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

<ol>
	<li>Siegel, R. L., Miller, K. D., Wagle, N. S., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 73 (1), 17-48 (2023).</li><li>Berek, J. S., Renz, M., Kehoe, S., Kumar, L., Friedlander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+of+the+ovary,+fallopian+tube,+and+peritoneum:+2021+update.">Cancer of the ovary, fallopian tube, and peritoneum: 2021 update.</a> Int J Gynaecol Obstet. 155 (Suppl 1), 61-85 (2021).</li><li>Ledermann, J. 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=Newly+diagnosed+and+relapsed+epithelial+ovarian+carcinoma:+ESMO+clinical+practice+guidelines+for+diagnosis,+treatment+and+follow-up.">Newly diagnosed and relapsed epithelial ovarian carcinoma: ESMO clinical practice guidelines for diagnosis, treatment and follow-up.</a> Ann Oncol. 24 (Suppl 6), vi24-32 (2013).</li><li>Jelovac, D., Armstrong, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+diagnosis+and+treatment+of+ovarian+cancer.">Recent progress in the diagnosis and treatment of ovarian cancer.</a> CA Cancer J Clin. 61 (3), 183-203 (2011).</li><li>Morgan, R. J., 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=Ovarian+cancer.+Clinical+practice+guidelines+in+oncology.">Ovarian cancer. Clinical practice guidelines in oncology.</a> J Natl Compr Canc Netw. 6 (8), 766-794 (2008).</li><li>Nieman, K. M., 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=Adipocytes+promote+ovarian+cancer+metastasis+and+provide+energy+for+rapid+tumor+growth.">Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.</a> Nat Med. 17 (11), 1498-1503 (2011).</li><li>Motohara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evolving+story+of+the+metastatic+voyage+of+ovarian+cancer+cells:+cellular+and+molecular+orchestration+of+the+adipose-rich+metastatic+microenvironment.">An evolving story of the metastatic voyage of ovarian cancer cells: cellular and molecular orchestration of the adipose-rich metastatic microenvironment.</a> Oncogene. 38 (16), 2885-2898 (2019).</li><li>Di Nicola, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omentum+a+powerful+biological+source+in+regenerative+surgery.">Omentum a powerful biological source in regenerative surgery.</a> Regen Ther. 11, 182-191 (2019).</li><li>Meza-Perez, S., Randall, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+functions+of+the+omentum.">Immunological functions of the omentum.</a> Trends Immunol. 38 (7), 526-536 (2017).</li><li>Liu, M., Silva-Sanchez, A., Randall, T. D., Meza-Perez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+immune+responses+in+the+peritoneal+cavity+and+omentum.">Specialized immune responses in the peritoneal cavity and omentum.</a> J Leukoc Biol. 109 (4), 717-729 (2021).</li><li>Lee, W., 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=Neutrophils+facilitate+ovarian+cancer+premetastatic+niche+formation+in+the+omentum.">Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum.</a> J Exp Med. 216 (1), 176-194 (2019).</li><li>Cardenas, C., 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=Adipocyte+microenvironment+promotes+Bclxl+expression+and+confers+chemoresistance+in+ovarian+cancer+cells.">Adipocyte microenvironment promotes Bclxl expression and confers chemoresistance in ovarian cancer cells.</a> Apoptosis. 22 (4), 558-569 (2017).</li><li>Wu, Q., 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=Cancer-associated+adipocytes:+key+players+in+breast+cancer+progression.">Cancer-associated adipocytes: key players in breast cancer progression.</a> J Hematol Oncol. 12 (1), 95 (2019).</li><li>Zhang, Z., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue:+The+dysfunctional+adipocyte+-+a+cancer+cell's+best+friend.">Adipose tissue: The dysfunctional adipocyte - a cancer cell's best friend.</a> Nat Rev Endocrinol. 14 (3), 132-134 (2018).</li><li>Alvero, A. B., 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=TRX-E-002-1+Induces+c-Jun-dependent+apoptosis+in+ovarian+cancer+stem+cells+and+prevents+recurrence+in+vivo.">TRX-E-002-1 Induces c-Jun-dependent apoptosis in ovarian cancer stem cells and prevents recurrence in vivo.</a> Mol Cancer Ther. 15 (6), 1279-1290 (2016).</li><li>Alvero, A. B., 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=Novel+approach+for+the+detection+of+intraperitoneal+micrometastasis+using+an+ovarian+cancer+mouse+model.">Novel approach for the detection of intraperitoneal micrometastasis using an ovarian cancer mouse model.</a> Sci Rep. 7, 40989 (2017).</li><li>Craveiro, V., 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=Phenotypic+modifications+in+ovarian+cancer+stem+cells+following+Paclitaxel+treatment.">Phenotypic modifications in ovarian cancer stem cells following Paclitaxel treatment.</a> Cancer Med. 2 (6), 751-762 (2013).</li><li>Sumi, N. J., 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=Murine+model+for+non-invasive+imaging+to+detect+and+monitor+ovarian+cancer+recurrence.">Murine model for non-invasive imaging to detect and monitor ovarian cancer recurrence.</a> J Vis Exp. (93), e51815 (2014).</li><li>Agarwal, R., 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=Macrophage+migration+inhibitory+factor+expression+in+ovarian+cancer.">Macrophage migration inhibitory factor expression in ovarian cancer.</a> Am J Obstet Gynecol. 196 (4), 348.e1-348.e5 (2007).</li><li>Kelly, M. G., 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=TLR-4+signaling+promotes+tumor+growth+and+paclitaxel+chemoresistance+in+ovarian+cancer.">TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer.</a> Cancer Res. 66 (7), 3859-3868 (2006).</li><li>Li, J., 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=CBX7+binds+the+E-box+to+inhibit+TWIST-1+function+and+inhibit+tumorigenicity+and+metastatic+potential.">CBX7 binds the E-box to inhibit TWIST-1 function and inhibit tumorigenicity and metastatic potential.</a> Oncogene. 39 (20), 3965-3979 (2020).</li><li>Tedja, R., 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=Generation+of+stable+epithelial-mesenchymal+hybrid+cancer+cells+with+tumorigenic+potential.">Generation of stable epithelial-mesenchymal hybrid cancer cells with tumorigenic potential.</a> Cancers (Basel). 15 (3), 15030684 (2023).</li><li>Dauleh, S., 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=Characterisation+of+cultured+mesothelial+cells+derived+from+the+murine+adult+omentum.">Characterisation of cultured mesothelial cells derived from the murine adult omentum.</a> PLoS One. 11 (7), e0158997 (2016).</li></ol>

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?...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 cm2 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?...

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

Research

JoVE Journal

Cancer Research

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

2.4K 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 ...

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

神经入侵的体内小鼠坐骨神经模型

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

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

斑马鱼神经母细胞瘤转移模型

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Here, we describe a method of generating glioblastoma (GBM) organoids from primary patient specimens or patient-derived cell cultures and maintaining them to maturity. These GBM organoids contain phenotypically diverse cell populations and recreate tumor microenvironments ex vivo.

使用三维类器官培养物离 体 维持人胶质母细胞瘤细胞多样性

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

离体 小鼠尿路上皮细胞腺病毒-Cre介导的基因缺失的类器官模型

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

心内注射人前列腺癌细胞以创建骨转移异种移植小鼠模型

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is ...

基于人网膜的卵巢癌腹膜转移离 体 模型

基于人网膜的卵巢癌腹膜转移离体模型