这项工作得到了补助R01-CA169354支持&#160;和R01-047822 &#160;来自美国国立卫生研究院和退伍军人事务部（ECB）优异奖。 RKP是由NIH T32 CA009287-30A1，一个ASCO青年研究者奖，加州乳腺癌研究项目奖学金，和美国癌症协会的指导式研究学者格兰特的支持; BAZ是由美国国立卫生研究院资助AI079320支持。 JM是由美国国立卫生研究院T-32培训资助T32-AI07290- 25，T32-AI07290-24和美国癌症协会博士后奖学金PF-12-052-01-CSM奖学金支持。

注:所有的动物实验均按照批准的斯坦福大学，Palo Alto的VA HCS，以及卫生机构AnimalCare和使用委员会的指导方针国家机构进行。 1.准备样品采集和处理（所需时间:〜10-15分钟） <ol><li>如先前所描述的6接种鼠B16F0黑素瘤细胞（0.5-1×10 6）皮下处或附近在雌性C57BL / 6小鼠的腹部正中线。可替代地，接种的肿瘤细胞，无需额外的解剖位点（ 如侧翼，乳腺脂肪垫，等等）。 注意:任何数量的肿瘤（植入或自发）可以使用该协议被使用和评价。例如，其它常用的同基因肿瘤的C57BL / 6包括结肠线MC38，Lewis肺癌（LLC），和前列腺TRAMP-C2线。同时接种到免疫缺陷小鼠的人异种移植物也可在此方式被评估，但应注意的是，在p肿瘤浸润白细胞rofiles必然会改变按照宿主小鼠的免疫缺陷状态。 <ol><li>使用完全培养基（含10％FBS和添加剂如 RPMI）或其他适当的含蛋白的缓冲液（ 例如，HBSS或PBS + 1％FBS或1％BCS）。寒意媒体或缓冲至4℃之前安乐死。 </li></ol></li><li>建立收集管和过滤器切除肿瘤;放置这些冰上。 <ol><li>使用1个50毫升的锥形管每肿瘤切​​除。将在每一个40-70微米的细胞悬液过滤器。另外，使用的15毫升锥形管与金属滤网。使用5ml的注射器柱塞或粗笔在15ml试管的顶部，以创建一个杯形的过滤器。 </li></ol></li><li>准备切除肿瘤的处理手段;用手术镊子和剪刀精切除和处理肿瘤。用70％的乙醇清洗切除/肿瘤样品之间的仪器。对于较大数量Ø˚F样品（ 如 &gt; 10），我们建议由多个样本的研究人员同时处理，以避免肿瘤样本之间的处理时间显著的差异。 </li></ol> 2.肿瘤切除术（所需时间:约5分钟/肿瘤） <ol><li>安乐死单独使用二氧化碳或其他批准的方法的B16肿瘤的小鼠。 </li><li>确保鼠标在手术阶段。用70％乙醇喷雾肿瘤区域和擦拭过量关闭;这有助于防止任何毛发目前蔓延。用于非裸鼠中，根据需要剃肿瘤周围的皮肤区域之前切除。 </li><li>使用镊子和剪刀，切除皮下肿瘤整块切除 ，包括覆及周围皮肤。所采取的"缘"皮肤的量可以变化，但典型地切除2-3毫米肿瘤周围的皮肤。 <ol><li>在这一点上权衡切除肿瘤（+/-周围的皮肤）。将切除的皮肤/肿瘤节内的PL保护过滤器ASTIC锥形管。 </li></ol></li></ol> 3.准备一个单细胞瘤悬架（所需时间:5-7〜最小/肿瘤） <ol><li>使用镊子和剪刀直言不讳肿瘤及覆/周围皮肤。百果使用，直到组织各大部分被加工成1-2毫米的部分剪样品。 注:通常情况下，整个肿瘤进行处理;可替代地，对于较大的肿瘤，肿瘤的代表性部分可被切碎，并进行处理。 </li><li>从一次性5或10毫升注射器使用柱塞，机械地分解对担保滤波器的切碎的肿瘤组织。 </li><li>使用移液器，用5-10毫升冷介质或缓冲洗涤处理肿瘤组织。这将通过过滤器冲洗单细胞。 </li><li>重复上述两个步骤几次，以确保总人数和洗液的体积大致相同，同样大小的肿瘤。 <ol><li>清洗处理肿瘤组织后，观察皮肤和/或其他结缔组织的小片段应在滤波器被留下。 </li><li>另外，对于肿瘤，其中单独的机械分解可能并不足够，消化用胶原酶和/或其它酶可以在切碎过程之前进行。这将允许进行更彻底的单细胞悬浮液。但是，应该指出的是，这样的消化协议可以影响表面抗原表达20，所以应该小心在解释这些结果。 </li></ol></li><li>洗，并通过添加媒体/缓冲液在4℃下500×g离心离心5分钟沉淀细胞。 </li></ol>的单细胞悬液4. FACS染色（所需时间:〜30-60分钟） <ol><li>重悬的细胞在冰冷的FACS缓冲液（PBS + 1％FBS）中。 </li><li>使用计数台盼蓝和血球，或自动细胞计数活细胞。 </li><li>确定每卷总活细胞的产量。这个数据可以用来推断绝对数字与FACS数据相结合肿瘤浸润的白细胞。 </li><li>确定细胞的数目，以进行染色并通过FACS分析。这将通过几个因素（ 例如肿瘤组织学类型，肿瘤的年龄和大小，浸润白细胞的平均百分比等）的影响。 注意:在B16黑色素瘤中，我们发现肿瘤浸润白细胞的低百分比（TIL;通常&lt;5％）相比，肿瘤细胞。因此，至少1×10 6个总肿瘤（肿瘤+白细胞浸润）细胞通过流式细胞仪通常收集用于分析。 <ol><li>使用锥虫染色计数数据，被染色用于FACS的细胞的计算量。例如，如果典型的TIL构成总肿瘤细胞的5％（许多肿瘤细胞会死亡锥虫染色），因子到这一点的肿瘤的单细胞悬浮液的总数被染色。 </li><li>对于不太常见的白细胞亚群，例如 CCR9 + PDC）使用总肿瘤细胞的更大数目（例如2-3×10 6），用于FACS染色，鉴于这些细胞在单细胞悬浮液的相对低的频率。 </li></ol></li><li>染色肿瘤单细胞悬液的FACS。 <ol><li>对活/死判别，使用可固定染色或未染色可固定（ 例如碘化丙啶）都可以使用。如果细胞染色无活/死的歧视，必须小心谨慎考虑到潜在的对抗体的非特异性结合。 </li><li>染色的细胞悬浮液用于使用标准FACS染色协议特定白细胞亚群。块的细胞，以减少非特异性抗体用含1％大鼠血清和Fc嵌段（抗CD16 / 32），以便事先为10分钟至染色与抗原特异性抗体的PBS / FBS的约束力。包括正确的同型对照抗体，以保证染色是具体的。 </li><li>在这一点上，后在FACS染色完成后，固定用4％甲醛或其他类似的固定剂的样品。在4°C的大存储样本RK（以避免荧光团的淬灭），直到收集和分析的时间。 注:存储固定的样品进行长时间可导致改变的荧光团21的信号和强度。 </li><li>收集样品上的流式细胞仪。最佳流式细胞仪的设置将根据仪器和激光/检测器设置的类型不同而不同。 注:此外，可以存在实验之间变化，即使使用相同的机器。因此，进行适当的机器设置（ 例如使用校准珠），以确保前FACS收集和每个实验的分析，适当的补偿设置。 </li></ol></li><li> leukoctye亚群分析<ol><li>使用FACS数据分析软件，分析数据。对于活/死染色，通过门控出死细胞开始。使用抗体对白细胞特异性标记（ 例如抗CD45）的活细胞内识别白细胞。 <ol><li>在此之后，使用各种趣纳克计划，以确定具体的子集，这取决于染色方案。例如，为了找出CD4 + T细胞，然后从CD45 +人口SSC 罗区，并选择CD19-CD3 +细胞;从该CD4 + T细胞可以识别22。 </li></ol></li><li>分析数据并将其显示在许多不同的方式，以确保一致性和识别的模式或趋势（ 即使用总数量，百分比，或比率来量化子集）。 注:例如，我们看住总CD45 +细胞共收集肿瘤细胞（ 图1）的百分比。此外，我们然后计算肿瘤内各白细胞亚群（例如CD4 + T细胞）的百分比，并比较这些作为对照组和试验组（ 图3）之间的比率。 </li></ol></li></ol>

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The authors have nothing to disclose.

在进行肿瘤微环境的详细分析在确定的机制和免疫调节的影响是至关重要的。与免疫治疗的增加存在于人类的临床领域，了解这些药物对肿瘤浸润白细胞的影响变得必要在确定它们的作用机理。在人类中，有经常存在在获得和分析肿瘤组织对白细胞子集分析，并且因此外周免疫应答的分析临床和/或后勤困难常常执行。进行临床前研究的动物模型使研究人员更充分地发掘免疫调节剂，肿瘤本身对免...

肿瘤的生长和促进和回归之间的平衡是，部分地依赖于存在于所述微1,2-亲和抗肿瘤浸润白细胞的平衡。为了研究肿瘤微环境（TME），并具体指明浸润白细胞亚群，我们在小鼠肿瘤模型开发用于皮下肿瘤的评估的方法。研究肿瘤微环境的重要性是众所周知的，并且在文献中的支持。大量研究表明，亲和抗肿瘤浸润免疫细胞的平衡可影响肿瘤生长的结果，不仅在小鼠而且人类研究（在3,4综述）。例如，Curiel 等。显示恶化的卵巢癌患者的临床结果均与增加的肿瘤浸润调节性CD4 + T细胞（Treg细胞）5百分比的存在。我们自己的工作也呈无效果对白细胞亚群在小鼠黑素瘤模型6，它也与比VEL白细胞趋化减少肿瘤的生长。因此，在一个肿瘤的白细胞亚群的详细分析，现在更广泛的认可和越来越重要。 有许多种方法来评价肿瘤微环境对白细胞浸润;例如团体已经工程化的转基因小鼠来表达各种荧光蛋白，以图像在TME 7，经典免疫组化和保存部8，包括各种成像模态，例如MRI，PET共聚焦显微镜9-11的免疫荧光-一些与能力监控intravitally 10,12。这些可与各种分子成像剂，如纳米颗粒13或新颖造影剂14，其标记的免疫细胞。我们的方法是一种流式细胞仪为基础的流动的方法和具有若干优点。首先，整个肿瘤微环境进行采样;在分析时，整个皮下肿瘤且环绕周边被手术切除处理。这消除了在一个单一的肿瘤的任何潜在的抽样偏差，并给出一个更"全球性"分析的肿瘤的整体。其次，使用多色流式细胞仪来分析白细胞亚群可以让我们更具体地衡量浸润白细胞的表型。取决于所使用的颜色的数量，很具体的子集可被识别。这是很重要的，因为有在一个特定的细胞类型数白细胞亚群 - 或甚至在一般亚型分类 - 具有不同的功能，即是在确定肿瘤的命运可能显著。例如，浆细胞样树突细胞（PDC）有牵连的抗肿瘤免疫15。然而，中pDCs的CCR9 +子集已被证明是致耐受性16和移位巴喷枪这样的一个子集可以具有对肿瘤生长的影响。 我们的方法是适合于皮下或能被切除整块其他肿瘤。在我们手中，肿瘤均匀地安乐死的时间切除。然而，可以想到，作为已经在一些研究中已经完成，即皮下肿瘤可以完全切除，在一个存活手术17周围皮肤的封闭，从而使动物的其他评估。然后在切除肿瘤进行分析。因此，该结果代表在肿瘤的发展的单个时间点。虽然这允许详细研究进入微环境，它也是什么无疑是一个动态过程，静态图片。然而，分离的白细胞（ 例如，通过磁分离或密度梯度）然后可以从肿瘤上皮细胞和间质单独分析，或在其他，功能测定法中使用，以进一步确定它们的表型，因为一直PREVIously描述18。该方法中，然后，将是有兴趣了解肿瘤微环境内的白细胞的组合物在给定时间点的任何调查是有用的，无论是在自然病程的设置，或之后的特定治疗扰动。虽然不是由我们完成的，该过程的变化可能也可能被用来分析在隔离肿瘤的特定部分。例如，给定的肿瘤的大小，所述周边区（S）可被解剖远离肿瘤的中心，可能坏死核心，得到研究者更空间隔离视图肿瘤微环境的。 在肿瘤免疫的新兴领域，无疑将是一个指数越来越多的研究评估在小鼠肿瘤模型新型免疫调节剂。几份报告都强调肿瘤与周围设有内特定的白细胞功能的差异vironment。例如，谢弗-Weaver 等人。表明在抗原特异性T效应CD8 +细胞，而活跃在外围，被转化到的CD8 + T抑制基因细胞，一旦他们贩卖到肿瘤微环境19的小鼠模型。这是由于TGF-β部分，但其他因素是可能的介入。因此，评估白细胞亚群 - 数字和比率，以及功能状态 - 肿瘤本身内将给出一个特定的免疫调节对肿瘤命运的影响的更准确的表示。 我们的技术允许肿瘤的详细分析，并提供了研究者的机会更密切地确定改变白细胞种群比以前的方法。

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			<td height="21" style="height:21px;width:229px;">Name of the Material/Equipment</td>
			<td style="width:229px;">Company</td>
			<td style="width:229px;">Catalog Number</td>
			<td style="width:229px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Cellgro</td>
			<td>10-040</td>
			<td>http://cellgro.com; keep on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Cellgro</td>
			<td>35-011-CV</td>
			<td>http://cellgro.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml conical tubes</td>
			<td>Falcon</td>
			<td>14-432-22</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 micron filter</td>
			<td>Falcon</td>
			<td>08-771-1</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 ml syringe</td>
			<td>BD</td>
			<td>14-823-35</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">surgical scissors/forceps</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td>roboz.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Cellgro</td>
			<td>MT-21-030-CM</td>
			<td>http://cellgro.com; keep on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypan blue</td>
			<td>Cellgro</td>
			<td>MT-25-900-CI</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hemacytometer</td>
			<td>Hausser Scientifice&#160;</td>
			<td>02-671-54&#160;</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live/Dead stain</td>
			<td>Life Technologies</td>
			<td>L34957</td>
			<td>lieftechnologies.com</td>
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		<tr height="21">
			<td height="21" style="height:21px;">FlowJo software</td>
			<td>TreeStar, Inc</td>
			<td></td>
			<td>flowjo.com</td>
		</tr>
	</tbody>
</table>

evaluation of tumor-infiltrating leukocyte subsets in a subcutaneous tumor model

专门免疫细胞浸润肿瘤微环境调节肿瘤的生长和存活。恶性细胞必须逃避或颠覆抗肿瘤的免疫反应，为了生存和繁荣。肿瘤采取了一些免疫不同的机制"逃逸"，其中包括的致耐受性DC的招募，免疫调节性T细胞（Treg细胞），和髓源抑制细胞（MDSC）抑制细胞毒性抗肿瘤反应的优势。相反地​​，抗肿瘤效应的免疫细胞可以减缓肿瘤的生长和扩增:免疫刺激性树突细胞，天然杀伤细胞，怀有先天抗肿瘤免疫和细胞毒性T细胞都可以参与肿瘤抑制。亲和抗肿瘤白细胞之间的平衡，最终确定的行为和转化细胞的命运;众多的人体临床研究已经证明了这一点。白细胞亚群内。因此，详细的分析肿瘤微环境已经变得越来越重要。在这里，我们描述了用于分析存在于在小鼠肿瘤模型中的肿瘤微浸润白细胞亚群的方法。小鼠B16黑色素瘤肿瘤细胞皮下接种于C57BL / 6小鼠。在指定的时间，肿瘤和周围皮肤手术切除了整块并加工成单细胞悬浮液，然后将其染色的多色流式细胞术。使用各种白细胞子集标记，我们能够比较浸润控制和凯莫瑞表达肿瘤之间白细胞亚群的相对百分比。研究者可以使用这样的工具来研究免疫存在于肿瘤微环境，当与肿瘤生长的传统的卡钳尺寸测量结合时，将有可能让他们阐明变化对肿瘤生长的影响，在免疫组合物中。这样的技术可以应用于任何肿瘤模型，其中肿瘤及其微环境Ç一个被切除和处理。

我们的研究结果表明，被迫过表达凯莫瑞小鼠的B16肿瘤的增强肿瘤浸润白细胞（的TIL）的百分比。此外，更改为与凯莫瑞表达相关的肿瘤微环境内表示白细胞亚群的相对比率被确定。与许可Pachynski 等 6重新打印。 图1示出，有该表达凯莫瑞与对照组相比，如通过切除天17肿瘤的FACS分析测定中，在肿瘤中显著增加总CD45 +细胞（肿瘤浸润淋巴细胞）。使?...

Watch this Scientific Journal Video about Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model at JoVE.com

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

evaluation-tumor-infiltrating-leukocyte-subsets-subcutaneous-tumor

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

20.0K 次观看.  Washington University School of Medicine. This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses. 

视频: Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

肾小囊和异种移植皮下颗粒植入前列腺癌发生和良性前列腺增生症的评价

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

使用渗透皮下血管紧张素Ⅱ输液泵诱导主动脉瘤小鼠

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

毛里岛皮下白脂肪组织（ISWAT）移植模型

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

手术小鼠模型中骨关节炎的标准化形态学评价

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...