这项工作是由美国国立卫生研究院R21 CA143505克里斯蒂娜Voelkel - 约翰逊的支持。

所有涉及动物的程序进行了审查和批准的机构动物护理和使用委员会在南卡罗来纳医科大学。该协议是根据美国农业部的D类止痛批准。 1。细胞移植<ol><li>在执行程序前两天，板1×10 6 MB49细胞转化为T-25瓶。使用添加有10％FBS（和抗生素，如果需要的话）高糖DMEM。一个烧瓶中足以为每个组最多5只小鼠将被麻醉并植入平行。 </li><li>在手术当天，通过用无菌DMEM基础培养基（1:2稀释）稀释无菌0.25％组织培养级胰蛋白酶至0.125％胰蛋白酶制备用于滴注。 1.2毫升足以用于10只小鼠。放置在37℃水浴中平衡。 </li><li> Prewarm加热垫和其下的麻醉系统将用于提供一本nosecones定位soflurane。将干净的吸水垫在加热垫。 </li><li>把小鼠放入麻醉系统的感应腔和诱导麻醉，用3％异氟烷。当老鼠已经失去了知觉，这是由脚趾捏反射消失核实，将它们转移到nosecones。确保每个动物是在仰卧位，并正确放置在加热垫，以保持体温。 </li><li>减少异氟醚的浓度为2％维持麻醉。 </li><li>应用眼药膏，以双眼，以防止干燥。 </li><li>可选步骤：删除腹毛。如果在体内生物发光成像，将执行头发放在下腹部，必须清除。 <ol><li>套用一个慷慨层的脱毛霜​​，如薇婷，最低1毛皮在腹部的广场上，注意不要接触外生殖器。等待3分钟。 </li><li>用干海绵擦拭的面积去除毛发，然后用湿海绵来清洁皮肤。如果需要的话重复一次。 </li></ol></li><li>导尿，膀胱<ol><li>润滑新，无菌24政小儿静脉导管用无刺激性的润滑凝胶，如肯塔基州。取下并丢弃针头。如果流体的导管周围泄漏是常见的问题， 即得 。它发生比在10小鼠1比较频繁，较大孔的导管（20 G）都可以使用。这可能在老年超过8周的小鼠是一个重要的考虑因素。 </li><li>握住导管的枢纽，用你的另一只手的拇指和食指传播鼠标的后腿和暴露尿道口。轻轻将导管插入尿道以45°角，改变角度，以一个并列的板凳完全插入。 </li><li>完全插入后，提起导管轻轻，保持它平行于工作台，并在视觉上确认，它被放置在尿道和不阴道，这是低于它。如果正确地放置，所述导管将能够在serted完全自阴道比尿道短。 </li><li>降低了异氟醚的浓度为1％。 </li></ol></li><li>附加的前端至P200移液器和通过施加抽吸到导管的外端从膀胱中移除尿。从导管毂除去任何剩余的尿液。尿倒掉，留下导管的位置。 </li><li>小心吸取80微升0.125％胰蛋白酶溶液进入导管的轮毂，避免产生气泡。附1毫升空气充式注射器的导管套筒，慢慢地压低柱塞0.1-0.2毫升交付胰蛋白酶进入膀胱。 </li><li>留在原地的注射器和导管组件15分钟。为任何剩余的麻醉小鼠（最多4个）重复步骤1.8-1.11。请继续等候期间的下一个步骤。提示：最初，它可能是有帮助的别人准备的细胞（步骤1.12）。 </li><li>准备MB49细胞植入。细胞铺板在1×10 6每T-2的5瓶48小时前注入（步骤1.1）。 <ol><li>除去介质并加入500微升0.25％胰蛋白酶的烧瓶中。当细胞分离加入5 ml完全DMEM重悬细胞。取出50微升等分试样进行计数，并且离心剩余在1,000 rpm离心5分钟。在离心步骤继续步骤1.12.2和1.12.3。 </li><li>使用血细胞计数器计数细胞。计算细胞/ ml。然后计算以使细胞沉淀，以4×10 6个细胞/ ml（每50μl的2×10 5个细胞）所需的体积。 </li><li>从离心细胞倒出上清液，并用中止细胞至4×10 6个细胞/ ml（如计算步骤1.12.2）所需的体积重悬在DMEM基础培养基中沉淀。保持细胞在室温下。 </li></ol></li><li>当达到用于气囊的胰蛋白酶处理15分钟时间点，从导管离开导管的位置分离的空气填充的注射器。花了胰蛋白酶将正常LY流回了通过导管。通过抽吸与P200的移液器除去从膀胱任何剩余的胰蛋白酶如上述步骤1.9和丢弃。 </li><li>立即吸取50微升MB49细胞悬液到导管的轮毂。附加1毫升空气充式注射器的导管的轮毂和通过慢慢压下柱塞0.1-0.2毫升递送细胞至膀胱。重复此步骤，最多四次，植入所有五只小鼠。丢弃任何未使用的单元格。 </li><li>离开导管，注射器组装到位，使细胞在膀胱停留50分钟。 </li><li>轻轻地从尿道撤出导管，注射器组装。关闭异氟醚浓度为零，每次移动鼠标一个或两个客场从鼻锥恢复加热垫英寸。 </li><li>当鼠标已经恢复了翻正反射，将其放回笼子。 </li></ol> 2。腺病毒的膀胱内交货（8天细胞移植后） </p&gt; 小心：这部分协议使用腺病毒，这是一种传染性剂，在严格BSL2准则应处理（http://oma.od.nih.gov/manualchapters/intramural/3035/）。 与传染性病原体进行的所有程序批准了南卡罗来纳医科大学的生物安全委员会。 <ol><li>植入小鼠MB49细胞后八天，检查血尿。拿起动物个体在一个白色的一张吸水纸，轻轻按在腹部涂抹的尿液滴在纸张上。尿液是粉红色到红色表示血尿是肿瘤已经建立的标志。肿瘤接受率至少为80％，具有优良的技术可以高达100％。 </li><li>解冻腺病毒库存在冰上并在无菌，室温PBS稀释至10 9 PFU/50微升（2×10 10 PFU /毫升）。 </li><li>如上面的步骤1.3-1.6中所述麻醉小鼠。 </li><li>导尿小鼠按照步骤1.8中所述，取出尿如步骤1.9 </li><li>尿除去后，立即吸取50微升病毒悬液到导管的轮毂。附加1毫升空气充式注射器的导管的轮毂和通过慢慢压下柱塞0.1-0.2毫升递送病毒膀胱。 </li><li>离开导管，注射器组装到位，让病毒在膀胱停留40分钟。 </li><li>轻轻地从尿道撤出导管，注射器组装，并丢弃到10％的漂白粉溶液灭活任何残留的病毒。 </li><li>关闭异氟醚浓度为零，每次移动鼠标一个或两个客场从鼻锥恢复加热垫英寸。 </li><li>当鼠标已经恢复了翻正反射，将其放回笼子。 </li><li>马克笼表明，老鼠已经被灌输与腺病毒。病毒会脱落入笼床上用品数天，所有的笼子里，床上用品必须是处理并适当消毒。 </li></ol>

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C., Shetty, A., 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=Validating+bladder+cancer+xenograft+bioluminescence+with+magnetic+resonance+imaging:+the+significance+of+hypoxia+and+necrosis.">Validating bladder cancer xenograft bioluminescence with magnetic resonance imaging: the significance of hypoxia and necrosis.</a> BJU Int. 106 (11), 1799-1804 (2010).</li></ol>

作者宣称，他们有没有竞争的财务权益。

在本协议中所述的主要方法是导尿鼠标膀胱，这对细胞的灌注或用于本地传递到膀胱上皮任何代理广阔的应用前景。上文所述的特定的协议进行了优化的短期研究（〜10日）。植入细胞的精确数目是至关重要的，因为更高的细胞数，将导致更快速的肿瘤生长和可能的动物的损失是由于肿瘤负荷大。使用200,000 MB49细胞滴注可能需要高达实施安乐死的动物的25％由14天因过度肿瘤负荷。根据我们的经验?...

膀胱癌是泌尿生殖道有近75,000预计在2012年1新病例和15,000死亡的第二位最常见的癌症。高复发率需要终身随访，这使得膀胱癌治疗最昂贵的癌症之一。膀胱癌已侵及肌层可能转移到肝脏，通过淋巴系统肺或骨。晚期肿瘤导致仅5年后20-40％的生存多式联运治疗。因此，迫切需要有效的治疗策略旨在减少浅表性膀胱癌的复发和进展，以及改善治疗效果的晚期患者。 新型疗法的发展需要临床前模型下初步的体外评估，以评估疗效。肿瘤微环境中可以显著影响癌症的发展和响应能力，从而强调了需要preclinic在肿瘤的发生或人的模型可以建立在器官来源。一种方法是转基因模型中的肿瘤自发产生的或可诱导的器官特异性的方式发展。转基因膀胱癌模型的一个很好的协议，已出版2。转基因模型的缺点是肿瘤倾向于慢慢地和以比希望的少的均匀发展。此外，保持了繁殖群体的成本，必须考虑到。另一种转基因模型是肿瘤细胞原位移植，其中有很短的时间帧的肿瘤成立市售小鼠的利益。虽然一些人膀胱癌细胞系可原位生长（我们已成功地使用UM-UC-3），它​​可能是可取的，以建立肿瘤的免疫活性小鼠。两种鼠膀胱癌细胞系，其中原位生长是MBT-2和MB49 3。由于MBT-2细胞被污染的复制C型逆转录病毒4，我们选择MB49细胞进行我们的研究。要注意，MB49细胞从雄性小鼠中分离和原位植入是在雌性小鼠中进行解剖结构的原因是重要的。这有便于识别的植入细胞的Y染色体标记的好处，但性别不匹配可能是一个缺点的免疫学研究。 膀胱上皮是由葡糖胺聚糖（GAG）层，其功能是作为由微生物屏障感染内衬。此屏障也可以用肿瘤细胞和几种方法植入干扰已被开发来克服这一困难（ 表1）。电灼已被广泛用作物理手段破坏GAG层5-13和协议证明电灼最近已公布在朱庇特的14。但是，如果一个电灼单元不可用，化学手段破坏对GAG层如硝酸银或聚-L-赖氨酸也可以使用15-24。肿瘤是由膀胱 ​​的短暂暴露于少量体积的硝酸银有效地建立（5-10微升，0.15-1.0 M，〜10秒），或用聚-L-赖氨酸的较长的接触（100 微升的0.1毫克/毫升为20分钟）（ 表1）。在这里，我们描述了使用胰蛋白酶，以促进MB49细胞植入的方法。 在试图改善的治疗方法为膀胱癌，基因疗法已经获得了显著关注。从临床上看，膀胱癌基因治疗由于容易获得器官的能力和在本地提供有效载荷的理想目标。已被研究用于膀胱癌基因治疗的病毒载体包括一种溶瘤单纯疱疹病毒25，逆转录病毒26，金丝雀痘病毒27，牛痘病毒，腺相关病毒，和adenoviru第28号。在我们的协议的第二部分中，我们描述了用于病毒递送，几乎是相​​同的肿瘤细胞的灌注的方法。在我们的实验室中感兴趣的是新颖的方法，以基因递送，我们使用表达萤光素酶转基因的腺病毒载体通过生物发光评估的发展。然而，可用于输送各种试剂膀胱导管的方法，因此具有广泛的适用性。

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="height:25px;">
			Name of reagent
			</td>
			<td style="height:25px;">
			Company
			</td>
			<td style="width:188px;height:25px;">
			Catalog number
			</td>
			<td style="width:193px;height:25px;">
			Comments
			</td>
		</tr>
		<tr>
			<td style="height:40px;">
			6-8 week old female mice
			</td>
			<td style="height:40px;">
			Jackson Laboratories
			</td>
			<td style="width:188px;height:40px;">
			Strain Name: C57BL/6J
			Stock Number: 000664
			</td>
			<td style="width:193px;height:40px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			Trypsin*
			</td>
			<td style="height:25px;">
			MediaTech
			</td>
			<td style="width:188px;height:25px;">
			MT25-053-CI
			</td>
			<td style="width:193px;height:25px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			DMEM*
			</td>
			<td style="height:25px;">
			MediaTech
			</td>
			<td style="width:188px;height:25px;">
			MT10-017-CV
			</td>
			<td style="width:193px;height:25px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			FBS
			</td>
			<td style="height:25px;">
			Hyclone
			</td>
			<td style="width:188px;height:25px;">
			SH30071.03
			</td>
			<td style="width:193px;height:25px;">
			Heat-inactivated
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			T25 flasks*
			</td>
			<td style="height:25px;">
			Corning Costar
			</td>
			<td style="width:188px;height:25px;">
			Corning No.:3056
			Fisher: 07-200-63
			</td>
			<td style="width:193px;height:25px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:42px;">
			MB49 cells
			</td>
			<td style="height:42px;">
			N/A
			</td>
			<td style="width:188px;height:42px;">
			N/A
			</td>
			<td style="width:193px;height:42px;">
			Obtained from Dr. Boehle (see reference11)
			</td>
		</tr>
		<tr>
			<td style="height:46px;">
			Puralube Vet Ointment*
			</td>
			<td style="height:46px;">
			Pharmaderm
			</td>
			<td style="width:188px;height:46px;">
			Henry Schein Company
			No.:036090-6050059
			Fisher: NC9676869
			</td>
			<td style="width:193px;height:46px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:53px;">
			Depilatory cream: Veet
			</td>
			<td style="height:53px;">
			local pharmacy
			</td>
			<td style="width:188px;height:53px;">
			
			</td>
			<td style="width:193px;height:53px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:53px;">
			Lubricant:
			K-Y Jelly
			</td>
			<td style="height:53px;">
			local pharmacy
			</td>
			<td style="width:188px;height:53px;">
			
			</td>
			<td style="width:193px;height:53px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:72px;">
			Catheters*
			</td>
			<td style="height:72px;">
			Exel International
			</td>
			<td style="width:188px;height:72px;">
			Exel International
			No.:26751;
			Fisher: 14-841-21
			</td>
			<td style="width:193px;height:72px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:45px;">
			Isoflurane
			</td>
			<td style="height:45px;">
			Terrell
			</td>
			<td style="width:188px;height:45px;">
			NDC 66794-011-25
			</td>
			<td style="width:193px;height:45px;">
			Obtained though hospital pharmacy
			</td>
		</tr>
		<tr>
			<td style="height:52px;">
			1 ml slip tip TB syringes
			</td>
			<td style="height:52px;">
			Becton Dickinson
			</td>
			<td style="width:188px;height:52px;">
			BD309659
			Fisher:14-823-434
			</td>
			<td style="width:193px;height:52px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:27px;">
			D-Luciferin
			</td>
			<td style="height:27px;">
			Gold Biotechnologies
			</td>
			<td style="width:188px;height:27px;">
			L-123-1
			</td>
			<td style="width:193px;height:27px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:76px;">
			Ad-CMV-Luc
			</td>
			<td style="height:76px;">
			VectorBiolabs
			</td>
			<td style="width:188px;height:76px;">
			1000; Request large scale amplification and CsCl purification for in vivo use
			</td>
			<td style="width:193px;height:76px;">
			Infectious agent that requires BSL2 containment
			</td>
		</tr>
		<tr>
			<td style="height:76px;">
			Steady-Glo&#160;Luciferase Assay System
			</td>
			<td style="height:76px;">
			Promega
			</td>
			<td style="width:188px;height:76px;">
			E2510 (10 ml), E2520 (100 ml), or E2550 (10 x 100 ml)
			</td>
			<td style="width:193px;height:76px;">
			
			</td>
		</tr>
	</tbody>
</table>
*available through multiple vendors



EQUIPMENT
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:97px;">
			Material name
			</td>
			<td style="width:114px;">
			Company
			</td>
			<td style="width:330px;">
			Catalog number
			</td>
			<td style="width:97px;">
			Comments
			</td>
		</tr>
		<tr>
			<td style="width:97px;">
			Anesthesia system
			</td>
			<td style="width:114px;">
			E-Z Systems, Euthanex Corporation
			</td>
			<td style="width:330px;">
			Anesthesia system: EZ7000
			5-port mouse rebreathing device: EZ109
			</td>
			<td style="width:97px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="width:97px;">
			Xenogen IVIS 200
			</td>
			<td style="width:114px;">
			Caliper Life Sciences
			</td>
			<td style="width:330px;">
			http://www.caliperls.com/products/preclinical-imaging/ivis-imaging-system-200-series.htm
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:97px;">
			FLUOstar Optima
			</td>
			<td style="width:114px;">
			BMG Labtech
			</td>
			<td style="width:330px;">
			http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
	</tbody>
</table>

an orthotopic bladder cancer model for gene delivery studies

膀胱癌是泌尿生殖道和新的治疗方法，可以减少复发和进展所需要的第二种最常见的癌症。肿瘤微环境中可以显著影响肿瘤的发展和治疗反应。因此，它通常是可取的生长的肿瘤细胞中生成它们的器官。本协议描述膀胱癌原位模型，其中MB49小鼠膀胱癌肿瘤细胞通过导管灌入膀胱。在这个模型中成功的肿瘤细胞植入需要保护的葡糖胺聚糖层，其可通过物理或化学的方法来完成的中断。在我们的协议膀胱用胰蛋白酶处理前细胞滴注。膀胱的导管也可用于递送治疗剂，一旦肿瘤被建立。这个协议描述，表达荧光素酶报告基因的腺病毒构建体的传递。虽然Ø乌尔协议进行了优化的短期研究，侧重于基因传递，小鼠膀胱导尿的方法具有广泛的应用。

血尿是在几乎所有的小鼠中观察到内200,000 MB49细胞植入后8天。在非肿瘤小鼠为87.5±19.2毫克（范围77-120毫克，N = 10） 如图1，膀胱重量比双打更从34.7±3.3毫克（范围31-37毫克，N = 4），在小鼠已经植入MB49细胞。在基因传递方面，我们发现，小鼠成像24小时后病毒滴入产生更强的信号超过48小时（ 图2）之后。腺病毒转染的动物，它应该用于统计目的的策划组大小时，必须考虑...

Watch this Scientific Journal Video about An Orthotopic Bladder Cancer Model for Gene Delivery Studies at JoVE.com

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

植入癌细胞进入器官来源可以作为一个有用的临床前模型来评估新疗法。 MB49膀胱癌细胞可在膀胱内以下膀胱灌注生长。该协议表明了鼠标膀胱肿瘤植入和腺病毒转染的目的导尿。

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

an-orthotopic-bladder-cancer-model-for-gene-delivery-studies

Research

JoVE Journal

Medicine

12.3K 次观看.  Medical University of South Carolina. 植入癌细胞进入器官来源可以作为一个有用的临床前模型来评估新疗法。 MB49膀胱癌细胞可在膀胱内以下膀胱灌注生长。该协议表明了鼠标膀胱肿瘤植入和腺病毒转染的目的导尿。 

视频: An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

患者组织及基因传递考试前的体外培养

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

科研

医学

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

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 ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

在原位小鼠脑肿瘤模型结合放疗

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

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 ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

甲状腺未分化癌的原位小鼠模型

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

生物荧光原位模型胰腺癌进展

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.