This work was supported in whole by funding from the NIH through the R21 grant, "Mechanisms of Perineural Invasion in Head and Neck Cancer" and the NCI T32 training grant, "Post-Doctoral Research Training in Head and Neck Oncology (2T32CA060397-21)." Thank you to Richard Steiman, MD, PhD and lab staff.

1.培养基的制备和食品（10分钟） <ol><li>添加的Dulbecco改良的Eagle培养基（DMEM）和10％胎牛血清（FBS）到96孔V型底板的孔中微升的100。 </li><li>从20℃冰箱中取出大约100半固体基质的微升预等分小瓶，并直接将其放置在冰上。 注意:做这个的背根神经节之前（DRG）收获，因为半固体基质大约需要30分钟，在冰上，这是必要的后续步骤达到液体状态。未能保持半固体基质冰在任何时候都将导致质量差的矩阵滴。 </li><li>标签玻璃底与永久墨水培养板，在板的下创建的每个四角的唯一标识符。将平板右侧朝上冰上冷却板面。 注意:每个小鼠收率32 - 40 DRG，所以从1标记它们以40这是没有必要预先寒意圆周宠物提示，冷冻提示将在温暖放置矩阵液滴，可能引入矩阵一致性差异的过程。 </li></ol> 2.鼠DRG的夹层（45分钟） <ol><li>安乐死的无胸腺裸小鼠按特定实验室协议，例如通过使用在 CO 2室和开胸。 </li><li>成立了解剖区域和显微镜。使用聚苯乙烯容器的清洁，灭菌，和平面底面为15或50毫升锥形管中。 注:此外，它们适合用作解剖表面，因为它们是便宜的，一次性的，并且允许使用销或针的脊椎固定。只用消毒器械和针头。 </li><li>在自然的视野，使沿着从尾部到鼠标的头部有一对直线或曲线精剪根脊柱的纵向切口。 </li><li>使用相同的剪刀横向鸿沟下面SACR人的脊椎。剖析脊柱两侧一路用剪刀颅底。在这一点上，确保该颈椎被颅尽量横断尽可能用剪刀。 </li><li>观察在低功率在手术显微镜下的脊柱的颈椎端。白色脊髓处于骨环，它是由不同量的椎旁肌肉和软组织包围的中间显而易见。 </li><li>驳头2的背部或上级的方面 - 与微观弹簧剪刀3椎体。使用剪刀的弹簧作用，打开切断，以确保在椎体解剖发生在中线该初始骨。继续在朝向骶棘小增量，然后通过完成对椎体的腹侧或劣方面相同的切口对开脊椎。 注意:如果不确保骨脊柱中线切开，将导致一个较低的DRG收率。 </li><li>在THI角度看，它的脊柱被分成两半。放置一个半脊椎一边，与脊髓到位，向下置于无菌板。 注:在地方离开脊髓将防止的DRG变干，作为病种深至脊髓。 </li><li>固定其他半脊柱两端与聚苯乙烯解剖平台上的两个18号针头。在颈端开始（这是显而易见的，因为脊椎是窄，DRG更接近彼此，和有肋插入），轻轻剥离背部约4椎骨水平的脊髓。 </li><li>观察连接DRG的感觉神经（通常为两个）。在神经插入到DRG的区域，轻轻抓住与显微镊周围筋膜。解剖和修剪这筋膜等神经组织与微观弹簧剪刀以释放DRG。温和回缩将带来DRG其位置的脊柱骨内。 注:增加在此步骤显微镜功率是有益的。挤压伤到DRG将显著限制突起的增长。这是通过从不直接抓DRG避免，而是通过抓住周围的筋膜。 </li><li>切周围神经（DRG的通常有一个外周神经，这是相对于深解剖视图）与微观弹簧剪刀释放DRG。 注意:这是最简单的，以确定在那里神经末端和背根神经节开始，而在张力，所以使该切口尽可能接近到DRG在这一点是理想的。一旦该远侧分支被切断，近侧分支被修整为好。 </li><li>修剪任何杂散神经纤维或筋膜附件中微观弹簧剪刀的DRG。后足够的隔离，将DRG到96孔V形底板中的室温介质。 注:放置在深色背景下的板使得可视化分毫米的白色病种付费更容易。只有放在EAC 1 DRGħ好;这样，它们可占更容易在随后的步骤。 </li><li>重复此过程，一路向下半脊椎，然后其他。每一方产生16 - 20病种付费，总额为32 - 40。 注意:如果有的DRG比这更少，需要进行进一步的头侧和/或尾端脊柱的解剖。按病种付费将变得越来越不那么定义为一个收益尾部。 </li></ol> 3.半固态基质液滴制备（&lt;每平板1分钟） <ol><li>从冰中取出一块玻璃以及底板，并将其放置冰块，在手术显微镜下的。确保矩阵的等分在任何时候都保持在冰上。 </li><li>放置基质的各玻璃底板的四个角的1.5微升液滴具有2μL或10微升micropipetter，留下的距离至少一样大液滴本身从玻璃的良好的边缘。 <ol><li>放置吸管尖上直接该玻璃以45度角。慢慢地吸取矩阵。在矩阵上板接合后，慢慢地从玻璃底搬走。 注:基体与玻璃之间的表面张力应创建一个完美的半球各一次。 </li><li>停止吸移之前的前端是空的，因为意外注入空气进入矩阵液滴使得矩阵液滴更大的直径，并且难以去除。 注:使用二手稳定吹打手利于更准确的位置。 </li></ol></li></ol> 4.插入DRG成半固体基质液滴（&lt;每盘2分钟） <ol><li>与简单地在室温（〜1分钟）的矩阵的液滴离开板。这略微僵硬的矩阵，使它更容易为DRG的精确位置。 </li><li>勺（不抓）轻轻地用左手封闭微观钳DRG。同样，对小黑色背景，白色DRG方便的可视化。转移DRG到21号针头的尖端在右手。这种转移将离开镊子残留介质。 </li><li>轻轻插入DRG到使用21号针头（ 图1）的矩阵液滴的中间。大部分时间，背根神经节将很容易释放到基质，然后可以与针中心定位。 <ol><li>如果DRG粘在针，使用微观镊子推DRG落针并进入基质液滴。从实验室显微镜镊子吸走多余的介质擦拭。 注:在介绍媒体到矩阵将改变直径，体积和测定的一致性。冰块用颜色或颜色的书写提供了背景的对比度，从而简化了这一微妙的过程可视化。 </li></ol></li><li>板具有所有四个的DRG之后，执行最终检查以确保该DRG是在基质液滴的中心。进行调整根据需要用21号针头。 </li><li>已完成的板转移到37℃培养箱。这将巩固半固体基质和固定DRG的位置。 </li><li>重复此过程为所有的按病种付费的。放置空白基质液滴未经DRG作为阴性对照。 </li><li>有10％FBS培养基中添加4毫升的DMEM每个玻璃底板完成所有板，并将它们暴露于37℃培养箱中至少3分钟后。放置板以微小的角度，慢慢添加介质，使得其逐渐进入与基质DRG单元接触。 注意:如果媒体被添加太快或大力，矩阵和/或DRG可以移位。 </li><li>存放在37℃培养箱的测定为未来48 - 72小时。 注:测定的周期检查将显示向基质边缘（ 图2）突起的圆周向外生长。当轴突比的方式四分之三更大的矩阵，它适合于板上的细胞。 </li></ol> 5.制备头颈癌细胞注:比头和颈部鳞状细胞癌细胞的其它细胞系可以在这个实验设计中使用。 <ol><li>保持在37℃培养箱中在DMEM鳞状细胞癌的细胞系用10％FBS的优选烧瓶或培养皿。为了准备实验，抽吸从培养瓶或培养皿所有的细胞培养基，用PBS洗两次。通过添加0.025％胰蛋白酶的5分钟适量，随后含10％FBS的悬浮细胞。吸取4毫升细胞和媒体混合成6毫升的培养皿中。 注:一个6毫升培养板提供了超过足够的细胞，即使当小于50％汇合。 </li><li>露出头颈部鳞癌细胞系24小时之前，染色和电镀的DRG分析不同的条件。重剂量的条件下，一旦将细胞铺板到保持一致的environmeNT。 注:加入的抗体，生长因子，细胞因子或其它分子与细胞培养皿1 - 以下小鼠解剖和背根神经节到矩阵的植入2天。 </li><li> （ - 背根神经节的收获和植入基质后3天2）电镀细胞之前添加荧光细胞染色1小时。 注:此处使用通过细胞膜自由通过特定污渍;然而，与细胞内硫醇基团反应之后，污渍保持在细胞内，并在子细胞内传递。染色极大地方便了实验中的可视化。这些临时污渍是理想的这些测定，因为荧光的存在为2 - 3天，这是这些实验的时间范围。它也允许使用的许多不同的颜色并避免了对创建荧光蛋白转染的细胞系的需要。 <ol><li>通过混合2 10微升10mM储备细胞污渍在2mL无血清DMEM的制备10μM的染料溶液对于每一个细胞的条件。将其添加到细胞前温暖这个至37℃。 </li><li> 6毫升板内取出培养基，留下粘附HNSCC细胞在平板上。添加2毫升磷酸盐缓冲盐水（PBS）中，并除去2毫升10μM的细胞染色/无血清DMEM加入到每个板</li><li>与细胞染色返回6毫升培养板于37℃培养箱中40分钟。 </li><li> 40分钟后，吸出培养基。加入2毫升的PBS，然后吸它。添加1毫升0.025％胰蛋白酶的对各板，并在37℃培养箱替换它们。 </li><li>加入2毫升的DMEM含10％FBS后3 - 5分钟，悬浮的细胞转移到15毫升锥形管中。旋转细胞，并重新悬浮他们在1毫升的DMEM含10％FBS。 </li><li>计数与血球或自动细胞计数器的细胞，然后用10％的FBS添加DMEM培养基，以创建每毫升300000细胞的最终细胞浓度。 </li></ol></li></ol> 6.电镀头和N埃克癌细胞<ol><li>与从培养箱基质-DRG测定中取出玻璃底板。 注: - 1天同样，当突起是的方式向基体，这通常发生后2的边缘至少75％它们是合适的。这是通过使用至少一个10X物镜最清楚地看到。 </li><li>吸出玻璃底板内的网上平台。完全吸出玻璃内的介质以及不去除矩阵DRG单元。 </li><li>制定使用200微升吸管30万细胞/ mL培养基200μL。放置细胞的两滴在每个矩阵DRG测定。执行此步骤每个矩阵液滴，在一个板上创建每个细胞条件4个重复。 注:基质的半球形状允许细胞在沿测定（ 图3a，图3b）的外周的环沉降。找到一个吸管具有光滑的触发使得统一安置下降容易得多。 </li><li>确认类似细胞数和二下4X显微镜各种细胞条件stribution。 </li><li>放置玻璃底板到37℃培养箱中培养大约60分钟。注意:虽然只有细胞和培养基（〜150微升）中的一小体积，测定法不干燥直到孵化时间几个小时已经过去了。 </li><li> 60分钟后，轻轻地用10％FBS的沿着玻璃底板的侧壁加入4毫升的DMEM。 注:经过一段时间，将细胞部分地附着到板底，并添加培养基不干扰细胞的方法。不同类型的细胞使采取更多或更少的时间来开始粘附到玻璃底板。 </li><li>在这个时候更换任何外源性药物或生长因子保持了以往的浓度水平。 </li><li>返回检测到37℃培养箱，显微镜时进行检查，他们除了。量化该测定通过显微成像的结果。简单地说，算上与神经浸润的股在四个象限使用4X显微镜图像。 注:带有4X目标和荧光功能的显微镜是足够的照片记录了实验。该测定的尺寸非常适合一个4X物镜，其详细足以捕捉神经浸润的各个区域的视野内。神经浸润变得在24小时内显现，但超过48小时，该实验的完整性开始减弱，因为肿瘤细胞沿着矩阵的周边划分和侵入。超过48小时，也有从背根神经节，采用明场显微术这掩盖可视成纤维细胞的大量流出。因此，这个窗口期间是可取的照片文档与4X显微镜的结果的至少两倍（ 例如，在电镀的肿瘤细胞后24和48小时）。牢记这些是3维测定法，它是难以完全图像整个测定法。大多数神经浸润的平面中发生从板的底部，其中t他的细胞包埋在基质中的板的底部略高于。因为这架飞机是接近水平，绝大多数肿瘤细胞轴突都在4X单级图像捕获。 </li></ol>

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O., 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=Neural+invasion+in+pancreatic+cancer:+a+mutual+tropism+between+neurons+and+cancer+cells.">Neural invasion in pancreatic cancer: a mutual tropism between neurons and cancer cells.</a> Biochem Biophys Res Commun. 374 (3), 442-447 (2008).</li><li>Gil, Z., 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=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102 (2), 107-118 (2010).</li><li>He, S., 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=GFRalpha1+released+by+nerves+enhances+cancer+cell+perineural+invasion+through+GDNF-RET+signaling.">GFRalpha1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling.</a> Proc Natl Acad Sci U S A. 111 (19), 2008-2017 (2014).</li><li>He, S., 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=The+chemokine+(CCL2-CCR2)+signaling+axis+mediates+perineural+invasion.">The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.</a> Mol Cancer Res. 13 (2), 380-390 (2015).</li><li>Bakst, R. L., 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=Radiation+impairs+perineural+invasion+by+modulating+the+nerve+microenvironment.">Radiation impairs perineural invasion by modulating the nerve microenvironment.</a> PLoS One. 7 (6), 39925 (2012).</li><li>Ayala, G. E., 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=In+vitro+dorsal+root+ganglia+and+human+prostate+cell+line+interaction:+redefining+perineural+invasion+in+prostate+cancer.">In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.</a> Prostate. 49 (3), 213-223 (2001).</li><li>Na'ara, S., Gil, Z., Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Modeling+of+Cancerous+Neural+Invasion:+The+Dorsal+Root+Ganglion.">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion.</a> J Vis Exp. (110), (2016).</li></ol>

The authors have no competing financial interests.

该议定书中的关键步骤 该协议中最重要的步骤是精确的解剖和背根神经节的提取。脊柱和中线纵分工合理横切为两半刺都获得大量的DRG的关键。在按病种付费个人的解剖，神经节不能直接处理，而是周围筋膜应与微观镊子来把握。不这样做将导致DRG，这很可能失败的主要原因为神经突长出的挤压伤。这是再好不过的解剖过程中欠修剪周围的神经组织，而不是过?...

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the US, with 10,000 deaths per year nationally and 300,000 deaths per year worldwide1. The overall prognosis for HNSCC has remained unchanged at 50% for the past several decades. Perineural invasion (PNI) is one of the most prominent pathological features that portend a poor prognosis in patients with HNSCC. Unfortunately, PNI is a frequent occurrence in HNSCC and can be found in up to 40% of HNSCC patients2,3. 
PNI is the process by which malignant cells track along nerves to adjacent tissues, allowing for higher rates of local and distant spread. Accordingly, PNI-positive HNSCC tumors have higher rates of locoregional recurrences and distant metastases, resulting in lower overall survival compared to HNSCC patients without PNI4-8.
Although the treatment of patients with PNI is typically maximized by employing surgery, radiation, and chemotherapy, the overall survival rates of these patients are still decreased by up to 40% compared to patients without PNI9-11. Thus, it is clear that the current treatment modalities for HNSCC are ineffective in improving the adverse prognosis associated with PNI. The approach of developing targeted therapy against PNI in HNSCC has been hindered by the poor understanding of the factors that regulate this process. This is, in part, a consequence of the lack of a consistent in vitro model for the study of PNI in HNSCC. 
In recent years, several groups have been utilizing an in vitro model for studying PNI in predominantly pancreatic and prostate cancers12-19. This model uses the neurites generated from dorsal root ganglia isolated from mice or rats as a surrogate for large-nerve invasion. The dorsal root ganglia are fixed in a factor-depleted semisolid matrix, which is a solubilized basement membrane protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. This matrix allows for the outgrowth of the neurites and the tracking of single cancer cells along these neurites. Described here is the adaption of this model for the examination of PNI in HNSCC.

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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.

背根神经节的解剖和基质液滴中放置后，测定的外观应该类似于图1。注意，在DRG不完全是圆的，但它是在基质液滴内居中。这允许在360度突起的生长，在图2中部分示出。请注意，DRG的某些部位发出的神经突更快，数量更多比别人，通常对应到传出和传入神经分支进入和退出的DRG，分别为。我们考虑到这一点，并为病种之间大小的差异通过随机电?...

Watch this Scientific Journal Video about A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma at JoVE.com

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

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, ...

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JoVE Journal

Cancer Research

10.5K 次观看.  University of Pittsburgh Medical Center. 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.

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

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

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 diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

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 ...

科研

癌症研究

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

神经入侵的体内小鼠坐骨神经模型

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

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 ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

舌细胞外基质的制备与舌鳞癌体外重建

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

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 ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

比较在小鼠肾脏和鸡胆膜上建立的转移性透明细胞肾细胞癌模型

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.