TGF β, 荧光素酶的记者活动增加1200倍的基础未经治疗的 MDA-MB-231 水平。这一反应被12µg/毫升的 TGF β抑制剂 (图 4A) 显著阻断。CAGA12-卢克试剂随后在乳腺肿瘤原位植入动物模型中进行试验。控制和 TGF 抑制剂治疗组显示类似的荧光素酶的记者活动前治疗。然而, 对于对照组, 在 PBS 治疗后, 荧光素酶信号没有改变, 而 TGF β抑制剂治疗的小鼠显示约3倍的信号减少 (图 4B, 4C)。总的来说, 这些结果表明, 在不需要动物牺牲的情况下, TGF β信号活动的实时和实时监测的潜力。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57926/57926fig1v2.jpg"> 图1。腺病毒试剂的测定.MDA-MB-231 细胞在不同的语言中感染了Ad-CMV-Td-汤姆, 并将其播种成12口水井板块。感染后24小时, 细胞被固定, 赫斯特核染色。(A) 图像被用来可视化 Td-Tom 阳性细胞 (红色) 和核 (蓝色), 并通过 ImageJ 量化。(B) Td-Tom 阳性细胞的百分比。(C) Td-Tom 像素强度/细胞正常化为背景。这些数字代表至少3项独立实验。显示的数据是 triplicates 的方法. 刻度条 = 50 µm (200X)。<a href="/files/ftp_upload/57926/57926fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57926/57926fig2.jpg"> 图2。双感染的单细胞成像.MDA-MB-231 细胞双感染ad CMV-GFP和ad CAGA12-Td-汤姆在语言 (2500) 和种子成12口水井板块。24小时感染后, 细胞接受治疗, 或无 5 TGF β。(A)治疗后24小时, 细胞被实时成像, 以可视化 GFP 阳性 (绿色) 和 Td-汤姆阳性 (红) 细胞。细胞被固定和核染色的赫斯特为量化。(B) 图像被用来可视化 GFP 阳性 (绿色), Td-汤姆阳性 (红色) 细胞和细胞核 (蓝色)。(C) 用 ImageJ 量化 GFP 或 Td-Tom 阳性细胞, 计算阳性细胞百分比。这些数字代表至少3项独立实验。所显示的数据是 triplicates 的方法. 统计学意义是由学生的 t 检验 (*** p ≤0.0001) 决定的。刻度条 = 50 µm (200X)。<a href="/files/ftp_upload/57926/57926fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57926/57926fig3.jpg"> 图3。活细胞 TGF β信号促进细胞迁移.MDA-MB-231 细胞感染了Ad CAGA12-Td-汤姆在语言 (2500) 和播种到6口水井板块。24小时感染后, 细胞被划伤使用 P200 吸管尖端创建一个伤口和媒体取代 5 ng/毫升 TGF β。再过24小时, 细胞被固定和核染色的赫斯特的视觉表征。(A) 伤口闭合的相比较, 鳞片棒 = 100 µm (100X)。(B) Td-汤姆阳性 (红) 细胞和细胞核 (蓝色)。鳞片棒 = 50 µm (200X) (C) Td-Tom 阳性细胞通过 ImageJ 量化, 并计算阳性细胞的百分比。这些数字代表至少3项独立实验。所显示的数据是 triplicates 的方法. 统计学意义是由学生的 t 检验 (*** p ≤0.0001) 决定的。<a href="/files/ftp_upload/57926/57926fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57926/57926fig4.jpg"> 图4。活体TGF-β信号成像.(A) MDA-MB-231 细胞感染了Ad CAGA12-卢克, 然后将其播种96个水井板24小时。此后, 细胞在一夜之间存在或没有12µg/毫升 TGF 抑制剂的情况下接受或不使用 1 TGF β。以全细胞裂解酶为基础, 测定荧光素的活性。荧光素酶活性作为诱导的褶皱规范化为无治疗控制。显示的数据是 triplicates 的手段. (B) MDA-MB-231 细胞在植入前感染了Ad CAGA12-卢克24 小时。细胞被植入到乳房脂肪垫两侧的免疫缺陷小鼠。72小时后, IVIS 成像术前和后24小时治疗 (PBS 为对照组和50µg/肿瘤 TGF β抑制剂治疗组)。(C) 用活体图像软件对光子通量进行量化。所显示的数据是每个治疗组的手段 (n = 12). 统计学意义由学生的 t 检验 (** p ≤0.01, *** ≤0.0001) 决定. p/s 是指光子/s.<a href="/files/ftp_upload/57926/57926fig4large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>

Watch this Scientific Journal Video about Live Cell Imaging of the TGF-	ÃŽÂ²/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System at JoVE.com

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

在这里, 我们提出了一个使用腺病毒记者系统的 TGF-β/Smad3 信号活动的活细胞成像协议。该系统实时跟踪转录活性, 可用于体外和活体动物模型中的单细胞.

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 ...

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Nanyang Technological University

Research

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Cancer Research

10.3K 次观看.  University of Melbourne. 在这里, 我们提出了一个使用腺病毒记者系统的 TGF-β/Smad3 信号活动的活细胞成像协议。该系统实时跟踪转录活性, 可用于体外和活体动物模型中的单细胞.

视频: Live Cell Imaging of the TGF-	β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

科研

癌症研究

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

长期活细胞成像对紫杉醇反应细胞命运的评估

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

基于荧光素酶的大抑癌 (拉图) 生物传感器监测河马信号通路活动

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

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.