Name: an in vivo murine sciatic nerve model of perineural invasion
Uploaded: 2018-04-23T13:00:00
Description: Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

作者承认分子细胞学设施和纪念斯隆凯特林癌症中心的动物设施提供的技术服务。这项工作得到了 NIH 赠款 CA157686 (R.J.) 和 P30 CA008748 (纪念斯隆凯特林癌症中心支助补助金) 的支持。

所有的程序与动物的对象是批准的机构动物护理和使用委员会在纪念斯隆凯特林癌症中心。
1. 肿瘤细胞的制备
<ol><li>收获亚汇合 Panc02-H7 细胞与0.25% 胰蛋白酶为5分钟在37°c。收集15毫升离心管中的细胞。 注: 细胞生长在 T-225 烧瓶中, 它包含大约 12 x 106细胞每瓶在80% 融合和4毫升胰蛋白酶/烧瓶使用。</li>
	<li>离心机在900克的细胞在 4°c 5 分钟, 通过 resuspending 细胞颗粒?...

<ol>
	<li>Liebig, C., Ayala, G., Wilks, J. A., Berger, D. H., Albo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+in+cancer.">Perineural invasion in cancer.</a> Cancer. 115 (15), 3379-3391 (2009).</li><li>Bapat, A. A., Hostetter, G., Von Hoff, D. D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+and+associated+pain+in+pancreatic+cancer.">Perineural invasion and associated pain in pancreatic cancer.</a> Nat Rev Cancer. 11 (10), 695-707 (2011).</li><li>Deborde, S., Wong, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Schwann+cells+facilitate+cancer+progression+in+nerves.">How Schwann cells facilitate cancer progression in nerves.</a> Cell Mol Life Sci. 341 (177-186), 1236361-1236416 (2017).</li><li>Abiatari, I., 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=Consensus+transcriptome+signature+of+perineural+invasion+in+pancreatic+carcinoma.">Consensus transcriptome signature of perineural invasion in pancreatic carcinoma.</a> Mol Cancer Ther. 8 (6), 1494-1504 (2009).</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>Gil, Z., Cavel, 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=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>Deborde, S. T., 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=Schwann+cells+induce+cancer+cell+dispersion+and+invasion.">Schwann cells induce cancer cell dispersion and invasion.</a> J Clin Invest. 126 (4), 1538-1554 (2016).</li><li>Pour, P. M., Egami, H., Takiyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+growth+and+metastases+of+induced+pancreatic+cancer+in+relation+to+the+prognosis+and+its+clinical+implications.">Patterns of growth and metastases of induced pancreatic cancer in relation to the prognosis and its clinical implications.</a> Gastroenterology. 100 (2), 529-536 (1991).</li><li>Eibl, G., Reber, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+xenograft+nude+mouse+model+for+perineural+invasion+and+recurrence+in+pancreatic+cancer.">A xenograft nude mouse model for perineural invasion and recurrence in pancreatic cancer.</a> Pancreas. 31 (3), 258-262 (2005).</li><li>Stopczynski, R. 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=Neuroplastic+changes+occur+early+in+the+development+of+pancreatic+ductal+adenocarcinoma.">Neuroplastic changes occur early in the development of pancreatic ductal adenocarcinoma.</a> Cancer Res. 74 (6), 1718-1727 (2014).</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=Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion.">Nerve-sparing therapy with oncolytic herpes virus for cancers with neural invasion.</a> Clin Cancer Res. 13 (21), 6479-6485 (2007).</li><li>Cardiff, R. D., Miller, C. H., Munn, R. 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在本协议中, 我们描述了一个在体内小鼠神经入侵模型, 它允许量化的坐骨神经侵袭的胰腺癌细胞。该模型能够研究神经入侵的分子机制。成功的实验使用这项技术需要谨慎的方法, 在三关键步骤的过程中: 1) 注射癌细胞 (步骤 2.7, 2.8), 2) 提取的侵入神经 (步骤 3.4) 和 3) 处理收获的神经 (步骤 4.1)。
注射应该非常小心, 以避免刺穿神经后壁, 导致癌细胞的损失。一旦针到位, ...

神经形成特定的肿瘤微环境, 刺激癌症的生长和迁移1,2,3。神经入侵 (PNI) 是癌细胞在神经和周围侵袭的过程。它可能被认为是一种独特的转移途径, 因为癌症入侵从起源的地方沿神经。PNI 有多种癌症类型, 包括胰腺、前列腺、头 & 颈部、唾液腺、宫颈癌和结直肠癌, 发病率从22% 到 100%1,2不等。PNI 与疼痛相关, 预后较差, 存活率更差1,2。
神经入侵的发展模式对于阐明这一过程的细胞和分子机制, 以及检测候选治疗剂以减少 PNI 是至关重要的。体外方法研究癌症和神经之间的相互作用, 包括与神经外植体的癌细胞共培养4, 背根脊神经5,6,7, 或特定细胞从神经微环境例如雪旺细胞7。然而,体内方法在生理上是相关的, 包括使用癌症小鼠模型, 在这种模式中, 癌症已经被诱导或移植, 并具有占整个神经微环境的优势。在胰腺或前列腺癌的原位模型中, PNI 报告了8,9,10 , PNI 的发病率可能会被记录下来, 但是由于这些器官神经的体积小, 很难看到整个神经, 因此量化 PNI 的程度。我们在这里描述的模型是一个在体内模型中的 PNI, 其中癌细胞通过一个简单的手术程序11注入到小鼠的坐骨神经中。异位移植在神经内侵袭脊髓。从注射部位到脊髓的神经侵入的长度可以测量, 以及神经内的肿瘤体积。重要的是, 侵入神经也可以收集各种化验, 包括显微, 分子分析。可以对多种癌细胞进行检测, 并使用特定化合物进行基因修饰或治疗的宿主老鼠。这一强有力的化验允许肿瘤细胞和宿主微环境被修改, 以调查的机制, PNI。

<table border="0" cellpadding="0" cellspacing="0" style="width:973px;" width="973">
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		<col />
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	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Mouse</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;">Number and age variable depending on experimental needs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture media (PBS, Trypsin, and DMEM+10% FBS)</td>
			<td>Any</td>
			<td></td>
			<td>Steps 1.1, 1.2, 1.3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tube, 50 mL</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Step 1.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge tube 1.5 mL</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td>Step 1.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electric razor</td>
			<td>WAHL</td>
			<td>9962</td>
			<td>Step 2.1. Can be substituted with commercial hair removal agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, 250 mL</td>
			<td>Baxter</td>
			<td>1001936060</td>
			<td>Step 2.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hypoallergenic surgical tape</td>
			<td>3M Blenderm</td>
			<td>70200419342</td>
			<td>Step 2.3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Betadine Swapsticks</td>
			<td>PDI</td>
			<td>SKU 41350</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>5110</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile surgical tools (scissors and forceps)</td>
			<td></td>
			<td></td>
			<td>Steps 2.4, 2.5, 3.3, 3.4, 3.5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10 &#956;L Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Steps 2.7, 2.8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Micro spatula</td>
			<td>Fisher Scientific</td>
			<td>S50823</td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bupivacine, 1 g</td>
			<td>Enzo Life Sciences</td>
			<td>BML-NA139-0001</td>
			<td>Step 2.9. Reconstitute to 0.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>698H</td>
			<td>Step 2.9</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek O.C.T. Compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Step 4.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek Cryomold Molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Step 4.1</td>
		</tr>
	</tbody>
</table>

an in vivo murine sciatic nerve model of perineural invasion

癌细胞通过称为神经侵袭 (PNI) 的过程侵入神经, 癌细胞在神经微环境中增殖和迁移。这种类型的入侵是由多种类型的癌症所表现出来的, 而且在胰腺癌中也经常出现。小鼠胰腺内神经纤维的显微大小使 PNI 在原位鼠模型中的研究更加困难。在这里, 我们描述一个异位的在体内模型的 PNI, 我们注射自体胰腺癌细胞系 Panc02-H7 到小鼠坐骨神经。在该模型中, 麻醉小鼠的坐骨神经被暴露并注入癌细胞。癌细胞从注射点到脊髓的神经向下部侵袭。侵入的坐骨神经, 然后提取和处理与 OCT 的冰冻切片。H & E 和免疫荧光染色这些部分允许量化的入侵程度和变化的蛋白质表达。该模型可应用于 PNI 的多种研究, 考虑了其通用性。使用不同基因修饰的小鼠和/或不同类型的癌细胞, 可以调查 PNI 和不同癌症类型的细胞和分子机制。此外, 治疗药物对神经侵袭的影响可以通过对这些小鼠的治疗来研究。

该方法描述了胰腺癌细胞移植到小鼠坐骨神经中的外科植入, 以建立一个可量化的神经入侵的体内模型。图 1说明了坐骨神经和注射部位的解剖位置。图 2显示了裸鼠的两个坐骨神经。PBS 注入神经 (左) 可与 MiaPaCa-2 癌细胞 (右) 注入的神经进行比较。癌细胞注入的神经被肿瘤浸润, 比用 PBS 注入的神经更厚、更暗。
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Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

神经入侵的体内小鼠坐骨神经模型

我们描述了一个体内小鼠神经入侵的模型, 注射自体胰腺癌细胞进入坐骨神经。该模型允许定量的神经入侵程度, 并支持调查的细胞和分子机制的神经入侵。

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

Research

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An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

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

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, ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

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

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

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. ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

矫形子宫内膜癌模型，具有从 Vivo 传播和培养的 VX2 细胞中制成的再生淋巴病

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Machine Learning Algorithms for Early Detection of Bone Metastases in an Experimental Rat Model

实验鼠模型中骨代谢的早期检测机器学习算法

Machine learning (ML) algorithms permit the integration of different features into a model to perform classification or regression tasks with an accuracy ...