这项研究的资金来源:特拉维夫大学的技术转移公司，拉莫特和示巴医学中心（M. CA和SI），ICRF - 以色列癌症研究基金会（M. CA）和以色列科学基金会的联合基金（ SI）。

1。细胞培养制备 MDA-MB-231细胞购自美国典型培养物保藏中心 （ATCC），并保存于液氮中。 <ol><li>种子10 6个MDA-MB-231细胞在92毫米直径的陪替氏培养皿在10毫升完全培养基中Dulbeco改进的Eagle培养基（DMEM），10％马血清，1％L-谷氨酰胺和1％两性霉素B。Penstrep允许细胞的激增至约80-100％汇合。 </li><li>从盘取出培养基并丢弃。 </li><li>简单细胞层洗净，用0.25％的胰蛋白酶-EDTA溶液（W / V）的血清清除所有痕迹。 </li><li>添加2.0毫升胰蛋白酶-EDTA溶液菜倒置显微镜观察细胞，直到细胞层分散（通常在5至15分钟内）。 </li><li>加入18毫升完全培养基，用吸管轻轻吸出细胞。转移到管。 </li><li>在1,200 rpm离心细胞悬浮液。 </li><li>重悬细胞沉淀在24毫升的铜介质lture。 </li><li>加入2 ml的细胞悬液，以35毫米的玻璃底菜（约25％汇合），在培养箱（5％CO 2，37℃）。 </li><li> 解决方案: <ol><li>的完整的细胞的增殖培养基:DMEM培养基含10％FBS，1％抗生素（100单位/毫升青霉素G，100微克/毫升链霉素，笔链球菌AMPHO的溶液）和2mM L-谷氨酰胺。 </li><li>胰蛋白酶-EDTA溶液中，含有0.25％的胰蛋白酶-EDTA。 </li></ol></li><li> 菜: 92毫米直径的培养皿。 直径35毫米，聚-D-赖氨酸镀膜玻璃底培养皿。 </li></ol> 2。现场共聚焦成像单元的制备<ol><li>种子2×10 5 MDA-MB-231细胞的玻璃底培养皿中第1节中提到的2毫升完全培养基。当细胞培养达到60-70％汇合（约3-4×10 5细胞每盘），继续转。 </li><li>与ENCOD两个质粒转染的细胞ing的融合蛋白，γ-微管蛋白（GFP荧光检测中心体）和组蛋白红色（H2B红色，荧光检测染色体）采用脂质体转染试剂喷射PI，制造协议。简言之，混合2微克从每个质粒在管用100微升氯化钠（150毫米）。用100μl150mM的氯化钠（）的第二管中的转染试剂（100微升）混合，并在室温（RT）下孵育5分钟。然后将二者结合起来的解决方案，混合（使用温和涡）和自旋向下。在RT下孵育30分钟。 </li><li>在温育的转染混合物，洗涤细胞一次，用PBS和替换的细胞培养基中，用2毫升温暖的DMEM培养液（37℃）没有补充。 </li><li>轻轻加在DMEM培养液中的细胞的转染混合物，然后返回的细胞培养箱中（37℃，5％CO 2）8小时。 </li><li> 8小时的潜伏期后，更换2毫升完全培养基DMEM细胞在孵化器和孵化24小时。 </li><li>转染后24小时，更换的培养基中的细胞，用2毫升完全培养基中含有20μMPJ-34。 </li><li>孵育细胞18小时（37°C，5％CO 2）。 </li><li>主题的细胞生活共焦成像的细胞在5％CO 2和37℃下保持至少16小时，在成像室</li><li>同时，检查转染效率36小时转染后用荧光显微镜如下: <ol><li>种子2×10 5的MDA-MB-231细胞在6孔板含有1盖玻片，在2毫升完全培养基中每孔的。 </li><li>转染的细胞中提到的第2.2-2.5。 </li><li>转染后36小时固定安装在盖玻片上培养转染的细胞在冷甲醇:丙酮（1:1）溶液，7分钟，-20℃下</li><li>吸出固定的解决方案，让安装的细胞，干化学罩盖玻片。 </li><li>申请延长Gold抗淬灭试剂用DAPI，让日É盖玻片干燥6小时在黑暗中。 </li><li>在荧光显微镜下检查的幻灯片，并计算转染的细胞（红色和绿色信号）从总人口的细胞（DNA用DAPI染色）的百分比。 100-200细胞计数时所需的转染百分率大约是20％至40％。 </li></ol></li></ol> 3。现场共聚焦成像扫描仪设置的技术参数<ol><li> ScanMode XYZT;针孔通风] 1.00;变焦3.5;分辨率8位，561纳米激光DPSS氩气，可见激光488纳米激光他/ NE可见的633纳米目的HCX PL APO CS 63X 1.40油抗紫外线;数值孔径1.4;扫描速度700赫兹;折射率1.52。 </li><li> 3-D图像演示制备Imaris里成像软件7.0。 </li></ol> 4。固定细胞有丝分裂的共聚焦成像<ol><li>种子为2×10 5 MDA-MB-231乳腺癌细胞（ATCC），正常小鼠胚胎成纤维细胞（MEF），或PARP1缺陷MEF（PARP - / - ，制备议员弗朗索瓦丝Dantzer）上在2毫升完全培养基的6孔板中的盖玻片。盖玻片，用96％乙醇洗涤后，用无菌DD水洗涤，干燥2小时，并放置在6孔盘的每个孔中。 </li><li> PJ-34（10-30微米）添加到培养基中，室温孵育规定时期内（通常可达96小时）。 </li><li>一旦清洗盖玻片，用PBS（磷酸盐缓冲液），并固定细胞，用孵化，在冰冷的甲醇:丙酮（1:1）溶液，-20℃下，7分钟</li><li>吸固定溶液，并让盖玻片化学罩中干燥（在此阶段，盖玻片可在-20℃保存数周）。 </li><li>用PBST（PBS含0.1％Tween-20的补充），一次清洗盖玻片通透细胞膜，并阻止细胞在RT下的1小时，用10％在PBST NDS（正常驴血清）（封闭溶液）。 </li><li>孵育通透固定细胞原发性antibodiES的RT 2小时（主轴和中心体染色）。如下:抗α微管蛋白（1:250稀释）和抗γ-微管蛋白（1:200稀释）在封闭液稀释抗体。一抗的应用方式如下:适用于将100μl（在下拉式菜单中）为每个盖玻片的6孔​​板盖（盖是上下颠倒）的封闭溶液中的抗体的混合物。轻轻把盖玻片上的抗体下降，面临着下降的种子细胞。孵育面临的抗体在室温下搅拌2小时的盖玻片。 </li><li>将盖玻片孔中，并用PBST洗涤细胞3次。然后用4.6所述的相同步骤，用荧光二级抗体标记细胞在盖玻片上。二次抗体1小时，RT孵育的细胞在盖玻片上，在黑暗中。该抗体稀释如下:封闭液的Alexa Fluor 488（1:1,000稀释，绿色）的Alexa Fluor 568（1:1,000稀释;红色）。 </li><li>安装盖玻片使用延长黄金的抗淬灭试剂用DAPI（染色体染色），在室温孵育过夜，在黑暗干燥。 </li><li>通过共聚焦显微镜检查盖玻片。 </li></ol> 5。 ATP的测量细胞活力 ATP产量测定发光ATP检测试剂盒。 <ol><li>种子细胞的96孔培养板中，在各孔中的800微升培养基中大约20,000细胞。应该用于测定介质的背景发光的三个空白孔。 </li><li>准备约10〜100μm的ATP标准品稀释系列，并保持在冰上。 </li><li>向每孔中加入50μl的洗涤剂和为5分钟，每分钟700转的轨道摇床中摇板。 </li><li>复溶试剂盒中的底物缓冲液，用5ml的的冻干基板的每个管形瓶中。 </li><li>加入50微升底物溶液的重组到各孔中，并摇动板轨道摇床，700转5分钟。 </li><li>保持板在黑暗的10分钟。 </li><li>测量每孔luminiscence用ELISA酶标仪。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

实时共焦成像提供了一种实时PJ-34的细胞毒作用在活的多中心体的细胞在有丝分裂过程中（ 图3和补充信息）的文档。这是第一次现场文档PJ-34在人类癌细胞中的细胞毒性归因额外中心体聚类和细胞死亡，表明诱导细胞死亡的有丝分裂灾难PJ-34 5-9。与此相反，双焦聚类数目的超中心体中观察到活的未处理的MDA-MB-231细胞进行正常的有丝分裂与双向focali的群集的多余的中心体（ <str...

菲衍生PARP1抑制剂，包括PJ-34，旨在保护耗能PARP1介导的DNA修复应力条件下（中风或心肌梗死）1静止期细胞诱导细胞凋亡。然而，最近我们发现，PJ-34，比诱导PARP1抑制浓度高出一倍，完全可以导致细胞死亡在人类癌细胞中2,3。更快速的细胞增殖，细胞更有效地消灭了。 PJ-34的细胞毒活性归因于额外中心体聚集在有丝分裂2。许多人类癌细胞海港multicentrosomes 4,5。孵化人类乳腺癌细胞MDA-MB-231，怀有编外中心体，为20μmPJ-34有效地根除这些细胞在72-96小时内不损害静止细胞或一些良性增生的细胞，窝藏两个中心在有丝分裂<suP&gt; 2,3。良性的细胞包括人类乳腺上皮细胞MCF-10，人类内皮细胞（HUVEC），从人胸腺制备的原代骨髓间充质细胞。这些细胞耐PJ-34的细胞毒活性。 PJ-34没有干扰他们的细胞周期过程中培养96小时，或影响其中心体和双焦主轴形成2,3。 双极双极4,5在有丝分裂纺锤体的形成是至关重要的中心体组装。因此，有两个以上的中心体的细胞已经开发了一种几乎不理解的分子机制，多余的中心体，在两极4-9聚类。双极其中心体组装的失败可能会导致多极化扭曲纱锭和异常染色体分离逮捕细胞周期G2 / M期阻滞，并导致细胞死亡归因于有丝分裂失败4,5。额外中心体聚类的分子机制的深入研究&lt;SUP&gt; 10。了解这个死亡机制将启用独占消灭癌细胞的同时保留健康组织5,10。 因此，激活有丝分裂灾难细胞死亡的化合物有选择性的癌症治疗提供了一种新的模式，这可能是有效率的在广泛的人类固体cancers.Our结果的建议聚焦成像，可用于识别分子影响额外中心体聚类2,3有丝分裂，使这些化合物癌症靶向药物候选人。 我们已经证明通过扫描固定的和活的人体癌症细胞（额外中心体在有丝分裂的发生率较高），相对于正常细胞的细胞毒活性对菲啶PJ 34。一步一步描述用于识别PJ-34在人类癌症细胞的细胞毒活性的成像程序包括下面。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen (GIBCO)</td>
			<td>41965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine Serum)</td>
			<td>Invitrogen (GIBCO)</td>
			<td>12657</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep-Ampho solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-033-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25030-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Tripsin-EDTA</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92 mm Petri dishes</td>
			<td>Nunc,Thermo scientific</td>
			<td>150350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-D-lysine coated glass bottom culture dishes</td>
			<td>MatTek Corporation, USA</td>
			<td>P35GC-0-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminescent ATP detection assay kit</td>
			<td>Abcam</td>
			<td>ab113849</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NDS (Normal Donkey Serum)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti &alpha;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>1:250 dilution (IF)</td>
		</tr>
		<tr>
			<td>Anti &gamma;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5192</td>
			<td>1:200 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Donkey Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-10042</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI (mounting)</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>JetPEI (liposomal transfection reagent )</td>
			<td>Polyplus</td>
			<td>101-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica (Mannheim, Germany)</td>
			<td>TCS SP5II</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells

菲衍生物作为有力PARP1抑制剂阻止编外中心体多中心体的人类癌症细胞有丝分裂的双焦聚类。菲啶PJ-34是最有力的分子。额外中心体的分簇多中心体细胞有丝分裂失败的原因和细胞死亡。最坚实的人力癌症有额外中心体的发生率较高。 PJ-34的活性共聚焦成像活的人乳腺癌MDA-MB-231细胞转染载体编码荧光γ-微管蛋白，这是非常丰富的中心体和荧光蛋白H2B目前在实时记录染色体。转染与PJ-34处理后的MDA-MB-231细胞中检测到异常的染色体安排和γ-微管蛋白聚集疫源代表declustered中心体。非集群额外两个纺锤体两极的中心体细胞死亡之前。这些结果与FOr的第一次检测独家额外中心体的聚类在有丝分裂，有丝分裂失败导致细胞死亡在人类癌细胞中的PJ-34的细胞毒活性。根据以前的研究结果固定细胞共聚焦成像，PJ-34独家消灭癌细胞的多中心体，而不损害正​​常细胞发生有丝分裂，两个中心和双焦主轴的观察。这PJ-34的细胞毒活性不共享等烈性PARP1抑制剂，观察，表明其独立性PARP1抑制PARP1缺失的MEF窝藏extracentrosomes。现场共聚焦成像提供了一个有用的工具，用于识别新的分子，消除细胞在有丝分裂过程中。

PJ-34是一种稳定的水溶性菲啶1（ 图1）。我们先前的研究发现细胞死亡和集群额外中心体在几种类型的固定的多中心体肿瘤细胞，分别用PJ-34。相比之下，正常增殖细胞没有受损2,3。针对centrine1在固 ​​定的额外中心体细胞2和γ-微管蛋白的抗体双标记进行鉴定中心体。 在这里，记录在这些现场额外中心体细胞的杀伤活性PJ-34实时通过现场...

Watch this Scientific Journal Video about Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells at JoVE.com

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

菲PJ-34在肿瘤细胞进行有丝分裂的细胞毒活性，实时记录现场共聚焦成像。 PJ-34根除人类乳腺癌MDA-MB-231细胞有丝分裂窝藏额外中心体。额外中心体不像正常的双向焦点有丝分裂，不聚集在两个纺锤体的两极存在的PJ-34。

共聚焦成像在肝癌细胞中观察到的异常有丝分裂细胞死亡相关

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

cell-death-associated-with-abnormal-mitosis-observed-confocal-imaging

Research

JoVE Journal

Medicine

14.9K 次观看.  Sheba Medical Center. 菲PJ-34在肿瘤细胞进行有丝分裂的细胞毒活性，实时记录现场共聚焦成像。 PJ-34根除人类乳腺癌MDA-MB-231细胞有丝分裂窝藏额外中心体。额外中心体不像正常的双向焦点有丝分裂，不聚集在两个纺锤体的两极存在的PJ-34。

视频: Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

高效率转导肝癌细胞的重组腺相关病毒血清型3载体

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

科研

医学

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

前溶瘤病毒的活组织的体内感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

癌症干细胞迁移的使用进行划分的微流控芯片和活细胞成像的评价

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

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

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

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

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

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

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

建模结肠炎相关的癌症与​​氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

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

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.

评估细胞死亡的吞噬细胞清除由Ligatable荧光探针试验行程

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...