作者感谢Jo Hill的组织病理学帮助，John Gleysteen博士在外科技术方面的教学，Tessa Diebel的摄影辅助，Wong和Coussens实验室的所有成员进行重要的洞察和讨论，以及OHSU Knight Cancer Institute的财务支持。作者承认T32GM071388-10和T32CA106195-11支持CEG，NCI / NIH，国防部乳腺癌研究计划，苏珊·戈曼斯基金会，乳腺癌研究基金会，以及一个支持癌症 - Lustgarten基金会胰腺癌融合梦想团队转化研究基金（SU2C-AACR-DT14-14）向LMC提供MHW颁发基金奖的妇女健康圈，以及Brenden-Colson对MHW和LMC的胰腺健康中心。

以下协议中使用的动物由俄勒冈州健康与科学大学机构动物护理和使用委员会（IACUC）涵盖，该委员会旨在符合"动物福利法"条例和公共卫生服务（PHS）政策。 维持无菌条件:应使用灭菌仪器，小鼠之间应用无菌纱布擦拭，用PBS冲洗，然后用消毒剂70％乙醇灭菌至少15分钟。外科手术帽，面罩，长袍和手套应佩戴进行生存手术。用于生存手术的动物的手术前准备包括在以下方案中。有关试剂和设备的清单，请参阅表1。 1.从初级乳腺肿瘤中分离和制备单细胞悬液<ol><li>麻醉供体雌性100天龄转基因MMTV-PyMT（FVB / n）小鼠并维持在c通过麻醉面罩给予2％异氟烷的连续镇静。确认鼠标已被适当地麻醉，没有足部反射。 </li><li>在无菌环境中，通过使用无菌剪刀从上覆皮肤和周围脂肪组织和/或淋巴结中分离乳腺肿瘤，从100天龄的转基因雌性MMTV-PyMT（FVB / n）小鼠切除原发性乳腺肿瘤。通过颈椎脱位安乐死麻醉的小鼠。 </li><li> 用无菌剪刀或手术刀将手术切成小块（〜1.0 mm 3 ）。将肿瘤片放在3.0mg / mL胶原酶A和4.0U / mL DNase I中溶解于DMEM中。每1.0厘米直径肿瘤使用〜10毫升上述消化培养基。 <ol><li>用无菌搅拌棒在无菌的25mL瓶中以〜125rpm和37℃进行消化40分钟。 </li></ol></li><li>通过加入胎牛血清（FBS）至10％的最终稀释度来停止消化，并将整个混合物置于w上并保持冰。 </li><li>将消化的肿瘤悬浮液通过0.7μm尼龙过滤器过滤到50mL锥形管中，并丢弃残留在过滤器中的任何肿瘤。在4℃下以300xg离心上清液。 </li><li>将沉淀重悬于10ml DMEM / 1.0cm肿瘤中，并通过0.7μm尼龙过滤器再过滤。计数细胞浓度，然后在4℃下以300xg离心。 </li><li>将沉淀物重悬于10％二甲基亚砜与90％FBS浓度为2×10 7活细胞/ mL。将整个原发肿瘤的单细胞悬液储存在-80°C。 </li></ol> 2.原位乳头肿瘤注射<ol><li>部分解冻冷冻的原代肿瘤悬浮液在37°C，直到冷冻丸粒可以从低温管释放到20 mL DMEM中并计数细胞。在4℃下以300xg离心，并将细胞重悬于1:1 DMEM:生长因子减少的溶解基底膜制剂e以1×10 7个细胞/ mL的浓度从Engelbreth-Holm-Swarm（EHS）小鼠肉瘤中减量（参见材料表 ）。 </li><li>将麻醉的受体雌性同基因小鼠腹侧向上放置，并通过麻醉面罩施用2％异氟烷，保持连续镇静。 </li><li>使用雾化的70％乙醇灭菌右侧第4个乳腺注射部位，并用无菌棉签涂覆聚（乙烯基吡咯烷酮）碘。 </li><li>使用29 G 0.3 mL胰岛素注射器将100μL（来自冷冻原发性肿瘤悬浮液的1×10 6个活细胞）倾斜侧向上注入6至10周龄雌性FVB / n小鼠的未清除右侧第4个乳腺。 </li></ol> 3.手术切除原位乳腺肿瘤<ol><li>肿瘤细胞注射后38-60天，安乐死不显示体积范围在172至450mm 3之间的原位肿瘤体积的长度x（宽度2 </sup&gt;）/ 2]（约75％的注射小鼠）。 </li><li>通过麻醉面罩给予2％异氟烷，将连续镇静下的适当肿瘤大小腹侧放置麻醉小鼠，并确认小鼠用不足的脚捏反射适当地麻醉。在镇静时应用兽药软膏防止干燥。 </li><li>喷雾70％乙醇，对原发肿瘤周围的手术区进行消毒，然后用无菌棉签涂敷聚（乙烯基吡咯烷酮）碘。 注意:手术部位的脱毛导致〜5％小鼠的皮肤剥离和/或感染，因此被排除在该步骤之外（数据未显示）。 </li><li> 如图1C所示，使用钝刀中间尾部对肿瘤进行初始皮肤切口。 </li><li>接下来，使肿瘤内侧的皮肤（ 图1C ）更好地切除。注意灼烧任何脉管系统的需要在延伸切口之前，将位于皮肤上的肿瘤编织。 </li><li>继续皮肤切口（肿瘤后），然后进行优异的皮肤切除（肿瘤外侧）和内侧切除（优于肿瘤）（ 图1C ）。 </li><li>皮肤周围切除肿瘤周围（ 图1C ）后，使用镊子将附着于肿瘤的覆盖皮肤抬起，同时将肿瘤从腹壁肌肉组织中分离出来，并保持乳腺不受影响。 </li><li>识别（通过钝器解剖）和烧灼通过第 4和第 5乳腺运行大型船只。 </li><li>在烧灼部位消耗大约一半的第4和第 5 个乳腺，以释放肿瘤，覆盖的皮肤和乳腺的部分（ 图1C ）。 </li><li>出血时，积极发现出血立即烧灼他们。如果超过250μL的血液丢失，请将小鼠从学习中排除并安乐死。 </li><li>使用伤口夹具将伤口夹紧闭切割部位（ 图1C ）。 注意:手术后10天用伤口夹去除剂取下伤口夹。 </li><li> 皮下施用温暖的无菌生理盐水（占动物体重的4％），并用热灯保持动物温暖。在麻醉恢复期间每5-10分钟检查动物。 注意:施用布比卡因或利多卡因会增加术后死亡率（数据未显示）。手术后1小时，出现疼痛迹象（臀部，不愿意移动，无法修复）的小鼠安乐死。 <ol><li>一旦鼠标完全恢复，返回到其他老鼠的公司。 </li></ol></li></ol>血液和肺的分离和处理用于流式细胞术和组织学 &lt;OL&gt; <li>在研究终点，如果需要，准备小鼠进行各种组织病理学评估。在安乐死前90分钟，给予每只小鼠腹膜内注射浓度为6.25μg/μL的1×PBS中的溴脱氧尿苷（BrdU，50μg/ g小鼠重量）。 注意:冷冻储存的溶解的溴脱氧尿苷在制备后1个月内使用。 </li><li>在安乐死前10分钟，麻醉小鼠，使用肝素化毛细管收集后蹄血（&gt; 500μL），随后转移到涂有冰上的EDTA二钾管。 </li><li>为了去除肺部和残留的乳腺组织，用剪刀从下腹部到口部进行中线切口以暴露胸腔和腹腔（ 图2A ），并横向剥离皮肤，还暴露剩余的右侧第 4和第 5 个乳腺腺体。 </li><li>消除剩余的乳腺组织，并检查我吨通过苏木精和伊红（H＆E）染色（ 补充 图1）评估串行切片福尔马林固定的石蜡包埋的（FFPE）组织，以排除原发性肿瘤再生长。 </li><li>为了清除肺，用剪刀打开隔膜的腹壁，然后通过切开腹主动脉来安乐死动物，在灌注肺之前排出血液（ 图2B -2C ）。 </li><li>从腹部方法切割隔膜，确保避免肺和心脏。然后，通过切穿肋骨的侧面暴露胸部（ 图2D ）。 </li><li>在20 mL注射器上使用23号针头，通过心脏右心室灌注〜5.0 mL DPBS（〜10 mL / min）的肺，直到肺完全变白（ 图2E -2F ）。立即切断主体的心脏因此，血液不会重新灌注到肺部。 </li><li>对于待组织的肺组织进行组织病理学评估，使用23号针头（ 图2G - 2H ），将4℃下的〜1.0mL 10％福尔马林注射到暴露的气管斜面侧朝向肺。一旦肺完全扩张并充满固定剂，停止注射（ 图2I ）。 </li><li>根据标准组织病理学程序，从气管切除肺叶，并将中性缓冲福尔马林的肺组织固定在随后的石蜡包埋或OCT-冷冻培养基中。 </li><li>通过FFPE肺组织的连续切片定量评估肺转移负荷，并通过H＆E染色（ 图1B ）评估切片切片（100μm，十三个切片）。 </li></ol>

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作者没有披露与本文提供的数据的冲突。

修改和故障排除: 当钝器从腹壁解剖肿瘤时，肿瘤可能仍然粘附在腹壁上。在&lt;5％注射肿瘤的小鼠中观察到这种情况（数据未显示）。对于具有粘附于腹壁的肿瘤的小鼠，应当对小鼠进行安乐死，因为切除难度而没有原发性肿瘤再生长。 模型/技术的限制: 除了肺外，其他研究者报告除了肺外，分布在肝脏?...

北美地区的女性患乳腺癌的终生风险约为12％ 2 ;大多数这些个体将通过手术去除原发性肿瘤，并且依赖于癌症亚型，然后将在佐剂设置中接受靶向的内分泌，化疗和/或放射治疗3 。诊断为接受抗雌激素治疗的激素受体阳性癌症妇女和接受HER2靶向治疗的HER2阳性肿瘤的妇女与放射治疗/化疗相关的实例包括:三重阴性肿瘤尚无靶向治疗3 。尽管在放疗，化疗，个性化和基于激素疗法的进步是补充手术切除，复发的疾病诊断为II期或III期疾病有4名妇女的30％-70％，由于治疗是在远处器官转移根除疾病很大程度上是无效的，包括肺，骨，b雨和/或肝脏5 。这是特别重要的，因为当没有原发性肿瘤再生长时发生转移性疾病时，这意味着在确定性手术时传播的恶性细胞可能已经存在于二级器官中。因此迫切需要能够消除或减缓转移性肿瘤生长的治疗方法。 虽然乳腺癌发生的从头鼠模型在揭示调节肿瘤进展1的机制方面已经非常显着，但现有模型也有一些限制。其中之一是事实， 从头转基因模型通常在多个乳腺中发展原发性肿瘤，其中原发性肿瘤负担限制了研究的持续时间。虽然原发性肿瘤细胞逃逸和转移性种子可能在这些模型的肿瘤进展早期发生，但是转移性肿瘤的坦率发展发生在晚期，而且依赖于n小鼠模型和应变背景，经常部分渗透1 。这进一步限制了从头模型发现调节继发器官转移的分子的效用，以及用于评估治疗剂在辅助治疗中的临床前功效。 为了规避这些局限性，我们开发了一种乳腺癌转移到肺的从头自体模型。携带晚期从头乳腺肿瘤的父母转基因雌性（ 即 ，本文所述用于研究的FVB / n株系背景上的MMTV-PyMT）老化至〜100天6 ，此时，其原发性肿瘤经手术切除并酶解解为单细胞悬液悬浮液（1×10 6个细胞）又原位移植到6-7周龄的受体同基因雌性小鼠中，其中单一原发性乳腺肿瘤发生在38至60天的时间段3 ）处，将受体小鼠麻醉并手术切除原发性肿瘤，使得在手术部位的肿瘤再生长被最小化，与女性的手术一致（ 补充图1 ）。在FVB / n株系背景下，小鼠在手术后〜115天内以45％的外显率在肺中发展组织学检测的转移灶（ 图1B ）。随着转移性肿瘤生长的这种延长的延迟，该模型在辅助治疗递送中是独特的，并且用于阐明和评估影响原发性肿瘤手术切除后的转移进展的潜在生物学。

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Isofluorane</td>
			<td style="width:140px;">Piramal Healthcare</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">Prescription order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Roche</td>
			<td>11088793001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>ThermoFisher</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 mL Pyrex bottle</td>
			<td>Sigma-Aldrich</td>
			<td>CLS139525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Atlanta Bio&#160;</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.7 &#181;m nylon strainer&#160;</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tube&#160;</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Growth factor-reduced Matrigel&#160;</td>
			<td>BD</td>
			<td>354230</td>
			<td>Growth factor-reduced solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(vinylpyrrolidone)&#8211;Iodine complex</td>
			<td>Sigma-Aldrich</td>
			<td>PVP1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">29 gauge 0.3 mL insulin syringe</td>
			<td>BD</td>
			<td>324702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Vessel Cauterizer Kit</td>
			<td>FST</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wound clips</td>
			<td>Texas Scientific</td>
			<td>205016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoClip wound clip applier&#160;</td>
			<td>BD</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoClip wound clip remover&#160;</td>
			<td>BD</td>
			<td>427637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromodeoxyuridine</td>
			<td>Roche</td>
			<td>10280879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparinized capillary tubes&#160;</td>
			<td>Fisher</td>
			<td>22362566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtainer tubes with dipotassium EDTA&#160;</td>
			<td>BD</td>
			<td>365974</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 mL syringe&#160;</td>
			<td>BD</td>
			<td>309661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS</td>
			<td>Thermo-Fisher</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT-freezing medium&#160;</td>
			<td>VWR</td>
			<td>25608930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formalin, Buffered, 10% (Phosphate Buffer)</td>
			<td>Fisher</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23g needle</td>
			<td>Fisher</td>
			<td>14-826-6B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FVB/n mouse</td>
			<td>Jackson Laboratories</td>
			<td>001800</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical procedures and methodology for a preclinical murine model of de novo mammary cancer metastasis

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

从衍生自MMTV-PyMT小鼠的原发乳腺肿瘤中接受1×10 6个细胞的大于75％的受体小鼠在38-60天内开发尺寸为172至450mm 3的单个乳腺腺癌（数据未显示）。符合随机分组的小鼠然后在手术切除原发性肿瘤后纳入研究组（ 图1C ）。在不到2％的原发肿瘤手术切除的小鼠中鉴定到原发性肿瘤再生长（ 补充 图1 ）。?...

Watch this Scientific Journal Video about Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis at JoVE.com

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

评估靶向乳腺癌转移的辅助治疗的临床前模型缺乏。为了解决这个问题，我们开发了从头肺乳腺癌转移，其特征在于，在辅助设定（后手术切除原发肿瘤的）给药的治疗可以为疗效影响先前接种的肺转移进行评估的鼠模型。

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

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

surgical-procedures-methodology-for-preclinical-murine-model-de-novo

Research

JoVE Journal

Cancer Research

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

13.7K 次观看.  Oregon Health & Science University. 评估靶向乳腺癌转移的辅助治疗的临床前模型缺乏。为了解决这个问题，我们开发了从头肺乳腺癌转移，其特征在于，在辅助设定（后手术切除原发肿瘤的）给药的治疗可以为疗效影响先前接种的肺转移进行评估的鼠模型。 

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

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

详细的定性C1498小鼠细胞系及其关联白血病小鼠模型协议

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

科研

癌症研究

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

原发肿瘤发展小鼠实验模型和腹膜转移<em&gt;通过</em&gt;原位接种卵巢癌细胞

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

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

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

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

开发和维护的临床前患者来源的肿瘤异种移植模型为新的抗癌疗法的调查

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

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

体内模型对缺氧对肿瘤转移的影响测试

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane permeabilization by means of &#34;electroporation&#34;. To achieve the &#34;electroporation&#34; of the cells, electric pulses are generated by a generator and delivered to the target tissue by the use of electrodes. Electroporation is a physical method which is used to introduce molecules, like cytostatic drugs, into the cells that could not pass the cell membrane on their own. In electrochemotherapy, currently, cisplatin and bleomycin are clinically used. Electrochemotherapy antitumor effectiveness is high, for example up to 100% complete response of canine mast cell tumors smaller than 2 cm3 was achieved. Additionally, electrochemotherapy can be used for the treatment of inoperable tumors. One of the important characteristics of electrochemotherapy is that it can be effective as a one-time treatment only. However, in the case of failure or partial tumor response it can be repeated several times with equal or improved effectiveness. Electrochemotherapy is already a standard treatment for cutaneous and subcutaneous tumors of various histologies in human and veterinary oncology. Furthermore, several clinical studies exploiting electrochemotherapy for deep-seated tumors are on-going.

Electrochemotherapy is the local treatment of solid tumors. The main mechanism is electroporation-mediated permeabilization of the tumor cells' membrane which enables increased internalization of cytostatics like bleomycin and cisplatin. Thus, antitumor effectiveness is potentiated at the site of the application of electric pulses to the tumors.

在电化学疗法的操作程序在肿瘤治疗中的狗和猫

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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 ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

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. ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

使用小鼠化疗引起的粘炎模型评估小肠损伤和适应的重要端点和增殖标记

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

临床前小鼠模型的外科手术和方法

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