我们感谢赵先生的细胞培养。这项工作得到了中国自然科学基金的资助 (81570403、81371284号赠款);安徽省自然科学基金 (批准号: 1408085MH158);安徽医科大学优秀青年研究员;安徽省高校优秀青年人才支持方案。

1. 细胞培养和转染
<ol><li>
细胞培养 注: HN4, HNSCC 细胞系, 来源于 HNSCC14的患者。<ol>
<li>Dulbecco 修饰鹰培养基中的培养 HN4 细胞 (DMEM, 4.5 克/升葡萄糖) 补充10% 胎牛血清和抗生素 (100 毫升/ml 青霉素和100µg/毫升链霉素)。孵育细胞在37°c 与 5% CO2。</li>
	</ol>
</li>
	<li>
细胞转染
	<ol>
<li>在转染前的一天, 5 x 105细胞在2毫升/井培养基中没有抗生素的 6-井板。</li>
		<li>稀释 100 pmol CLIC4 siRNA 或炒 siRNA 在250µL 减少血清介质, 轻轻地。稀释5µL 脂质体试剂250µL 减少血清培养基和孵育5分钟. 将稀释后的 siRNA 与稀释脂质体试剂结合, 轻轻搅拌, 室温下孵育20分钟。</li>
		<li>将混合物添加到每个含有细胞和培养基的井中。轻轻摇动盘子来回搅拌。在进行以下 Rh123 标记程序之前, 在 CO2孵化器中孵化37摄氏度的细胞, 24 小时。转染后用正常生长培养基4小时替换培养基。</li>
	</ol>
</li>
</ol>2. Rh123 标签
注: Rh123 用于测量 HN4 细胞中的基质金属蛋白酶1。
<ol><li>CLIC4 siRNA 治疗后 (1 节), 在6井板中使用 HN4 细胞进行以下治疗。每个井中 HN4 细胞的数量足以在盖玻片上重复实验10次。</li>
	<li>用镊子将圆形盖玻片放在新的12井板的底部。板的数量是根据实验设计 (图 1A) 设置的。</li>
	<li>把150µL 100 µg/毫升多聚赖氨酸在每个盖玻片中间的12井板和等待2分钟. 注意不要把多聚赖氨酸放在盖玻片外面。然后 pipettor 彻底删除多聚赖氨酸 (图 1B)。</li>
	<li>去除皿内 HN4 细胞的培养基, 加入1毫升0.25% 胰蛋白酶分离细胞。6分钟后, 加入2毫升培养基以中和胰蛋白酶。将 HN4 细胞通过 pipettor 轻轻混合5次, 在圆形盖玻片上种子 2-6 х 104细胞。安置在孵化器在37°c 与 5% CO2 (图 1C)。</li>
	<li>8小时孵化后, 用2µmol/升 Rh123 HN4 细胞孵化37摄氏度40分钟。轻轻地混合中上下几次, 以确保均匀分布在介质中的 Rh123 (图 1D)。</li>
	<li>准备足够的正常的生理盐水溶液 (NPSS);使 NPSS (在毫摩尔中): 140 氯化钠, 5 氯化钾, 1 氯化镁2, 1 CaCl2, 10 葡萄糖, 5 HEPES, pH 7.4)。在水浴预热到37摄氏度。</li>
	<li>使用镊子去除盖玻片和洗涤剩余的液体通过简要地安置盖玻片入预热的 NPBS 缓冲 (图 1E)。</li>
	<li>将盖玻片放在500µL 不锈钢腔的底部 (见材料表), 可更换的25毫米玻璃盖玻片底部密封到位的 o 形环。通过收紧房间的盖子来修复它 (图 1E)。</li>
	<li>将450µL 预热的 NPSS 缓冲器添加到组装腔内, 在荧光显微镜或共聚焦激光扫描荧光显微镜 (图 1E) 的视野下放置浴缸。</li>
</ol>3. 检测基质金属蛋白酶
<ol><li>
普通荧光显微镜
	<ol>
<li>按照制造商的说明打开荧光显微镜。 注: 共用荧光显微镜完成了汞灯光纤光源, 一个 FITC 过滤器设置为 Rh123 (480/40 励磁过滤器, 505 LP 分色镜和535/40 排放过滤器) 在室温下。20X. 目标被用来进行活细胞成像。</li>
		<li>打开成像系统软件, 从命令栏中选择 "新建" 按钮开始新的实验 (图 2a)。</li>
		<li>调整可视字段和焦点以在白光中查找单元格的位置。</li>
		<li>关灯, 按按钮关闭白光。单击命令栏上的 "焦点" 按钮, 然后选择 "开始聚焦" 以打开荧光。通过显微镜再发现细胞的位置与507毫微米励磁和529毫微米长的通行证发射波长 (在实验之前设置)。如果需要, 调整可视字段并重新对焦 (图 2B)。</li>
		<li>选择一个仅包含20到30个分离的 HN4 单元格的可视字段。每个细胞的细胞内荧光信号的变化将被准确记录。</li>
		<li>一旦实现了清晰的可视字段, 请单击焦点栏上的 "关闭焦点"。从命令栏中单击 "Acq 一" 以获取一组图像。单击命令栏中的 "区域", 然后单击 "确定" 按钮编辑度量区域。 注意: 从编辑区域栏, 不同的区域形状可以根据研究而使用。</li>
		<li>若要适合单个单元格的不同形状, 请选择 "跟踪区域" 工具, 并在单个单元格区域中循环并在完成后双击。重复该过程, 直到所有单元格都被圆圈 (图 2C)。单击 "完成" 并选择 "保存图像" 以查找保存的位置。</li>
		<li>点击 "Zeroclock", 快速推 F4 开始监测荧光强度的变化。每5秒记录一次荧光强度的实时变化5分钟 (图 2D)。</li>
		<li>当基线趋于稳定时, 将50µL NPSS 与0.5 µL 100 毫摩尔/升 ATP 混合在一起, 通过 pipettor。记住不要触摸房间以避免删除视觉领域 (图 2E)。 注: 荧光图像将记录20分钟, 然后实验可以通过手动操作结束。时间期限通常被安定为20分钟为了观察整个荧光变化。然而, 当意想不到的因素影响曲线的稳定性时, 或当整个过程少于20分钟时, 就应用了手动停止捕获的操作。</li>
	</ol>
</li>
	<li>
共焦激光扫描荧光显微镜
	<ol>
<li>根据制造商的说明打开共焦激光扫描荧光显微镜, 并开启捆绑的软件。 注: 在这里, Rh123 是兴奋的氩激光设置在 488 nm 和发射的信号是记录在 545-700 nm 的范围内通过应用的 TD488/543/633 过滤器。</li>
		<li>点击 "获取" 菜单下的 "目标", 选择 63 x/1.4 油物镜。观察标本, 在光场下找到一个清晰的视野。</li>
		<li>按下 "TL/IL" 按钮切换到荧光模式, 并选择正确的荧光过滤器, 以找到在黑暗中的细胞的位置, 通过显微镜。当观察完成时, 按 "快门" 来保护标本。</li>
		<li>在 "获取" 菜单中, 调整合适的 PMT 通道并单击 "实现" 以激活已结算的光路径 (图 3A)。点击 "配置" 菜单, 选择 "激光" 来确定所需的激光类型。本协议建议使用 "氩气" (图 3B)。</li>
		<li>单击 "实时" 以获取实时图像, 并确保可视字段仅包含20到30个分离的 HN4 单元格。每个细胞的细胞内荧光信号的变化将被准确记录。</li>
		<li>在 "获取" 菜单 (图 3C) 下的 "获取" 子菜单项中, 选择 "xyt"。分别设置 5 s 和20分钟的时间间隔和工期。当所有参数都解决时, 单击 "应用"。</li>
		<li>在 "量化" 菜单下, 使用 "绘制折线" 工具环绕每个单元格的记录区域。选择 "线条轮廓", 然后选择 "开始" 进行观察。记录荧光强度的实时变化为5分钟 (图 3D E)。</li>
		<li>当基线趋于稳定时, 将50µL NPSS 与0.5 µL 100 毫摩尔/升 ATP 混合在一起, 通过 pipettor。切记不要触摸房间, 以免移除视野。 注: 荧光图像将记录20分钟, 然后完成实验。</li>
	</ol>
</li>
</ol>4. 统计分析
<ol><li>
普通荧光显微镜
	<ol>
<li>按照操作员的手册, 定期打开荧光显微镜。打开成像系统软件, 然后从命令栏中选择 "打开" 按钮以打开保存的实验。单击 "命令栏" 中的 "区域", 然后单击 "确定" 按钮编辑度量区域。</li>
		<li>选择 "跟踪区域" 工具, 然后在单个单元格区域中循环, 完成后双击。重复该过程, 直到所有单元格都已被圆圈。单击 "完成" 并选择 "F4: 向前" 按钮以获得显示荧光强度的痕迹。</li>
		<li>完成后, 单击位于图表右下角的向下箭头按钮, 然后单击 "显示图形数据" (图 2F)。单击 "确定" 两次, 然后选择 "日志数据" 按钮打开数据处理软件的接口。再次点击 "记录数据", 发现电子表格上出现荧光强度实时波动的记录。 注: 对于每个细胞, 在应用 ATP 或 TG (F1/F0) 之前, 基质的变化显示为荧光相对于强度的比值。每个荧光强度数据平均为 20-30 细胞。Rh123 的数据显示为平均值. 2 组的结果由双尾独立t检验和P值 < 0.05 被认为是统计学意义。采用统计分析软件进行统计分析。</li>
	</ol>
</li>
	<li>
共焦激光扫描荧光显微镜
	<ol>
<li>打开共焦激光显微镜, 并按照制造商的说明登录到捆绑的软件。</li>
		<li>选择 "开火" 打开录制的实验。</li>
		<li>选择 "量化" 菜单下的 "堆栈配置文件" 工具, 选择 "全部在一个" 从 "按通道和 ROIs 排序图表"。</li>
		<li>使用 "绘制多边形" 工具来圆细胞区域进行观察。</li>
		<li>选择 "图形" 菜单, 然后单击鼠标右键, 然后选择 "导出到 excel", 将荧光强度值添加到电子表格中。下面的分析与正常的荧光显微镜 (图 3F) 是一致的。</li>
	</ol>
</li>
</ol>

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S., Ha, T. J., Yoon, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanidin-3-glucoside+Inhibits+ATP-induced+Intracellular+Free+Ca(2+)+Concentration,+ROS+Formation+and+Mitochondrial+Depolarization+in+PC12+Cells.">Cyanidin-3-glucoside Inhibits ATP-induced Intracellular Free Ca(2+) Concentration, ROS Formation and Mitochondrial Depolarization in PC12 Cells.</a> Korean J Physiol Pharmacol. 18, 297-305 (2014).</li><li>Paddock, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practices+of+laser+scanning+confocal+microscopy.">Principles and practices of laser scanning confocal microscopy.</a> Mol Biotechnol. 16, 127-149 (2000).</li><li>Paddock, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+boldly+glow...+applications+of+laser+scanning+confocal+microscopy+in+developmental+biology.">To boldly glow... applications of laser scanning confocal microscopy in developmental biology.</a> BioEssays : news and reviews in molecular, cellular and developmental biology. 16, 357-365 (1994).</li></ol>

作者没有什么可透露的。

有充分的文献表明, Cl−通道是必不可少的保持止血的内部环境, 并发挥重要作用, 细胞增殖和凋亡15,16。因此, 了解离子通道靶向干预与细胞凋亡之间的关系, 对于寻找一种更好的治疗方法对17种癌症有很大的必要性和意义。线粒体维持细胞正常的生物状态, 其功能与细胞膜通透性和跨膜电位密切相关。除了癌症, 积累病理调查记?...

Rh123 是一种阳离子荧光染料, 可作为跨膜电位的指标。Rh123 能够穿透细胞膜, 进入线粒体基质, 这取决于细胞膜内外电位的差异1。细胞凋亡导致线粒体膜完整性受损。线粒体通透性过渡孔隙 (MPTP) 将打开并导致基质细胞的崩溃, 从而使 Rh123 释放到线粒体的外部。最后, 在荧光显微镜下检测出更强的绿色荧光信号。有充分的证据表明, 细胞凋亡和高膜通透性的减少是2的早期迹象。因此, Rh123 可用于检测基质金属蛋白酶的变化和细胞凋亡的发生。
头颈癌作为世界上第六大常见的癌症, 严重恶化了一个人的健康3。虽然近年来发展了许多方法, 但治疗头颈部鳞状细胞癌 (HNSCC) 患者的临床疗效仍不理想4。探索新的治疗方法可以提高 HNSCC5的治疗效果。涉及许多生物过程的离子通道在不同的癌症的发展中显示了重要的作用6。氯离子通道的部分或全部参与高度参与了肿瘤转化的各种性质, 包括主动迁移、高增殖率和侵袭性。有鉴于此, 新的蛋白质家族点击已被列为癌症治疗6、7治疗靶点。最近的研究表明, 点击家族的成员, 包括 CLIC1, CLIC4 和 CLIC5, 定位到心脏线粒体和活性氧 (ROS) 水平上调的 CLIC5, 表明线粒体定位 Cl的功能作用-在凋亡反应中的通道8。CLIC4, 点击家族的一个成员 (也称为 mtCLIC, P64H1 和 RS43), 已被广泛研究, 其凋亡调节性质的癌细胞和亚细胞位置, 包括高尔基, 内质网和线粒体的人角质细胞7,9,10。CLIC4 的表达谱由肿瘤坏死因子α (TNF α)、P53 和外部刺激调节。CLIC4 的过度表达和下调引起的凋亡反应主要是通过线粒体通路伴随 Bcl-2 家族成员的不平衡, 活化 caspase 叶栅和释放细胞色素 C11,12,13. 因此, 基质金属蛋白酶的测量对探讨 CLIC4-related 细胞凋亡至关重要, Rh123 是理想的荧光指标。
本研究描述了一个详细的协议, 以检测基质干细胞研究 CLIC4 击倒诱导凋亡的 HN4 细胞。Rh123 用作荧光探针观察基质金属蛋白酶的变化。在普通荧光显微镜和共聚焦激光扫描荧光显微镜下, 可解决该基质金属蛋白酶的实时波动。本文详细讨论了共焦激光扫描荧光显微镜的优点和局限性, 并与其他方法进行了比较。该协议也可应用于其他与凋亡相关的研究。

<table border="0" cellpadding="0" cellspacing="0" style="width:1296px;" width="1294">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">HNSCC cells</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:183px;">CRL-3241</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polylysine</td>
			<td>Thermo Fisher Scientific</td>
			<td>P4981</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Specific siRNA for human CLIC4</td>
			<td>Biomics</td>
			<td>NM_013943</td>
			<td style="width:559px;">(accession numbers, NM_013943; corresponding to the cDNA sequence 
			5-GCTGAAGGAGGAGGACAAAGA-3) and scrambled siRNA (5 ACGCGUAACGCGGGAAUUU-3) were designed and obtained from Biomics Company</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamine 2000 Transfection Reagent&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000-015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Opti-MEM I Reduced Serum Medium, GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>51985-042</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rhodamine 123, FluoroPure grede</td>
			<td>Thermo Fisher Scientific</td>
			<td>R22420</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:372px;">Dulbecco&#8217;smodified Eagle medium (DMEM, 4.5 g/L glucose)</td>
			<td>Gibco</td>
			<td>11965-084</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">Fetal Bovine Serum, Qualified, Australia Origin</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">Trypsin-EDTA Solution</td>
			<td>Beyotime</td>
			<td>C0201</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">Antibiotic-Antimycotic, 100X</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser Scanning Confocal Microscopy</td>
			<td>Leica Microsystems GmbH</td>
			<td>LEICA.SP5-DMI6000-DIC</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nikon Eclipse TE300 Inverted Microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Metaflour, V7.5.0.0</td>
			<td>Universal Imaging Corporation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Leica application suite, v2.6.0.7266</td>
			<td>Leica Microsystems GmbH</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microsoft office Excel 2007</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sigma Plot 12.5</td>
			<td>Systat Software</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Attofluor Cell Chamber</td>
			<td>Thermo Fisher Scientific</td>
			<td>A7816</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of mitochondria membrane potential to study clic4 knockdown-induced hn4 cell apoptosis in vitro

线粒体膜电位的耗竭 (基质金属蛋白酶, ΔΨm) 被认为是凋亡级联的最早事件。它甚至发生在核凋亡特征之前, 包括染色质凝结和 DNA 断裂。一旦基质细胞崩溃, 癌细胞的凋亡就会不可逆转地启动。一系列的亲脂阳离子染料可以通过细胞膜和聚集在线粒体基质内, 作为荧光标记来评价基质金属蛋白酶的变化。CLIC4 作为 Cl 胞内通道 (点击)家族的六名成员之一, 主要通过线粒体通路参与细胞凋亡过程。在这里, 我们描述了一个详细的协议, 通过监测罗丹明 123 (Rh123) 的荧光涨落来测量基质, 通过它, 我们研究 CLIC4 的凋亡诱导。本文详细讨论了共焦激光扫描和正常荧光显微镜的优点和局限性, 并与其它方法进行了比较。

在本研究中, Rh123 用于检测基质金属蛋白酶。最初, HN4 细胞被培养为以下荧光染色实验。用镊子将圆形盖玻片放在6井板的底部 (图 1A)。盖玻片涂上多聚赖氨酸5分钟, 然后通过 pipettor 去除多聚赖氨酸 (图 1B)。然后, HN4 细胞被 trypsinized, 并在被放置在6井板底部的盖玻片上播种。接下来, 添加适量的 Rh123, 混?...

Watch this Scientific Journal Video about Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro at JoVE.com

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

在这里, 我们提出了一个详细的协议, 应用罗丹明123识别线粒体膜电位 (基质) 和研究 CLIC4 击倒诱导 HN4 细胞凋亡的体外。在普通荧光显微镜和共聚焦激光扫描荧光显微镜下, 记录了基质金属蛋白酶的实时变化。

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

detection-mitochondria-membrane-potential-to-study-clic4-knockdown

Research

JoVE Journal

Cancer Research

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

10.2K 次观看.  Anhui Medical University. 在这里, 我们提出了一个详细的协议, 应用罗丹明123识别线粒体膜电位 (基质) 和研究 CLIC4 击倒诱导 HN4 细胞凋亡的体外。在普通荧光显微镜和共聚焦激光扫描荧光显微镜下, 记录了基质金属蛋白酶的实时变化。

视频: Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

如何学习基底膜刚度生物物理触发在前列腺癌和其他与年龄相关的病理或代谢性疾病

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

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

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

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

门静脉注射模型来研究肝转移乳腺癌

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

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ability to accurately classify and quantify cell extensions and measure the effect on a cell&#39;s adhesive capacity is critical to determining how cell-signaling events impact cancer cell behavior and aggressiveness. Here, we describe the in vitro design and use of a cell extension quantification method in conjunction with an adhesion capacity assay in an established in vitro model for adrenocortical carcinoma (ACC). Specifically, we test the effects of DKK3, a putative tumor suppressor and a pro-differentiation factor, on the membrane extension phenotype of the ACC cell line, SW-13. We propose these assays to provide relatively simple, reliable, and easily interpretable metrics to measures these characteristics under various experimental conditions.

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

细胞膜延伸特性及其在癌细胞黏附动力学中的作用研究

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

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

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

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

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

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

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

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

将操纵的肿瘤相关中性粒细胞转移到肿瘤携带小鼠中,以研究其血管生成潜力在Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

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 ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.