The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.

注：病人组织采购是通过制度化审查委员会（IRB）批准-approved生物样本和临床协议（协议号09-121和11-041，分别）在纪念Sloan Kettering癌症中心。 1，组织采购<ol><li>患者原发肿瘤/转移采购 注：迄今为止，该协议已经在手术切除胰腺癌，胃癌和乳腺癌的肿瘤类型，以及，淋巴瘤转移进行。 <ol><li>直接手术团队提供样品通过快递或气动管道系统的病理学部门在无菌防溢容器密封和无菌防漏塑料样品袋。直接手术团队运送标本的新鲜状态，无甲醛固色剂或。 </li><li>直接病理学家或病理学家助理使用无菌技术收获的新鲜标本，其中公司ludes使用无菌手套和仪器在层流罩。 </li><li>记录采购的标本，这是严格根据30分钟从完成手术过程中不停的收割时间。考虑到冷缺血考虑效果，不采取标本切除标本&gt; 30分钟18冷缺血时间。 </li><li>引导病理科人员以除去以约0.5 cm 3的原发肿瘤样品至1.0 cm 3的在层流罩以保持无菌的环境。如果可能，请从指数病灶周围的肿瘤组织，以避免潜在的坦诚中心坏死（细胞死亡）。 注：坏死组织可以是通过以下任一条件严重识别：颜色或组织的苍白的损失;强度损失在坏死组织是软的，易碎的;坏死和可行的组织之间有明显的分界。 </li><li>直接pathologiST或病理学家助理提供外周组织是过量的（手术丢弃）肿瘤的诊断评估的即时取样后。将样品中含有约含1％抗生素（青霉素和链霉素）的最小必需培养基（MEM）5毫升15毫升的无菌离心管中。 </li><li>如果可用（ 例如，乳房切除标本腋尾部的淋巴结），购买相同尺寸（0.5 毫升 〜1.0 毫升 ）的极正面的关联淋巴结标本并比较原发肿瘤对药物的反应。象的原发肿瘤样品，将含有1％抗生素（青霉素和链霉素）的相关淋巴结样品在一个15毫升的无菌离心管中含有约最低必需培养基（MEM）中的5毫升</li><li>如果手术标本的许可证（ 例如，乳房切除标本），除去（从原发肿瘤的远处）的正常组织的样品（ 即大小，正常密/纤维乳腺实质内），置于含有约含1％抗生素仅传送目的的最低必需培养基（MEM）的5毫升15毫升的无菌离心管中。继转移到实验室设施，在“正常”样本转移到离心管，&#39;捕捉&#39;冻结和存储-80℃冷冻以备将来的分子分析。 </li><li>置于湿冰上所有标本，并立即运送到实验室空间用于离体新鲜组织切片。不要一节正常的标本，但在-80，而存储℃冰箱为未来的分子分析。在-80℃的冷冻以备将来分子分析转移，原发肿瘤的一个离心管，&#39;捕捉&#39;冻结和存储部分和相关的转移。 </li></ol></li><li>从临床研究组织采购（ 如可选的空芯针穿刺活检（CNB）） 注：我们获得的肿瘤组织从患者就读于谁给他们的同意接受之前的疗法开始无障碍肿瘤转移的CNB临床方案。 CNBS，迄今为止，已在患者的乳房，边缘区淋巴瘤，套细胞淋巴瘤，转移性神经节瘤，子宫颈（鳞状细胞癌）和卵巢癌进行。 <ol><li>有从介入放射学或相关部门临床人员进行预处理粗针切片，然后将样品在一个15毫升的无菌离心管中含有约含有1％的抗生素（青霉素和链霉素）最低基本培养基（MEM）的5毫升。 </li><li>置于湿冰上活检标本，并立即运送到实验室空间用于离体新鲜组织切片。 </li></ol></li></ol> 2，准备材料的切片和Vibratome设置<ol><li>在切片准备材料<ol><li>做一个4％的琼脂糖溶液加入4g阿加上升到100毫升的PBS。高压釜（周期设置：液体，121℃，30分钟，15磅每平方英寸）的首次使用之前的溶液。对于以后的使用，将4％的琼脂糖在微波炉液化在每次使用前（大火煮30秒的间隔），并适当地冷却到约25℃（ 即温暖的触摸）铭记琼脂凝固在室温。 </li><li>使用4％琼脂糖作为非永久性组织包埋媒体。当在4％的琼脂糖充分冷却并仍处于液体状态时，用无菌镊子将组织标本中的嵌入模具和倒在4％的琼脂糖。 </li><li>直接在嵌入模具的切削表面壁定位在组织标本接近（〜2毫米的距离），但并非如此。在4％琼脂糖将开始迅速固化，因此适当地快速定位在组织标本。在切片，保持4％琼脂糖在55℃水浴，保持在溶液中。 </li><li>到24孔板的截面分析，加1毫升邻˚F完全培养基（ 即 MEM + 10％胎牛血清+ 1％抗生素）到每个孔中。保持在24孔板上于室温（〜25℃），在整个切片的持续时间。 </li></ol></li><li> Vibratome设置的制备<ol><li>使用振动切片机中，设置在所述叶片接近试样的速度[0（0.00毫米/秒）至10（2.5毫米/秒）]和叶片[0（0赫兹）的频率为10（100赫兹） 。根据组织（ 即密度/硬度）的质地调整设置。 </li><li>为浸润性导管癌原发性乳房肿瘤，调整设置到6和图3，分别速度和频率。对于密度较小/事务所肿瘤组织，降低速度，并相应地提高频率。 </li><li>设置仪器范围内的切片厚度（1 - 999微米）。对于这些分析，将部分厚度为200微米。 </li><li>用一薄层液体粘合剂的在试样圆盘塔装入琼脂糖包埋的标本t被淹没在媒体水库被包裹在湿冰。 </li></ol></li></ol> 3，组织切片，处理及分析<ol><li>紧接在使用时在一个多井板用1毫升完全培养基的无菌镊子和地方分段移除精确切片部分。当获得足够的部分为响应和相关的分析，将多孔板用于在组织培养箱设定在标准组织培养条件的治疗反应。 </li><li>取多个控制部件的书夹的处理部分，以努力确定剂量依赖性响应，而不是培养的工件。进行组织切片快速（〜30 - 40分钟）内的冷缺血时间的任何不利影响以及（〜2 - 4小时）18。进行处理的部分，只有下面有1小时的适应期。 </li><li>继1小时适应期，加上增加剂量（治疗相关; <em&gt;例如，0 - 10μM的浓度）的药物中的部分和内部标准组织培养条件下，用于治疗周期下。对于本研究中，使用24和/或48小时评估周期。然而，控制（车辆只）的组织切片将保持存活，如在本研究中直至并包括96小时评估周期测试的期间。 </li><li>治疗后，通过将组织切片中含有为10％福尔马林，地点在室温Ø管取出无菌条件和福尔马林固定下的所有路段/ N或含4％多聚甲醛管（从瓶中稀释16％的多聚甲醛用PBS的4％的最终浓度），并置于室温2小时。 <ol><li>固定较大的组织切片（ 例如 ，〜的6毫米x 4毫米），200微米厚的10％福尔马林缓冲液中O 2 / N在RT。固定较小的部分（ 例如为1mm×1mm）上200微米厚于4％多聚甲醛进行2小时，在室温。 注：对于固定剂f的体积的有效渗透ixative应20X组织的体积。鉴于此协议的结果非常小的组织的大小（ 例如，6毫米组织3 = 6μL体积），为保证适当的固定使用1毫升固定液为所有规模的组织。 </li><li>以下有固定的组织标本石蜡包埋的病理科人员。部分标本蜡块上收取的幻灯片是苏木精和曙红染色，免疫组化评估（ 如细胞凋亡）。 </li></ol></li></ol>

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No conflicts of interest disclosed.

试图制定有效的治疗策略时，癌症生物学家面临显著挑战。在开发上建立肿瘤细胞系测试药物不能准确地反映体内响应和在上PDX模型体内实验是劳动密集型的并且非常昂贵。鉴于上述情况，原发性患者的体外技术的应用肿瘤14，15现在位于建立肿瘤细胞系和患者来源的异种移植物（PDX）的分子分析的旁边。 本文以体外技术的应用推向?...

发展有效的癌症疗法已被证明是极具挑战性的。肿瘤细胞系和肿瘤外植体-以及异种移植物已被用于癌症研究的半个多世纪1,2,3。迄今为止，药物敏感性和抗性均建立肿瘤细胞系和来自患者的异种移植物中的分子分析（PDX）是不可缺少的。然而，在建立的肿瘤细胞系的化合物的检测是不经常的预测在体内的药效，并对应在体内研究中的动物，特别是在PDX模型，是非常昂贵和费时的。这些模型系统，即无法告知的原生微环境中肿瘤进展和应对治疗策略的影响的局限性，导致了研究领域，开发更多的方法来赞美这些分析。近期，高度地注意对病人TUM的体外分析或外植体4,5由于较大的理解是癌的治疗反应是不排斥对癌细胞的内在分子组成，而是由肿瘤细胞微环境6,7一项功能，不能用传统的培养方法和可概括的影响很大/或PDX。 体外分析在上述情况下（ 即 ，相邻的周围的肿瘤细胞微环境的影响）意味着可行原发性肿瘤/转移部分的评估，而不是细胞株8,9的体外分析。 我们在这里报告的离体技术（ 即 ，精确切片新鲜组织切片）既患者的原发肿瘤和转移相关（ 即 ，淋巴结），忠实地通知关于反应（IC 50），脱靶效应，并允许分子分析性和反馈机制。此外，相关therape分析UTIC敏感性/抗性与生物标记物和基因表达图谱可以在努力确定患者更可能对实验药物的兴趣响应来执行（ 即 ，高的药物响应相匹配的患者与特定生物信息）。在体外的技术和评估的多参数的方式中的应用是对病人的选择和临床疗效的整体改善运动。 体外治疗反应的分析可以成为癌症治疗的临床前和临床开发的标准工具，并设想为对治疗性发展战略的一个个性化医疗方法的一个步骤。

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:96px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome Leica VT1000s</td>
			<td style="width:96px;">Leica</td>
			<td style="width:119px;">14047235613</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UltraPure agarose</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">16500500</td>
			<td style="width:167px;">Prepare 4% and 6% before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Injector blade</td>
			<td style="width:96px;">Ted Pella</td>
			<td style="width:119px;">121-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MEM with Penicillin&#160; + Streptomycin</td>
			<td style="width:96px;">Media Core Facilities (MSKCC)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The media is prepared at Memorial Sloan Kettering Cancer Center&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scalpel no. 10</td>
			<td style="width:96px;">Thermo Scientific</td>
			<td style="width:119px;">31-200-32</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable forceps</td>
			<td style="width:96px;">Cole-Parmer</td>
			<td style="width:119px;">84011182</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Embedding mold</td>
			<td style="width:96px;">Electron Microscopy Science</td>
			<td style="width:119px;">70181</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS (heat inactivated)</td>
			<td style="width:96px;">Gemini</td>
			<td style="width:119px;">100106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 well plates</td>
			<td style="width:96px;">Corning</td>
			<td style="width:119px;">3524</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formalin (10%)</td>
			<td style="width:96px;">Sigma Diagnostics</td>
			<td style="width:119px;">SDHT501128&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">16% Formaldehyde solution</td>
			<td style="width:96px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Embedding microsettes</td>
			<td style="width:96px;">Simport</td>
			<td style="width:119px;">M503-2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol (70%)</td>
			<td style="width:96px;">Fisher Scientific</td>
			<td style="width:119px;">A405P-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Waterbath</td>
			<td style="width:96px;">Fisher Scientific</td>
			<td style="width:119px;">15-462-2SQ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microwave</td>
			<td style="width:96px;">General Electric</td>
			<td style="width:119px;">ModelJES2051DNBB</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adhesive (Ethyl Cyanoacrylate)</td>
			<td style="width:96px;">Sigma-Aldrich</td>
			<td style="width:119px;">E1505-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10mm dishes</td>
			<td style="width:96px;">BD Falcon</td>
			<td style="width:119px;">353003</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">15ml tubes</td>
			<td style="width:96px;">BD Falcon</td>
			<td style="width:119px;">352096</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

在这项研究中， 体外技术，用在热休克蛋白90抑制剂（Hsp90i）治疗敏感性/抗性的相关性分析。在此Hsp90i，乳腺癌原发肿瘤中，ER +浸润性导管癌（IDC），以及相关的淋巴结转移的临床前评估，分析体外对治疗的反应&#160; （ 图1）。多个200微米的连续切片分别用车辆之用，增加剂量的Hsp90i（0.25，0.50，1.0和2.5μM）。 图1中的数据表示福尔马林固定，?...

Watch this Scientific Journal Video about Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics at JoVE.com

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

建立癌症细胞系和裸鼠已经癌症研究的中流砥柱，在过去的几十年。然而，最近的证据表明，治疗响应在很大程度上是通过肿瘤细胞微环境的影响。因此，我们开发了原发肿瘤标本进行药物开发目的的体外分析。

原发性肿瘤和/或转移相关的临床前和治疗的临床开发的离体处理响应

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

ex-vivo-treatment-response-primary-tumors-andor-associated-metastases

Research

JoVE Journal

Medicine

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

14.6K 次观看.  Memorial Sloan Kettering Cancer Center. 建立癌症细胞系和裸鼠已经癌症研究的中流砥柱，在过去的几十年。然而，最近的证据表明，治疗响应在很大程度上是通过肿瘤细胞微环境的影响。因此，我们开发了原发肿瘤标本进行药物开发目的的体外分析。 

视频: Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

热消融治疗腹部肿瘤

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

科研

医学

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

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 ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝转移的临床前小鼠模型

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

隔离肝脏灌注作为治疗葡萄膜黑色素瘤肝转移

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Treatment of Liver Metastases Using an Internal Target Volume Method for Stereotactic Body Radiotherapy

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For inoperable patients, local ablation therapies, such as stereotactic body radiotherapy (SBRT), can provide effective local tumor control with minimal toxicity. Because of its high precision and accuracy, SBRT delivers a higher radiation dose per fraction, is more effective, and targets smaller irradiation volumes than does conventional radiotherapy. In addition, steep dose gradients from target lesions to surrounding normal tissues are achieved using SBRT; thus, SBRT provides more effective tumor control and exhibits fewer side effects than conventional radiotherapy. The use of SBRT is prevalent for treating intracranial lesions (known as stereotactic radiosurgery); however, it is now also used for treating spinal and adrenal metastases. Because of advancements in image-guided assistance and respiratory motion management, several studies have investigated the use of SBRT for treating lung or liver tumors, which move as a patient breathes. The results of these studies have suggested that SBRT favorably controls tumors in the case of moving lesions.
Four-dimensional computed tomography (4D-CT) with an abdominal compressor (AC) is clinically convenient for effective respiratory motion management. Because this method is noninvasive and allows free breathing, its use reduces complications. Furthermore, patients consider this method convenient. Moreover, it is considered more efficient than other methods of respiratory motion management by physicians and therapists. The use of 4D-CT with an AC for treating pulmonary lesions has also been widely investigated, and the technique is gaining acceptance for treating hepatic lesions. However, the protocols for using 4D-CT with an AC for treating hepatic lesions are different from those used for treating pulmonary lesions. In this article, we describe a new protocol for SBRT with 4D-CT and an AC for treating liver metastases.

Stereotactic body radiotherapy (SBRT) requires rigorous accuracy and precision for delivering high radiation doses per fraction to small treatment volumes to improve tumor control and simultaneously reduce toxicity. Herein, we present a noninvasive and clinically convenient respiratory motion management protocol for SBRT for liver metastases.

内靶容积法治疗肝转移瘤的立体定向体放疗

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

温度控制384孔板中用 Ca 敏感荧光染料测定人 iPSC 的心肌细胞较多的收缩

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...