这项研究部分由珍妮特·伯罗斯纪念基金会资助。我们感谢患者和 Karmanos 癌症研究所妇科肿瘤科收集网膜样本。我们还感谢 Karmanos 癌症研究所的生物样本库和相关科学核心，以协调患者招募和病理切片的制备。生物样本库和相关科学核心部分由美国国立卫生研究院中心向韦恩州立大学卡马诺斯癌症研究所提供的 P30 CA22453资助。

以下研究方案已由韦恩州立大学机构审查委员会 （IRB） 审查和批准。手术前已获得所有患者的知情同意。 图1 说明了该协议中的三个一般步骤。
1.人网膜组织的制备
<ol><li>准备网膜培养基（DMEM / F12 + 10%胎牛血清+ 1%青霉素 - 链霉素）并储存在4°C。 将 30-40 mL 该培养基分装到无菌的 50 mL 锥形管或手术标本容器中。</li>
	<li>通过网膜活检或网膜切除术获取手术标本。在手术室中取出后，立即将无菌标本浸入网膜培养基中。如果工作流程不允许这样做，请尽快将标本放入网膜培养基中。通过将样品放在冰上转移样品并储存在4°C直至处理。 注意:切除的网膜标本的大小将决定可工作组织的数量。</li>
	<li>使用层流罩在收集后1-2小时内处理网膜标本。确保所有材料和工具都是无菌或无菌的。</li>
	<li>从收集容器中取出网膜并将其转移到 100 毫米培养皿中。将标本浸入1x磷酸盐缓冲盐水（1x PBS）中，轻轻洗涤标本以除去任何血凝块或碎屑。</li>
	<li>将组织切成碎片（0.5 cm x 0.5 cm或100-200 mg; 图2A）使用小手术剪刀或 10-15 毫米手术刀。使用小手术钳帮助操纵组织并避免压碎网膜。如果使用能量装置通过手术切除网膜标本，则避免使用密封边缘处的扭曲组织。</li>
	<li>将每块切割的网膜放入24孔培养板的单个孔中，并用500μL网膜培养基填充孔或填充到足以覆盖网膜的体积，而不会让网膜块漂浮在孔底上方（图2B）。</li>
	<li>将网膜块储存在4°C的新鲜培养基中，直至准备注射。</li>
</ol>2.卵巢癌细胞的制备
<ol><li>在75cm 2培养瓶中在37°C和5%CO2至网膜收集当天至少75%汇合度下培养人卵巢癌细胞。 注意:该协议利用先前描述的mCherry阳性OCSC1-F2人卵巢癌细胞15,16,17,18。它可以被修饰为任何标记有荧光信号的癌细胞系。</li>
	<li>收集网膜标本后立即在层流罩内制备细胞。确保所有材料和工具都是无菌或无菌的。</li>
	<li>向培养瓶中加入 10 mL 无菌 1x PBS，轻轻摇动培养瓶洗涤细胞。除去 1x PBS，然后加入 3 mL 0.05% 胰蛋白酶-EDTA。</li>
	<li>轻轻摇动培养瓶以覆盖所有细胞，然后将培养瓶置于培养箱（37°C和5%CO2）中不超过5分钟。敲击培养瓶以完全分离细胞，然后通过加入 3 mL 网膜培养基中和胰蛋白酶。</li>
	<li>通过移液混合细胞悬液，然后将悬浮液转移到 15 mL 锥形管中。将悬浮液在24°C下以1,200× g 离心5分钟。 除去上清液，将细胞沉淀重悬于 6 mL 新鲜网膜培养基中。</li>
	<li>使用血细胞计数器对细胞进行计数。将细胞悬液稀释至每 100-200 μL 悬浮液至少 100,000 个细胞。这是注射到每块网膜的体积。</li>
	<li>将适量的细胞转移到新的培养瓶中以继续细胞传代。</li>
</ol>3.卵巢癌细胞注射
<ol><li>使用 1 mL 注射器吸取细胞悬液，然后连接 26 G 针头。不要用针头抽出细胞悬液，因为这会使细胞破碎。</li>
	<li>使用小手术钳拿起一块切开的网膜。将网膜转移到单独的工作无菌培养皿中以方便注射。用针尖轻轻刺穿网膜，并将少量细胞悬浮液注射到组织中（图2C）。</li>
	<li>在网膜的几个区域重复注射至总体积至少为 100 μL 或 100,000 个细胞。请注意，大部分细胞悬液可能似乎聚集在标本周围。如果担心注射质量，则重复注射或将更大体积的细胞悬液注射到标本中。</li>
	<li>将注入的网膜样品放回 24 孔培养板的每个孔中。请注意，网膜培养基的体积达到覆盖网膜而不让其漂浮的水平。小心地处理板并将其放入培养箱（37°C和5%CO2）中。</li>
	<li>可选:使用基质胶（基底膜基质）将网膜组织悬浮在固体基质中，以便更容易注射和更浓缩的细胞悬液递送。<ol><li>在4°C下解冻基底膜基质，并在使用前立即与网膜培养基以1:1的比例混合。将基底膜基质混合物储存在冰上或4°C直至注射。用足够的基底膜基质混合物填充空孔，以浸入切好的网膜。</li>
		<li>将标本放入基底膜基质混合物中，然后将24孔培养板置于培养箱（37°C和5%CO2）中20分钟。混合物凝固后，继续如上所述注射。</li>
	</ol></li>
	<li>使用荧光成像确认注射成功。确保在注射部位看到荧光信号条纹（图2D）。请注意，注射后附着在网膜上的实际细胞数是不可预测的。</li>
</ol>4.人网膜和卵巢癌细胞的共培养
<ol><li>通过加入 500-2000 μL 新鲜网膜培养基，每 48-72 小时更换培养基一次。不要将培养基直接移液到网膜组织顶部，因为这可能会取代癌细胞。请注意，如果介质颜色变为黄色，则更改介质。 注意:培养基更换的频率将取决于使用的培养基体积和癌细胞生长的轨迹。一旦癌细胞播种了网膜，网膜块是否漂浮就不再重要了。</li>
	<li>可选:如果使用基底膜基质，则基质通常会在第一次更换培养基时溶解。</li>
	<li>使用荧光成像监测卵巢癌肿瘤的生长。影像学检查的频率由研究者自行决定。预计约14天出现小肿瘤的证据（图3A-C）。结果可能因注射质量而异。</li>
	<li>根据实验终结点终止实验。 注:过去 50 天观察到肿瘤生长和网膜组织的完整性。</li>
</ol>

建立卵巢癌-网膜相互作用的 3D 离体模型

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作者没有什么可透露的。

使用该方案，结合基本的体外和离体技术开发了卵巢癌腹膜癌的临床前模型。在用 mCherry+ OCSC1-F2 人卵巢癌细胞接种网膜标本后，在 50 天的共培养中观察到进行性肿瘤生长。该方法是在使用不同网膜标本的几次实验试验中开发和优化的。肿瘤的成功生长取决于网膜的质量、癌细胞的活力和注射的有效性。在本报告中，我们仅使用了 mCherry+ OCSC1-F2 细胞和 mCherry+ R182 人卵巢癌细胞系，这些细胞系先前在几篇出版物 12、15、16、17、18、19、20、21、22 中报道。使用这两种具有不同倍增时间的细胞系（OCSC1-F2为16小时，R182为36小时），注意到在网膜组织内实现对数生长的时间差异。尽管如此，我们还是成功地在共培养系统中建立了两种细胞系。
研究人员、研究协调员和手术团队之间的有效沟通对于网膜标本的收集和处理至关重要。研究者确定了方案候选者，研究协调员在手术前征得了患者的同意，研究者从手术室收集了标本。鉴于这种工作流程，研究者的日程安排必须灵活，因为许多影响收集的患者侧因素是不可预测的（例如，手术取消、手术时间改变、网膜切除不足或未切除）。从手术室直接观察和选择组织，确保了标本的完整性、手术无菌性和及时处理。最初，标本采集工作委托给研究协调员;然而，在收集了一些标本后，我们注意到处理时间延迟（即 >4 小时），网膜质量严重劣质（例如，组织是碎片）。在一项实验试验中，视网膜组织在注射前在4°C下储存48小时，因为癌细胞没有及时准备好。仍然观察到类似的肿瘤生长，但由于已知温度变化会影响肿瘤微环境（TME），因此没有重复该序列。
在采集标本之前，根据手术时间表让癌细胞充分融合是很重要的。由于每月收集的标本数量各不相同，因此并不总是保持卵巢癌细胞的活性培养。至少提前 1 周解冻冷冻细胞，使用标准方案接种，然后至少通过一次细胞以确保活力。该协议中使用的细胞系相对有弹性;然而，我们仍然遇到过细胞在手术当天没有恢复并且无法注射的情况。在这项研究中，没有尝试注射汇合度低于50%的癌细胞。通过使用良好的无菌技术和根据预期的标本采集时间表按比例分裂细胞以保持汇合，从而减轻了与细胞制备相关的后续延迟。我们将方案限制为仅两种荧光标记的癌细胞系，但重要的是要注意，也可以使用其他已建立的癌细胞系或没有荧光蛋白的原代癌细胞。
最后，肿瘤生长速率与注射后脂肪细胞附近和内部癌细胞的汇合相关。这是通过在注射后拍摄所有网膜标本的荧光图像来确定的。与实体器官或皮下组织的肿瘤细胞接种相比，将癌细胞注射到网膜组织中更具挑战性。网膜的松散结构和脂肪稠度使组织无法有效保持细胞悬液体积而不会泄漏。当使用较小的针头（即 30 G 或更窄）进行注射时，由于细胞增殖减少，因此存在细胞碎裂的担忧。我们测试了各种注射技术、细胞数量和细胞悬液体积，发现结果与上述方案一致。同时，我们承认，鉴于细胞的恶性，协议的这种变化可能仍会产生类似的结果。基质胶的加入使注射步骤更容易，因为细胞外基质更好地保持细胞悬液;然而，这种方法更昂贵，并且不能始终如一地在样本中产生更高的肿瘤负荷。总体而言，任何网膜标本都很少生长肿瘤，但肿瘤的数量和生长速度各不相同。
该模型通过复制早期转移到富含脂肪的腹膜组织来模拟卵巢癌肿瘤发生的标志。该协议允许在不需要技能的情况下进行变化，易于复制，并生成具有潜在转化价值的系统。与卵巢癌现有的体 外 和 离体 模型相比，这里描述的方法结合了多种技术来产生一种在较长寿命内保留肿瘤结构和TME的模型。此外，我们观察到，当将具有多个肿瘤的网膜标本转移到新孔中并用无癌网膜培养（即未注射）时，幼稚网膜在 7 天内接种了癌细胞。这一发现表明，复制的肿瘤通过上皮-间充质转化保留了转移潜力。在文献中，我们没有发现任何类似的卵巢癌模型可以长时间培养组织活检。这种方法得到了先前发现的支持，即从小鼠网膜中分离的间皮细胞在收集后可以培养超过 30 代23。
本研究中描述的模型可用于进一步了解卵巢癌细胞与富含脂肪的网膜内细胞之间直接相互作用的影响。癌细胞可以从网膜组织中解离出来，用于转录组学或免疫组化（IHC）分析。此外，细胞还可用于药物反应研究。
该模型的主要局限性是肿瘤数量、肿瘤生长速度和肿瘤位置是不可预测的。样本之间缺乏标准化和差异可能会限制该模型的应用。我们还提出了一些观察结果，这些观察结果需要在制定该协议期间进一步检查。例如，一项实验中的"对照"重复实际上并非无癌症，因为患者随后被诊断出患有网膜微转移;然而，未注射的肿瘤在整个实验期间仍然存活。该模型的未来应用将包括早期肿瘤发生过程中TME的分子研究，通过注射免疫细胞操纵TME以及药物反应研究。综上所述，我们相信本文描述的临床前模型将增加现有研究工具的库，并有助于阐明TME在卵巢癌中的分子机制。

卵巢癌是全球最致命的妇科恶性肿瘤1.罹患这种癌症的终生风险约为 1/70，中位诊断年龄为 63 岁2。原发性卵巢恶性肿瘤在组织学上分为上皮性和非上皮性。上皮性卵巢癌 （EOC） 占肿瘤的 90% 以上，最常见的亚型是高级别浆液性癌 （HGSC），约占 EOC 的 70%-80%。目前，尚无有效的筛查方法及早发现疾病。因此，大多数患者在癌症扩散到整个腹膜腔后被诊断为晚期（即国际妇科和妇产科联合会 [FIGO] III 期或 IV期）2。
标准的一线治疗是细胞减灭术，以去除所有可见的肉眼病变，然后进行辅助铂类化疗以消除任何残留的微观病变。虽然在过去二十年中卵巢癌治疗取得了许多进展，但大约 70% 的晚期疾病患者会在治疗后 3 年内复发3.鉴于这些患者的总体预后较差，EOC正在进行和未来的转化研究工作旨在确定早期检测的生物标志物，预防转移，改进目前的疗法以逃避耐药性，并开发新的个性化癌症治疗方法。
腹膜腔内的全身转移及其相关的化疗耐药性是改善卵巢癌患者治疗的两个主要限制因素4,5。网膜是一种脂肪围裙状结构，从胃垂下到肠道上方，是卵巢癌转移的主要部位 6,7。除了作为物理屏障的功能外，网膜还被证明具有再生和血管生成能力，并具有免疫活性，它们共同促进血管形成、加速伤口愈合和限制感染8。它含有高浓度的干细胞，可以分化成各种细胞类型，可以帮助修复受损组织。网膜会因损伤或感染而发炎，从而触发免疫细胞迁移到损伤部位9。这些免疫细胞释放生长因子和其他分子，有助于促进受损组织的修复和再生。位于网膜中的免疫细胞，如巨噬细胞、淋巴细胞和浆细胞，是被称为"乳白色斑点"的结构，负责检测和攻击病原体并调节腹膜免疫。网膜还被证明在诱导免疫耐受方面发挥作用10，免疫耐受是免疫系统耐受自身抗原而不攻击健康组织的能力。然而，相同的免疫相关活动也参与病理反应，例如网膜肿瘤的生长、转移和免疫监视的逃避 9,11。我们实验室和其他实验室先前的研究表明，脂肪微环境在抑制抗肿瘤免疫反应和获得化疗耐药性方面具有独特而积极的作用12,13,14。不幸的是，我们对网膜提供促肿瘤微环境的细胞和分子机制的信息有限。
为了更好地了解癌细胞与网膜之间的相互作用，开发了一种由人卵巢癌细胞和患者来源的网膜外植体组成的 3D 培养系统。这里描述的方案代表了一种新的腹膜癌的 离体 模型。该模型模拟了这种富含脂肪的组织中卵巢癌肿瘤发生的自然进展。所提出的模型易于生成，价格低廉，并且可能适用于卵巢癌研究中的转化研究。

<table><tbody><tr><td>0.05% Trypsin-EDTA (1x)</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>1 mL Insulin Syringe with 26 G detachable needle</td><td>BD</td><td>329652</td><td></td></tr><tr><td>10 mL Serological Pipets</td><td>CELLTREAT</td><td>229010B</td><td></td></tr><tr><td>100 mm Tissue Culture Dish</td><td>Fisherbrand</td><td>FB012924</td><td></td></tr><tr><td>15 mL Centrifuge Tube</td><td>CELLTREAT</td><td>229411</td><td></td></tr><tr><td>24 Well Cell Culture Plate</td><td>Costar</td><td>3524</td><td></td></tr><tr><td>50 mL Centrifuge Tube</td><td>CELLTREAT</td><td>229421</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Tissue Culture Flask</td><td>CELLTREAT</td><td>229341</td><td></td></tr><tr><td>Corning Cell Counter</td><td>Corning</td><td>9819000</td><td></td></tr><tr><td>Cytation 5 imager</td><td>Biotek</td><td></td><td></td></tr><tr><td>DMEM/F12 (1:1) (1x), +L-Glutamine, +2.438 g/L Sodium Bicarbonate</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>Fetal Bovine Serum, Qualified</td><td>Gibco</td><td>1043028</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356230</td><td>Basement membrane matrix</td></tr><tr><td>No. 10 Stainless Steel Disposable Scalpel</td><td>Integra-Miltex</td><td>4410</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffered Saline, pH 7.4 (1x)</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Revolve microscope</td><td>Echo</td><td></td><td></td></tr></tbody></table>

an ex vivo model of ovarian cancer peritoneal metastasis using human omentum

卵巢癌是最致命的妇科恶性肿瘤。网膜在为转移性卵巢癌细胞提供支持性微环境以及允许肿瘤耐受的免疫调节信号方面发挥着关键作用。 然而，我们只有有限的模型来密切模拟卵巢癌细胞和富含脂肪的组织之间的相互作用。为了进一步了解网膜提供促肿瘤微环境的细胞和分子机制，我们开发了一种独特的癌细胞-网膜相互作用的 3D 离体 模型。使用人类网膜，我们能够在这种富含脂肪的微环境中培养卵巢癌细胞，并监测负责肿瘤生长和免疫调节的因素。除了为研究这种富含脂肪的肿瘤微环境提供平台外，该模型还为开发和评估靶向该利基转移性癌细胞的新治疗方法提供了极好的平台。该模型易于生成，成本低廉，适用于转化研究。

到第14天左右，卵巢癌细胞成功在网膜标本中建立是显而易见的（图3A-C）。每个收集的标本至少制备并注射24个重复样品，以便进行进一步的实验。通过拍摄荧光图像来监测肿瘤生长（图3D，E）。图像必须仔细解释，因为每个未附着在网膜上的孔的底部也生长着单层癌细胞。我们更喜欢在网膜悬浮在培养基中时拍摄网膜图像，以避免与癌细胞单层重叠的荧光信号。
到第14天没有荧光信号可视为注射失败。据观察，癌细胞最初在前 7 天内扩散到网膜表面，但随着时间的推移，它们聚集形成肿瘤（图 3A-D）。样本之间肿瘤负荷的替代测量是网膜培养基颜色从红色变为黄色的速率（图3F）。我们利用荧光成像、组织学检查和大体检查来确认共培养 50 天后的肿瘤生长和网膜组织活力（图 4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66031/66031fig01.jpg"> 图 1:协议中一般步骤的图示。 （A）步骤1:网膜的制备;（B）第2步:注射癌细胞;（C）第三步:建立癌细胞-网膜微环境。（图于 BioRender.com 年编制）。<a href="https://www.jove.com/files/ftp_upload/66031/66031fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66031/66031fig02.jpg"> 图2:癌细胞的制备和注射到网膜中 。 （A） 将网膜切成 1x1 厘米的小块。（B） 将每块放入 24 孔板的孔中，并覆盖有 500 μL 培养基。（C）将癌细胞注射到网膜片中。（D） 注射后观察到荧光癌细胞的条纹。 <a href="https://www.jove.com/files/ftp_upload/66031/66031fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66031/66031fig03.jpg"> 图 3:共培养 14 天和 25 天后的代表性图像。 （A-C） 共培养 14 天后的代表性图像:（A） 阶段，（B） mCherry 通道，（C） 合并。（D-E）共培养 25 天后的代表性图像。（F） 培养基颜色从粉红色变为黄色是癌细胞注射成功和共培养建立的标志。<a href="https://www.jove.com/files/ftp_upload/66031/66031fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66031/66031fig04.jpg"> 图4:大体形态和苏木精和曙红（H&E）染色。 （A，B） 第 50 天共培养物的代表性大体形态;箭头指向 mCherry+ 卵巢癌细胞的生长部位。（C，D）第 50 天共培养物的代表性 H&E 染色。<a href="https://www.jove.com/files/ftp_upload/66031/66031fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction at JoVE.com

作者聚焦:用于研究癌症-脂肪微环境相互作用的高级离体模型 

该协议描述了癌细胞 - 网膜相互作用的三维（3D） 离体 模型的建立。该模型为阐明脂肪生态位内的促肿瘤机制和测试新疗法提供了一个平台。

基于人网膜的卵巢癌腹膜转移离 体 模型

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

目前的研究使用由分离的脂肪细胞和卵巢癌细胞组成的共培养系统。这些脂肪细胞在体外与前脂肪细胞分化。然后通过transwell插入物分离这些细胞，因此缺乏细胞间的直接接触。这种离体培养方法为研究癌细胞与脂肪微环境的直接相互作用提供了一个模型。我们的模型保留了人类动量的结构，因此保留了该器官中典型的各种细胞类型。除脂肪细胞外，这些细胞还包括支持成纤维细胞和免疫细胞。该模型还将这些细胞维持在其固有基质中。该模型为开发和评估靶向该利基市场转移性癌细胞的新治疗方法提供了一个极好的平台。

an-ex-vivo-model-ovarian-cancer-peritoneal-metastasis-using-human

Nanyang Technological University

Research

JoVE Journal

Cancer Research

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

2.5K 次观看.  Karmanos Cancer Institute. 目前的研究使用由分离的脂肪细胞和卵巢癌细胞组成的共培养系统。这些脂肪细胞在体外与前脂肪细胞分化。然后通过transwell插入物分离这些细胞，因此缺乏细胞间的直接接触。这种离体培养方法为研究癌细胞与脂肪微环境的直接相互作用提供了一个模型。我们的模型保留了人类动量的结构，因此保留了该器官中典型的各种细胞类型。除脂肪细胞外，这些细胞还包括支?...

视频: 作者聚焦:用于研究癌症-脂肪微环境相互作用的高级离体模型 

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 ...

科研

癌症研究

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 ...

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 ...

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

<meta charset="utf8"></head><body>使用人基质促进转移的卵巢癌原位小鼠模型

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

使用人基质促进转移的卵巢癌原位小鼠模型

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

医学

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

腹膜内卵巢癌转移的定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Orthotopic Inoculation of Ovarian Cancer Cells into Murine Ovary: A Procedure to Establish an Ovarian Cancer Mouse Model to Study Peritoneal Dissemination of Cells

Source: Koya, Y. et al. <a href="https://www.jove.com/t/54353">Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells.</a> J. Vis. Exp. (2016)
This video describes the protocol for creating a murine experimental model to study tumor development and peritoneal metastasis. This ovarian cancer model is generated by the orthotopic inoculation of human epithelial ovarian cancer cells into the ovaries of a female mouse.

卵巢癌细胞原位接种小鼠卵巢:建立卵巢癌小鼠模型以研究细胞腹膜播散的程序

Source: Koya, Y. et al. Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian ...

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Animal model studies

Modeling Ovarian Cancer Cell Colonization of Omentum: An Ex Vivo Method to Establish Milky Spot Colonization of Fluorescently Labeled Ovarian Cancer Cells in Murine Omentum Explants

Source: Krishnan, V. et al. <a href="https://www.jove.com/t/52721">In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we establish an ex vivo omental organ culture overlaid with GFP-expressing ovarian cancer cells to study ovarian cancer colonization within the omental explant. This protocol analyzes immune cell structures within the omentum known as milky spots that promote metastases.

模拟网膜卵巢癌细胞定植:一种在小鼠网膜外植体中建立荧光标记卵巢癌细胞银斑定植的离体方法

Source: Krishnan, V. et al. In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. ...

基于人网膜的卵巢癌腹膜转移离体模型

副本

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原发肿瘤发展小鼠实验模型和腹膜转移

体内模型对缺氧对肿瘤转移的影响测试

门静脉注射模型来研究肝转移乳腺癌

使用人基质促进转移的卵巢癌原位小鼠模型

使用人基质促进转移的卵巢癌原位小鼠模型

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

腹膜内卵巢癌转移的定量

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

卵巢癌细胞原位接种小鼠卵巢:建立卵巢癌小鼠模型以研究细胞腹膜播散的程序

模拟网膜卵巢癌细胞定植:一种在小鼠网膜外植体中建立荧光标记卵巢癌细胞银斑定植的离体方法

基于人网膜的卵巢癌腹膜转移离 体 模型

副本

摘要

探索更多视频

原发肿瘤发展小鼠实验模型和腹膜转移

体内模型对缺氧对肿瘤转移的影响测试

门静脉注射模型来研究肝转移乳腺癌

使用人基质促进转移的卵巢癌原位小鼠模型

使用人基质促进转移的卵巢癌原位小鼠模型

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

腹膜内卵巢癌转移的定量

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

卵巢癌细胞原位接种小鼠卵巢:建立卵巢癌小鼠模型以研究细胞腹膜播散的程序

模拟网膜卵巢癌细胞定植:一种在小鼠网膜外植体中建立荧光标记卵巢癌细胞银斑定植的离体方法

基于人网膜的卵巢癌腹膜转移离体模型