这项研究是由国家癌症研究所的院内研究计划中的一部分支持。

1。休眠和转移性肿瘤细胞株的细胞文化维护<ol><li>成长在10厘米的培养板包含贝科改良Eagle培养基（DMEM），高糖和10％胎牛血清休眠（D2OR / MCF7/K7M2-AS.46）和转移性肿瘤细胞（D2A1 / MDA - MB - 231 / K7M2）（ FB）和抗生素。一旦细胞达到70％至80％汇合，进行以下的检测。 </li></ol> 2。培养细胞处于休眠状态（静态）和转移（扩散）的肿瘤细胞增殖实验在一个三维生物医学工程系统 在三维系统休眠/转移性细胞培养 <ol><li>解冻Cultrex生长因子，减少基底膜提取物（BME）的4 ° C冰箱进行检测前一个晚上。请注意在冰上处理的生物医学工程应在任何时候都。 </li><li>翌日，地方上的一盘冰层流罩内的96孔板。大衣搭配使用饮水机用注射器冰冷的BME 50 -100μL每口井。确保无气泡形成在井。 96孔板中涂与生物医学工程与5％的CO 2在培养箱在37℃放置30分钟。 </li><li>在此期间，吸从休眠或转移性肿瘤细胞（第1条编制）媒体。冲洗培养板，用10 ml磷酸盐缓冲生理盐水，pH值7.4（PBS）的。吸的PBS和加入2ml胰蛋白酶预热在37 ° C，培养板。在饱和湿度，5％CO2孵育板在37℃，5分钟。 </li><li>转移细胞15毫升的锥形管中，辅以10％胎牛血清和抗生素的DMEM高葡萄糖5毫升和细胞计数。 </li><li>离心1500克速度在室温5分钟，培养一个组织文化的离心机的细胞总数。在我们的实验中，我们准备为每个单元格的行或时间点2X10 3细胞/孔进行审查。然而，这可能会有所不同，这取决于使用的细胞线。 </li><li>小心吸出上清液。请注意，在多数情况下是不可见的沉淀。因此，留下一些媒体的背后。用手指轻按15毫升锥形管的底部，以确保获得一个单细胞悬浮液。重新悬浮颗粒辅以抗生素用DMEM低血糖的2％FCS + 2％的BME（化验媒体）。应加100μL的检测媒体2X10每3细胞。磨碎的细胞多次与5毫升吸管，以确保保持单细胞悬液。 </li><li>板100μL的细胞混合，每孔96孔BME涂层板的顶部。背景评估（第2.8节）除了​​100μL，每板以及只有96 BME涂层板顶部的检测媒体。在加湿5％CO2培养箱孵育培养96孔板，在37 ° C 。细胞应该重新喂养，每4天检测媒体。 </li></ol> 增殖实验： <ol start="8"><li>细胞增殖实验：添加到所需的时间，井点20μL细胞滴度96水溶液的一个解决方案细胞增殖检测试剂盒。饱和湿度5％CO2培养箱中孵育在37 ° C为2h。使用ELISA酶标仪记录吸光度在490nm处。背景评价和减法，添加细胞滴度96水溶液的一个解决方案细胞增殖检测试剂盒20μL井预涂层与生物医学工程和检测媒体覆盖。使用ELISA酶标仪记录吸光度在490 nm。 </li></ol> 3。免疫荧光染色细胞信号分子，在休眠状态的肿瘤细胞（静态）和/或转移性肿瘤细胞（增殖） 在三维系统immunfluorescence染色培养休眠/转移性细胞 *以下协议是一个3D的文化Debnath 说 J 等 18公布的协议的修改。 <ol><li>准备在2.1节中所述的生物医学工程。第二天：8室载玻片系统放在一盘冰层流罩内。冰冷的BME使用200μLpipetman 50ul大衣每口井。确保生物医学工程是均匀分布，无气泡形成在井。 8室玻璃幻灯片涂在饱和湿度5％的CO 2与生物医学工程在37 ° C放置20分钟。 </li><li>收获的休眠状态，或转移性细胞从第1条和2.3 - 2.4节所述，准备文化。收集在15毫升的锥形管培养细胞的总数。我们准备5 × 10 3细胞/孔为每个细胞株和时间点进行检查。在组织文化的1500克速度离心机离心的细胞，在室温5分钟。仔细吸出上清液。注意，球团是不可见的，因此留下一些媒体背后。用手指轻按15毫升锥形管的底部，以确保获得单细胞悬液。重新挂起检测媒体沉淀。应增加5X10每3细胞检测媒体400μl。磨碎用5ml移液管细胞的许多倍。这一步非常重要，以确保保持单细胞悬液。 </li><li>细胞混合涂与生物医学工程8商会每板400μl。培养8商会载玻片系统在加湿5％的CO 2培养箱培养，在37 ° C。细胞应该重新喂养，每4天检测媒体。 </li></ol> 免疫荧光染色： <ol start="4"><li>在所需的时间点上，吸出上层的媒体和添加200μL含4％多聚甲醛（PFA），5％蔗糖和0.1％Triton X - 100的的固定液，在室温下孵育5分钟。吸出固定液，并添加4％的煤灰200μL含有5％的蔗糖和孵化，在室温下放置25分钟。 </li><li>吸出固定液，加400μL磷酸盐缓冲液（PBS），以每口井。孵育10分钟，在室温。吸出PBS，加入400μ用含0.05％吐温20在室温下10分钟。 </li><li>座为10％驴血清200μL或1小时（阻止使用的解决方案应确定为每个主抗体经验）3％的牛血清白蛋白在室温下固定细胞。 </li><li>吸出阻塞的解决方案，并添加200μL抗体（稀释应确定要使用的每所小学的抗体经验）。稀释10％驴血清抗体，如果10％驴血清阻塞，或在3％BSA的主要抗体，如果使用3％BSA封闭液稀释。孵育一抗4℃过夜° C。 </li><li>吸的抗体，用400μlPBS洗井15分钟，并重复两次。吸出PBS和添加驴200μL反各自的抗体结合罗丹明红色（稀释应凭经验确定），覆盖铝箔和1小时在室温下孵育8室幻灯片。 </li><li> 400μlPBS（3x15分钟洗），洗井。吸PBS。装用DAPI VECTASHIELD安装介质。 40分钟，在室温下在黑暗中的干幻灯片。幻灯片可以保持1周，在4 ° C商店在黑暗中的幻灯片。共聚焦显微镜图像幻灯片。 </li></ol> 4。代表性的成果： 一个休眠D2.0R在3D文化D2A1转移性肿瘤细胞的扩散分析的一个例子是在图1A所示。 D2.0R细胞处于休眠状态（静态）通过整个实验的14天的文化时期，而高转移D2A1细胞仍然蛰伏仅4至6天之后，他们开始增殖。在最初的休眠期，许多细胞保持的3 - D的文化孤立（ 图 1B，第4天），而其他非增殖细胞形成多细胞球体。在3 - D文化过渡D2A1从休眠细胞增殖状态（ 图1B; 12天）是与细胞形态的剧烈变化。因此，这个实验可以用来测试什么因素/ s可能触发休眠D2.0R细胞摆脱其休眠状态，什么因素/ S可能会阻止其休眠状态D2A1细胞过渡。 图2是一个代理的例子防止D2A1细胞从休眠增殖状态的过渡。正如图2所示，D2A1细胞的肌球蛋白轻链激酶（ML - 7）的特异性抑制剂治疗保持D2A1细胞处于休眠状态。 在三维系统中培养的处于休眠状态和增殖的肿瘤细胞中的细胞信号可通过免疫荧光染色细胞信号分子的研究。正如图3所示的一个D2A1细胞（红染色）肌球蛋白轻链磷酸化显着增加，其次是F -肌动蛋白细丝形成的肌动蛋白压力纤维（绿色染色）重组过程中出现的过渡，从休眠（1-4天）扩散（7天）。然而，阻断肌球蛋白轻链均有产品或特定的药物（ML - 7）在D2A1细胞激酶的活性保留D2A1细胞处于休眠状态，抑制肌球蛋白轻链磷酸化和F -肌动蛋白压力纤维组织（ 图4）的结果。 <img alt="图1" src="/files/ftp_upload/2914/2914fig1.jpg" /> 图1。 体外模型来研究孤立肿瘤细胞的休眠和转移性增长开关。一）休眠D2.0R扩散和转移D2A1的，在3 - D Cultrex BME，N = 8，平均（± SE ）。三个实验（* P≤0.05）的代表结果。二）培养D2.0R和D2A1细胞的光镜图像在3 - D Cultrex BME放大倍率X20 。图修改巴坎等17。 <img alt="图2" src="/files/ftp_upload/2914/2914fig2.jpg" /> 图2。防止在抑制肌球蛋白轻链激酶（MLCK）的三维培养系统的D2A1（静止）从休眠细胞增殖的开关。时间当然D2A1细胞培养增殖的3 - D，N Cultrex BME = 8，平均（± SE）。细胞未处理（对照组），或与MLCK的特异性抑制剂（ML - 7; 5微米）培养5天48小时开始治疗。图修改巴坎等17。 <img alt="图3" src="/files/ftp_upload/2914/2914fig3.jpg" /> 图3。其次是F -肌动蛋白重组的肌球蛋白轻链磷酸化在从休眠D2A1细胞增殖生长的开关。 D2A1细胞培养8室载玻片上，在3 - D Cultrex BME 。细胞的DAPI（蓝色）核本地化，鬼笔环肽（绿色）F -肌动蛋白与抗体对磷酸化形式的肌球蛋白轻链（MLC - P）（红色），表示在不同的时间点，固定和染色。 F -肌动蛋白的MLC - P染色（黄色）的合并。 D2A1（日数1 - 4）从休眠细胞增生性增长（7天），肌动蛋白应力纤维的形成（箭头）的过渡期间增加的MLC - P的表达。共聚焦显微镜，放大倍率x63。白条等于20微米。图修改巴坎等17。 <img alt="图4" src="/files/ftp_upload/2914/2914fig4.jpg" /> 图4。抑制肌球蛋白轻链激酶（MLCK）D2A1细胞介导的F -肌动蛋白应力纤维形成D2A1细胞未处理（对照组），或与MLCK的（ML - 7，5微米）抑制剂治疗，48小时开始对文化日5，或炒或MLCK的shRNA的治疗，并为磷酸化的肌球蛋白轻链（MLC - P）（红色），F -肌动蛋白（绿色），和细胞核（蓝色）形式染色。 F -肌动蛋白的MLC - P染色（黄色）的合并。共聚焦显微镜，放大倍率x63。白条等于20微米。

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没有利益冲突的声明。

的基本机制，保持传播的肿瘤细胞处于休眠状态或在其过渡到转移性增长的结果仍是未知。这种现象已经非常困难的研究在人类患者4,12和少数临床前模型已开发来解决这个问题。然而， 在体内和体外模型系统，为肿瘤休眠的 ​​一些特点 （1,12审查）。然而， 在体内肿瘤休眠模式主要用来验证规范肿瘤休眠的 ​​潜在机制，但不适合进行实时探索一个单一?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> DMEM高糖</td><td> Invitrogen公司</td><td> 11965-118 </td><td></td></tr><tr><td> DMEM培养液低血糖</td><td> Invitrogen公司</td><td> 11885-092 </td><td></td></tr><tr><td>胎牛血清（FBS） </td><td> Invitrogen公司</td><td> 10091-148 </td><td></td></tr><tr><td>生长因子的3 - D Cultrex减少基底膜提取</td><td> Trevigen公司</td><td></td><td>蛋白质浓度之间14-15mg/ml </td></tr><tr><td> D2.0R和D2A1细胞株</td><td></td><td></td><td> 5,19 </td></tr><tr><td> K7M2和K7M2AS1.46细胞</td><td></td><td></td><td> 20 </td></tr><tr><td> MCF - 7和MDA - MB - 231乳腺癌细胞</td><td> ATCC </td><td></td><td></td></tr><tr><td>一个8室载玻片系统</td><td> （实验室TEK，Thermo Scientific的） </td><td> 177402 </td><td></td></tr><tr><td>细胞滴度96水溶液的一个解决方案细胞增殖检测试剂盒</td><td> Promega公司</td><td> G3580 </td><td></td></tr><tr><td> VECTASHIELD安装用DAPI媒介</td><td>矢量安全检测实验室公司</td><td> H - 1200 </td><td></td></tr><tr><td>正常驴血清</td><td>杰克逊ImmunoResearch </td><td> 017-000-121 </td><td></td></tr><tr><td>酶标板阅读器</td><td>生物-​​ TEC </td><td></td><td>记录490nm处</td></tr><tr><td>共聚焦显微镜</td><td>蔡司LSM - 510 </td><td></td><td>放大倍率x63 </td></tr></tbody></table>

an in vitro system to study tumor dormancy and the switch to metastatic growth

乳腺癌复发往往遵循一个潜伏期长，其中有没有癌症的迹象，并转移可能不会成为多年后切除原发肿瘤的辅助治疗，直到临床明显。这种现象的一个可能的解释是，肿瘤细胞有种子的转移部位，耐常规疗法，并保持时间 1-4长时间处于休眠状态。 以前在辅助站点休眠癌细胞的存在已被描述为静态，既不增殖也不是孤立发生凋亡 5-7的细胞。此外，这些孤立的细胞已被证明传播从原发肿瘤的早期疾病进展8-10的阶段，并驻留在病人的骨髓，血液和淋巴结1,4,11中生长被捕。因此，了解的机制，规范增殖状态的休眠或交换机是发现新的目标和干预措施，以防止疾病的复发的关键。然而，揭开肿瘤休眠开关调节转移性增长的机制已经阻碍了缺乏可用的模型系统。 在体内和体外模型系统研究进展肿瘤细胞的转移已被描述以前 1,12-14 。然而，这些模型系统并没有提供实时和高通量的方式进入孤处于休眠状态的肿瘤细胞增殖转移性疾病的出现触发什么机械的见解。最近，我们在体外系统开发的一个三维模型 ， 在细胞体内生长的特点，表现出休眠（D2.OR MCF7，K7M2 - AS.46）或增生（D2A1，MDA - MB - 231，K7M2）转移性行为在体内 。我们证明了，表现在体内肿瘤细胞在转移部位的休眠保持静态时，在一个3维（3D）基底膜提取物（BME）培养，而高度在体内转移的细胞很容易在三维培养增殖后，变量，但相对较短时期的平静。重要的是利用体外模型系统中的三维，我们首次展示了ECM的成分，起着重要的调节是否处于休眠状态的肿瘤细胞会切换到增殖状态，并确认 在体内研究15-17作用。因此，本报告中所描述的模型系统提供了在体外培养的方法，以模型的肿瘤休眠研究的微环境诱导的增殖生长的过渡。

Watch this Scientific Journal Video about An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth at JoVE.com

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

修改后的3 - D在体外培养系统是其中的几个重组基底膜的肿瘤细胞株的生长特性与休眠或增生的肿瘤细胞转移的辅助站点的行为相关<em&gt;在体内</em&gt;。

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

an-vitro-system-to-study-tumor-dormancy-switch-to-metastatic

Research

JoVE Journal

Medicine

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

15.7K 次观看.  University of Haifa. 修改后的3 - D在体外培养系统是其中的几个重组基底膜的肿瘤细胞株的生长特性与休眠或增生的肿瘤细胞转移的辅助站点的行为相关在体内。

视频: An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

利用经颅磁刺激，以研究人类的神经肌肉系统

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

科研

医学

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

测量和制止在弱视治疗

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

同基因小鼠黑色素瘤细胞注射，以确定其在肺转移潜能

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

利用视网膜成像研究痴呆

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

大鼠前庭系统的外科迷路切除术研究

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

大鼠 DRG 压缩模型的标准化 Tuina 协议

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

作者聚焦： 推拿对压迫背根神经节疼痛的大鼠模型的镇痛作用  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

推拿对大鼠模型的镇痛作用，压迫背根神经节疼痛

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

一个系统来研究肿瘤休眠和转移性增长