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><tbody><tr><td>Vibratome Leica VT1000s</td><td>Leica</td><td>14047235613</td><td></td></tr><tr><td>UltraPure agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 4% and 6% before use</td></tr><tr><td>Injector blade</td><td>Ted Pella</td><td>121-4</td><td></td></tr><tr><td>MEM with Penicillin&amp;nbsp; + Streptomycin</td><td>Media Core Facilities (MSKCC)</td><td></td><td>The media is prepared at Memorial Sloan Kettering Cancer Center&amp;nbsp;</td></tr><tr><td>Scalpel no. 10</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Disposable forceps</td><td>Cole-Parmer</td><td>84011182</td><td></td></tr><tr><td>Embedding mold</td><td>Electron Microscopy Science</td><td>70181</td><td></td></tr><tr><td>FBS (heat inactivated)</td><td>Gemini</td><td>100106</td><td></td></tr><tr><td>24 well plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Formalin (10%)</td><td>Sigma Diagnostics</td><td>SDHT501128&amp;nbsp;</td><td></td></tr><tr><td>16% Formaldehyde solution</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Embedding microsettes</td><td>Simport</td><td>M503-2</td><td></td></tr><tr><td>Ethanol (70%)</td><td>Fisher Scientific</td><td>A405P-4</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Scientific</td><td>15-462-2SQ</td><td></td></tr><tr><td>Microwave</td><td>General Electric</td><td>ModelJES2051DNBB</td><td></td></tr><tr><td>Adhesive (Ethyl Cyanoacrylate)</td><td>Sigma-Aldrich</td><td>E1505-5G</td><td></td></tr><tr><td>10 mm dishes</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>15 ml tubes</td><td>BD Falcon</td><td>352096</td><td></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

14.7K 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

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 ...

科研

医学

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

癌症研究

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

使用结构DNA改变分析和患者衍生异种移植测试癌症的靶向疗法

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

测量组织肿瘤组织切片的实时药物反应

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

乳腺癌临床前模型乳腺肿瘤的矫形移植

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

患者源性肿瘤外植体作为预测患者耐药性的"实时"临床前平台

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

使用宽场活细胞成像进行批量和单类器官分析的多参数肿瘤类器官药物筛选

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

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

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