这项工作是由美国国家癌症研究所（R15CA173657到AW和R01CA109446到TSG），西蒙斯癌症研究所（至AW），以及Minnestoa大学助学金爬上4肾脏癌症基金会（TSG到）的支持。我们感谢克丽斯廷·安德森博士与免疫援助。

以下方案描述用于诱导和监测实验原位肾肿瘤和任何潜在自发转移的生长的实验程序。下面的所有程序都是按照有关人性化的使用实验动物的体制政策和批准的程序进行。 1.细胞系的维护<ol><li>维持鼠肾腺癌细胞系的Renca，在的Roswell Park Memorial研究所（RPMI）1640培养基补充有10％胎牛血清（FCS），1％青霉素 - 链霉素，和各1mM的非必需氨基酸，L-谷氨酰胺的和丙酮酸钠（简称完全RPMI）。培养细胞在培养箱中在37℃和5％CO 2。 注意:与任何建立的细胞系，则建议使用低传代数RENCA细胞用于转染和随后的注入，以最小化长期崇拜后可能发生的任何表型或遗传变化URE。 <ol><li>使用转座子系统工程师RENCA细胞稳定表达绿色荧光蛋白（GFP）和萤火虫荧光素酶。 <ol><li>使用阳离子基于脂质体的转染试剂，共转染的Renca细胞与转座酶表达载体（PPGK-SB11）和萤火虫萤光素酶的表达的转座子载体编码，以及一个编码GFP融合基因和zeocin抗性基因，两者的双向启动子（PKT2 / LuBiG）19的转录控制下。 </li><li>两天后，板转染的细胞以有限稀释并允许细胞扩大的完全RPMI补充有博莱霉素（300微克/毫升）。确认在通过流式细胞术每个克隆GFP表达。这些细胞被称为的Renca-GL。 注:限制稀释克隆将允许从单个细胞衍生的所选单元格。 </li></ol></li></ol></li><li>植入RENCA细胞肾内，制备RENCA细胞我n要在Hank氏平衡盐溶液（HBSS）以2×10 6个细胞/ mL的单细胞悬浮液。 <ol><li>使用0.25％胰蛋白酶在HBSS中的组织培养瓶中除去粘附在表面的Renca细胞。约5分钟后，完全RPMI添加到该烧瓶中以中和胰蛋白酶，将细胞转移到一个离心管中进行洗涤。离心将细胞在300相对离心克力（RCF×g）离心5分钟，在25℃下。 </li><li>倒出上清液和重悬细胞，在HBSS中。计数使用血球的细胞，并用HBSS调节音量，得到2×10 6细胞/ mL。 </li><li>绘制细胞成1毫升注射器装配有18号针头，以防止对细胞的剪切应力。替换前的肿瘤细胞的注射一个28号针头，18号针头。 0.1毫升的注射递送2×10 5个细胞入肾。 </li></ol></li></ol> 2.手术区准备<ol><li>准备无菌手术区在生物安全柜通过将无菌手术单过一个加热垫。收集所有用品，包括镊子，解剖刀，剪刀，滴眼剂，5％聚维酮碘防腐剂，和无菌纱布。高压灭菌器使用前所有仪器。 </li><li>执行使用无菌尖端技术的手术。动物之间重新消毒文书，珠灭菌。戴上无菌手套，白大褂和口罩。 </li></ol> 3.动物的制备<ol><li>麻醉用根据机构指南氯胺酮/赛拉嗪（87.5 mg / kg和12.5毫克/千克，分别，腹膜内）的小鼠。通过执行脚趾捏确定麻醉深度。 </li><li>当动物被认为是无反应，由拔除（用手指）或剃须（使用裁切机）的皮毛上准备Balb / c小鼠注射部位（ 例如，左侧面）。 </li><li>从手术准备区移动小鼠（ 例如，房栊）到手术区和报考兽医药膏给眼睛，防止干燥。宽松拭制备的切口部位用5％聚维酮碘防腐剂。 </li><li>注入盐酸布比卡因（0.1毫升2.5毫克/毫升溶液，SC的）插入其中的切口将被制成为手术前的镇痛的区域。 </li><li>使单个切口（〜1.0 - 1.5厘米）对动物的左翼使用无菌解剖刀或剪刀，小心以免穿透腹膜。用无菌剪刀腹膜分离真皮。民建联切口部位用无菌纱布，以消除任何血液。 </li><li>按住鼠标，用双手，用左手持有的头部和右手支撑侧翼。 </li><li>识别脾通过腹膜和，用右手的一个手指，从下方触诊鼠标;肾脏现在应该通过腹膜可见。保持动物的坚定持有，以提供跨腹膜和肾脏紧张。 </li><li>有第二人注入肿瘤CELLS（1.2节中制备的。）通过完整腹膜成使用1毫升注射器装配有28号针头的肾脏的中心。慢慢地递送细胞的0.1毫升（2×10 5）。一旦细胞注射，持针到位5 - 10秒，以减少细胞的回流。注意在此步骤非常谨慎的需要，因为注射细胞的人就会把针头插入接近按住鼠标的人的手指是很重要的。 </li><li>卸下针和用氰基丙烯酸酯组织粘合剂的薄层关闭切口。 </li><li>将动物放回一个干净的笼子消毒纸巾。让动物在加热垫上恢复。不要离开无人看管的动物在恢复期。不要将动物返回到标准的住房条件，直到他们正直和胸骨。按照使用实验动物的所有程序制度的指导方针。 </li></ol> 4.生物发光成像<ol><li>监测由生物发光成像的肿瘤生长。 注意:用于执行生物发光成像程序的细节可以从Lim 等来获得。 20荧光素酶信号可以在肿瘤细胞植入后早在24小时进行检测。由于有在该模型中没有外部可见的肿瘤，它周期性地监视肿瘤的生长是必不可少的。根据不同的设计的实验，原位肿瘤生长可以进行不同的时间长度。使用荧光素酶表达的Renca细胞的一个好处是，肿瘤的生长可反复在相同的动物定期监测在实验的持续时间，和荷瘤和无肿瘤的器官的特定询问可以在完成定义端点。 <ol><li>注入荧光素（0.1毫升在无菌PBS中有15毫克/毫升溶液）ip和允许其将鼠标到生物发光成像系统之前循环10分钟。将莫<html>其背部朝上使用，在其一侧略微卷起，才能有朝上注入的肾。 </li><li>测量使用图像分析软件确定的关注区域（ROI）的区域内的每秒（光子通量）发出的光的光子的肿瘤负荷。 </li></ol></li><li>在个别器官的肿瘤负荷的定量。 注:定量可以在确定的时间点在个别器官从安乐死的小鼠其取出后进行。 <ol><li>注入小鼠荧光素，如步骤4.1。 </li><li> 10分钟后，安乐死使用经批准的过程的小鼠。 </li><li>解剖出感兴趣的器官（ 例如，肾和肺;见下文第5节）。转移组织以35×10毫米的培养皿并将其放置在生物发光成像系统。 </li><li>测量使用图像分析软件定义的ROI内每秒（光子通量）发出的光的光子的肿瘤负荷。 </li></ol></li></ol><html> e_title"&gt; 5。器官收获<ol><li>在用于各个实验所定义的终点，根据优选机构准则（ 例如，CO 2，颈椎脱臼，麻醉过量）安乐死荷瘤小鼠。然而，要避免颈椎脱位，如果肺是印度墨通货膨胀之后被收集。 </li><li>通过测量湿器官重量执行在所注入的肾原发性肾肿瘤生长的毛评估。 <ol><li>为了确定肿瘤块（以克计），隔离21和称重荷瘤肾脏和从同一小鼠，其中在肾重量差定义了肿瘤质量的对侧肾。测量它们的重量之前，请仔细剖析远离切除肾脏任何多余的结缔组织。 </li></ol></li><li>通过用墨汁之前切除充气肺并立即固定在的Fekete的溶液肺评估肿瘤转移至肺的程度小姑娘= "外部参照"&gt; 22。然后可以执行的肺表面上的转移病灶的人工计算。 <ol><li>请使用开始于-腹部中期剪刀正中切口，通过胸腔，并且向上通过颈部/唾液腺。小心地取出用剪刀和镊子胸腔，而不会损害肺组织。 </li><li>通过去除结缔组织暴露气管。 </li><li>制备3mL注射器填充有在HBSS 10％印度油墨溶液的18号针头。 </li><li>使用镊子，小心翼翼地拧气管下丝线。这将用于打结在充气后的气管。 </li><li>小心地将针头顺着气管，慢慢压低注射器膨胀与印度墨液肺部。大约1 - 1.5毫升的印度油墨溶液将完全膨胀肺部。 </li><li>捏通过把几节，与以下针缝合，以抑制墨逆流关闭气管。 </li><li>祛瘀E中的针，小心地剪去结缔组织去除膨胀肺部。 </li><li>在化学通风橱中，将肺成含有5的Fekete的溶液中的溶液15毫升锥形管中（35毫升95％乙醇，15毫升卫生署2 O，5毫升的甲醛，和2.5毫升冰酸性酸的中一个100毫升的玻璃瓶）23。 </li><li>允许肺修复/脱色24 - 在室温下48小时之前手动计数脱色肿瘤结节。 </li><li>脱色后取出完整肺和仔细叶在35×10毫米的培养皿解剖固定肺。算肿瘤结节（白色结节）上在解剖显微镜下每个叶的数目。 </li></ol></li></ol>

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+intravascular+staining+redefines+lung+CD8+T+cell+responses.">Cutting edge: intravascular staining redefines lung CD8 T cell responses.</a> J. Immunol. 189, 2702-2706 (2012).</li><li>Anderson, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+staining+for+discrimination+of+vascular+and+tissue+leukocytes.">Intravascular staining for discrimination of vascular and tissue leukocytes.</a> Nat. Protocols. 9, 209-222 (2014).</li></ol>

The authors declare no conflicts of interest.

我们提出了一个原位RCC小鼠模型的协议。小鼠肾腺癌肿瘤细胞植入小鼠肾脏提供海上救援协调中心临床相关模型。该模型导致在肺中，这两者都是晚期RCC的标志的肾脏和远处转移中的原发肿瘤。本文中所呈现的描述是具体的在免疫活性Balb / c小鼠的的Renca细胞系。使用这种原位模型，我们已经表明的Renca肿瘤响应免疫，表明治疗性测试<sup class="xref...

肾细胞癌（RCC）占全世界大多数恶性肾肿瘤的和约3％所有成年恶性肿瘤的1，2。由于缺乏症状，误诊，和不足的筛选工具，用于RCC，患者几乎30％将呈现与转移性RCC（MRCC）在诊断时，与患者进展至转移期2 20-30％的额外。这些情况导致每年〜13500人死亡1，3。虽然早期检测和治疗原发性RCC的有所改善，RCC相关的死亡的速率持续增加，这表明转移性疾病是死亡3的主要原因。治疗发展RCC已经发展了与发现并实施有针对性的治疗和免疫治疗的最后十年。不幸的是，无进展surviv人与晚期病例总生存率仍远低于2年，大多数患者4，5，6。这些统计数据为保证进一步研究的识别和对RCC有效的治疗方法发展的需要。为了充分促进治疗性干预在一项针对mRCC，首先需要疾病的翻译相关的临床前模型。 相关的和一致的模型的发展概括晚期RCC人的条件应解决几个关键问题:1）是模型解剖相关; 2）是否肿瘤进展类似于人体病理学; 3）执行转移从原发肿瘤出现;及4）可原发性和转移性肿瘤进展随时间监测？根据治疗的类型被调查，小鼠RCC肿瘤细胞系，人肾细胞癌的肿瘤细胞系，和人RCC患者来源xenog筏可在免疫活性或免疫缺陷的小鼠中。具有完整和功能性免疫系统中的主机需要这些研究评估免疫疗法的一些方面中，需要使用来自Balb / c小鼠7的自发肾腺癌导出的充分描述的Renca细胞系。大多数研究注入RENCA细胞皮下（sc），其形成一个易于度量局部肿瘤，或静脉内（IV）到Balb / c小鼠，以产生实验性肺"转移" 8，9，10，11，12。的SC-植入的Renca肿瘤模型人RCC的使用具有许多限制，包括脉管系统13的不准确的神经支配，在微环境14，15，和缺乏器官/肿瘤细胞COMMUNIC差异通货膨胀13,16。此外，许多皮下肿瘤（特别的Renca）不会转移到远端器官，抑制这是一种常见的临床特征17事件的研究。为了研究mRCC的，RENCA细胞可以静脉注射以建立在RCC患者转移肺-主要位置肿瘤负荷。发起转移性肿瘤的静脉内注射的方法，然而，不允许的侦查如何，何时，或为什么细胞已经从初级器官（ 即，肾）到远端部位迁移。与原发性和转移性疾病明显在相同的动物模型是研究晚期疾病的进展和治疗是至关重要的，尤其是考虑到转移通常在这些患者中的死亡率的原因。 我们的实验室已开发海上救援协调中心的鼠模型，结合了所有上述功能。要建立公关imary，原位肿瘤，的Renca细胞直接植入到动物的透过半透明腹膜肾。在左侧面一个小切口允许脾（如地标）和左肾的可视化。使用小规格针，的Renca肿瘤细胞通过腹膜用于原位移植直接注射到肾脏。相比植入肾囊18的下方的Renca细胞的其它方法中，注入的这个方法允许更高的吞吐量，因为它是相当的非侵入性的，并不需要缝合，是一种低疼痛类的，而且是时间有效时实践。 这充分表征的模型的结果在注射肾再现的原发性肿瘤负担（〜99％的接受率），以及转移性肿瘤负荷于肺部。这种模式的优点显著包括它的同源性质，允许免疫治疗调查;其自发转移，以研究疾病的先进适用的;其原位移植，来模拟对疾病进展和治疗解剖的影响。旨在针对RCC的治疗会从这个模型的临床前开发过程中的利用率大大受益。

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a syngeneic mouse model of metastatic renal cell carcinoma for quantitative and longitudinal assessment of preclinical therapies

肾细胞癌（RCC）每年影响&gt; 60000人在美国，和RCC〜30％的患者在诊断时多发转移。转移性肾细胞癌（MRCC）是不治之症，与只有18个月的中位生存时间。基于免疫的干预（ 例如，干扰素（IFN）和白细胞介素（IL）-2）诱导的mRCC患者的一小部分持续反应，和多激酶抑制剂（ 例如，舒尼替尼或索拉非尼）或抗VEGF受体的单克隆抗体（单抗）是主要是治标不治本，因为完全缓解是罕见的。在目前的治疗对于患者mRCC的这些缺点提供的新颖的治疗方案的开发的理由。在新的疗法用于mRCC的临床前检测的关键部件是一个合适的动物模型。重演人的条件有益特征包括原发性肾肿瘤，肾肿瘤转移，和一个完整的免疫系统，以调查任何治疗驱动的免疫效:ř反应和肿瘤诱导的免疫抑制因子的形成。这份报告描述了具有所有这些特征的原位mRCC的小鼠模型。我们描述使用鼠标肾腺癌细胞系的Renca一个肾内注入技术，随后在肾（原发部位）肿瘤生长和肺（转移部位）的评估。

<html> RENCA细胞的成功植入会导致肿瘤的发展在肾脏和转移到小鼠肺。荷瘤和对侧肾脏切除后，湿组织重量（克）进行测定，以证实基于增加的重量（ 图1）的肿瘤负荷。 RENCA肿瘤可以通过免疫组化通过细胞角蛋白8和18染色（ 图2）来识别。对于纵向研究中，荧光素酶表达的Renca细胞系植入。使用体内成像系统（IVIS）来跟踪肿瘤负荷和对治疗?...

Watch this Scientific Journal Video about A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies at JoVE.com

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

在免疫活性小鼠肾细胞癌的原位模型的实现，得到研究者通过初级肾肿瘤和肺转移瘤的在相同的动物的存在所定义的临床相关系统。该系统可用于临床前测试各种体内治疗。

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

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Cancer Research

13.5K 次观看.  University of Minnesota. 在免疫活性小鼠肾细胞癌的原位模型的实现，得到研究者通过初级肾肿瘤和肺转移瘤的在相同的动物的存在所定义的临床相关系统。该系统可用于临床前测试各种体内治疗。 

视频: A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

开发和维护的临床前患者来源的肿瘤异种移植模型为新的抗癌疗法的调查

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

科研

癌症研究

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

临床前小鼠模型的外科手术和方法<em&gt; De Novo</em&gt;乳腺癌转移

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

在头颈部鳞状细胞癌的神经浸润模型

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

自体胰腺癌小鼠模型研究不可逆电穿孔的影响

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

雄激素依赖性和去势抗性前列腺癌的荧光自体小鼠模型

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

同源小鼠 B 细胞淋巴瘤模型对 CD19 轿车 T 细胞的临床前评价

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...