Name: a mouse model to investigate the role of cancer-associated fibroblasts in tumor growth
Uploaded: 2020-12-22T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth at JoVE.com

作者要感谢Coller实验室的所有成员的有益意见。H.A.C.是丽塔·艾伦基金会的米尔顿·E·卡塞尔学者。我们感谢NIH / NCI 1 R01 CA221296-01A1，NIH 1 R01 AR070245-01A1，黑色素瘤研究联盟团队科学奖，癌症研究所临床实验室整合计划奖，Iris Cantor妇女健康中心/ UCLA CTSI NIH Grant UL1TR000124，加州大学癌症研究协调委员会，大卫格芬医学院代谢主题奖，临床转化科学研究所和Jonsson综合癌症中心， 布罗德干细胞研究中心（Rose Hills和Ha Gaba）的创新奖，加州大学洛杉矶分校前列腺癌孢子奖（美国国立卫生研究院国家癌症研究所，奖项编号P50CA092131），布罗德干细胞中心的创新奖，加州大学洛杉矶分校Eli和Edythe Broad再生医学和干细胞研究中心， 肿瘤细胞生物学培训计划（USHHS Ruth L. Kirschstein 机构国家研究服务奖 # T32 CA009056）、加州大学洛杉矶分校 AR071307 的皮肤病学 T32 计划以及加州大学洛杉矶分校肌肉细胞生物学、病理生理学和治疗学 T32 培训计划 5 T32 AF 65972。

所有描述的实验都得到了加州大学洛杉矶分校动物护理委员会的批准。
注意:选择与宿主小鼠匹配的癌细胞和成纤维细胞进行小鼠品系。选择与宿主小鼠性别相匹配的癌细胞和成纤维细胞。从繁殖群体获得小鼠或从信誉良好的供应商处购买。将肿瘤引入~8-10周龄的小鼠体内。有毛的小鼠将处于毛囊周期的休止期或休息阶段。计划将成纤维细胞与癌细胞的比例定为 0.5-3。
1.确定用于实验的适当数量的小鼠
<ol><li>在进行研究之前，执行功效分析以确定用于研究的适当数量的小鼠。使用以下公式确定具有指定功效的试验的样本数量: n = （Z（1-α/2）+Z（1-β）/ES）2 如果α是所选的显著性水平（通常为 0.05），则 1-α/2 = 0.975 且 Z=1.960。 β是功率，如果需要 80% 的功率，则 Z=0.84。 ES，效果大小 = \μ1-μ 0\/σ 其中μ 0 是假设 H0 下的平均值，μ1 是假设 H1 下的平均值，σ是感兴趣结果22 的标准差。 注意:有关计算样本数量的更多信息，请参见22。</li>
</ol>2.产生注射用癌细胞
<ol><li>使用以下公式确定相关增长率: Gr = （ln （N（t）/N（0）））/t 注意:显示了在 24 小时内翻倍的单元格的示例 倍增时间 = ln（2）/增长率 24 小时 = ln（2）/增长率 24*增长率 = .693 增长率 = .693/24= .028875</li>
	<li>确定要铺板的癌细胞数量以及注射前细胞需要生长的时间。 注意:对于形成的每个肿瘤，注射0.25-1百万个癌细胞。</li>
	<li>根据公式计算生成足够数量的单元格所需的天数 N（t）=N（0）egr*t 其中 N（t） 是时间 t 的细胞数，N（0） 是时间 0 的细胞数，gr 是生长速率，t 是时间。 注意:这些计算的一个示例，用于生长 500，000 个细胞以在 12 个肿瘤中的每一个中生成10 个 6 个 细胞。根据这些计算，5天足以生长必要数量的细胞: N（t）=N（0）egr*t 12 x 106 = 500，000e（gr*t） 12 x 106=500，000e（.028875*t） 24=e（.028875*x） ln（24）=0.028875x 3.178=0.028875x X=110 小时 = 4.59 天 留出额外的时间让细胞附着在板上以及在收集过程中丢失的细胞。</li>
	<li>平板癌细胞。 注意:使用培养细胞时请戴上手套和实验室外套。 注意:在层流罩中进行细胞操作，以尽量减少微生物从空气中引入样品。使用前用紫外线处理组织培养罩。使用无菌技术，仅使用无菌细胞培养塑料器皿和耗材，例如组织培养处理的板、移液管和锥形管。<ol><li>根据小瓶中的细胞数量和接种后的所需细胞浓度计算组织培养板的正确数量和大小。</li>
		<li>标记组织培养板。</li>
		<li>用37°C的水准备水浴。</li>
		<li>将培养基分装到锥形管中，置于37°C的水浴中。 许多癌细胞可以在Dulbecco的改良鹰培养基（DMEM）中生长，并补充有10%胎牛血清（FBS）。</li>
		<li>从液氮冰箱中取出所需数量的冷冻细胞。 注意:在液氮冰箱中处理细胞时，请使用冷冻手套。</li>
		<li>在水浴中快速解冻细胞瓶，同时轻轻摇动。 注意:在处理小瓶时将乙醇添加到手套中以保持无菌。</li>
		<li>在组织培养罩中使用无菌技术，将细胞移液到含有 10 mL DMEM + 10% FBS 的 15 mL 锥形管中。</li>
		<li>将细胞混合物以180× g 离心5分钟。</li>
		<li>用无菌抽吸或无菌 10 mL 移液管轻轻取出上清液。</li>
		<li>轻轻地将沉淀中的细胞重悬到 1 mL DMEM + 10% FBS 中。</li>
		<li>将重悬的细胞移液到带有无菌p1000的标记组织培养板上。</li>
		<li>使用无菌 10 mL 移液管，将含有血清的培养基移液到组织培养板中。</li>
		<li>在37°C的组织培养箱中用5%CO2孵育组织培养板。</li>
	</ol></li>
	<li>扩增癌细胞以产生足够的癌细胞进行注射。 注意:用光学显微镜监测细胞的汇合度。每两到三天或根据需要胰蛋白酶消化一次，以防止细胞达到100%汇合。如果需要大量细胞，请使用超烧瓶（材料表）以空间和中等效率的方式生长大量细胞。每次需要传代细胞时，请按照以下步骤操作:<ol><li>用无菌抽吸或无菌 10 mL 移液管从板中取出培养基。</li>
		<li>通过移液温热的磷酸盐缓冲盐水（PBS）轻轻清洗板。然后用无菌抽吸或移液管取出PBS。</li>
		<li>将5mL的1x胰蛋白酶 - 乙二胺四乙酸（EDTA）移液于PBS中，用于10cm板到细胞上，并在37°C下孵育5分钟。</li>
		<li>用轻拍从平板上取出细胞，将细胞从平板上移开。</li>
		<li>使用无菌 10 mL 移液管，将细胞收集到 DMEM 中，在锥形管中含有 10% FBS 血清。</li>
		<li>以180 x g 离心锥形管5分钟。</li>
		<li>倒出上清液以除去上清液。细胞沉淀应该是可见的。观察它以确保它留在管中。</li>
		<li>通过加入 1 mL DMEM + 10% FBS 并轻轻上下移液来重悬沉淀细胞。</li>
		<li>将重悬细胞等分到具有DMEM + 10%FBS的适当尺寸的新组织培养板上（10 mL培养基适用于10 cm组织培养板）。</li>
	</ol></li>
</ol>3.产生注射用成纤维细胞
注意:原代成纤维细胞在过多的倍增/传代后会衰老。重要的是在有限的传代次数或倍数后使用原代成纤维细胞。跟踪成纤维细胞从小鼠或人类皮肤生长的传代次数或倍数。使用少于 15 代的成纤维细胞作为原代人真皮成纤维细胞。使用少于9代的成纤维细胞用于小鼠胚胎成纤维细胞。成纤维细胞在汇合时会发生变化。当成纤维细胞大约 90% 汇合时，将它们胰蛋白酶消化。成纤维细胞将具有不同的特性，具体取决于它们的培养方式。许多科学家提倡在比组织培养板（例如更有效地捕获组织样环境的3D胶原基质）更具生理相关性的基质上进行培养23，24，25。
<ol><li>使用第2.1节中的公式来确定成纤维细胞的生长速率。</li>
	<li>使用第2.2节中的公式来确定扩增成纤维细胞所需的天数。</li>
	<li>在适当的日期使用适当的培养基按第2.3节所述将成纤维细胞铺板，以确保癌细胞和成纤维细胞在同一天准备好注射。</li>
	<li>按照第2.4节中描述的程序，在适当的培养基中扩增成纤维细胞，以产生足够数量的成纤维细胞。</li>
</ol>4.剃掉小鼠以准备注射小鼠
注意:与小鼠一起工作时，请穿上实验室外套、发网、鞋套和手套。
<ol><li>注射前一至两天，根据机构动物护理委员会的规则，麻醉小鼠。如果使用异氟醚进行麻醉，请使用异氟醚和O2的混合物。将小鼠置于麻醉室中，并引入异氟烷（5%）/ O2 混合物以诱导麻醉。然后，将鼻锥放在麻醉动物上，以用恒定流量的异氟醚（2%）/ O2 混合物维持麻醉的手术平面。</li>
	<li>通过捏住小鼠的脚趾并确认小鼠没有移动来检查动物以确保它们处于适当的麻醉手术平面。</li>
	<li>使用带有手术刀片#40的动物剪刀，通过将动物剪刀多次穿过皮肤直到去除皮毛，从小鼠侧面的适当位置轻轻去除皮毛。</li>
	<li>监测小鼠，直到它们从麻醉中恢复。</li>
</ol>5.准备注射用癌细胞和成纤维细胞
注意:癌细胞和成纤维细胞应在收集后尽快注射，最好在30分钟内注射。在注射的早晨，从组织培养板中分别收获癌细胞和成纤维细胞。对于每种细胞类型，请执行以下步骤:
<ol><li>用温和的无菌抽吸或移走培养基从组织培养板中取出培养基。</li>
	<li>轻轻移取 5 mL PBS 而不破坏细胞。旋转 PBS。用吸力轻轻吸出PBS，或移出PBS。</li>
	<li>将PBS中的5mL胰蛋白酶-EDTA轻轻移液到细胞层上，并在37°C下孵育5分钟。</li>
	<li>用轻轻敲击从平板上取出细胞以去除细胞。用 10 mL 移液管多次移液细胞，并将细胞转移到含有含有 10% FBS 的 DMEM 的锥形管中。</li>
	<li>将锥形管以180 x g 离心5分钟。</li>
	<li>轻轻倒出上清液，保留细胞沉淀，以除去上清液。用PBS洗涤细胞沉淀两次。每次将 10 mL PBS 移液到细胞上。用 p1000 移液管轻轻混合。</li>
	<li>按照步骤5.5离心细胞。用移液管轻轻取出PBS并重复。</li>
	<li>用 p1000 将洗涤的沉淀细胞重悬于 1 mL PBS 中。</li>
	<li>用酒精清洁血细胞计数器载玻片并干燥。</li>
	<li>将 100 μL 细胞混合物移液到试管中，加入 400 μL 0.4% 台盼蓝，最终台盼蓝浓度为 0.32%。</li>
	<li>通过移液细胞混合物直到腔室充满，轻轻填充盖玻片下方玻璃血细胞计数器的腔室。</li>
	<li>使用显微镜，聚焦10倍物镜的网格线。</li>
	<li>将活的，未染色的细胞和蓝色细胞计数在一组16个正方形中。</li>
	<li>数所有 4 组 16 个正方形。</li>
	<li>平均 4 组 16 个正方形中透明和蓝色单元格的单元格计数，然后乘以 10，000。乘以 5 以校正台盼蓝的 1:5 稀释度。</li>
	<li>最终计数是活细胞和死细胞的细胞/mL浓度。乘以体积以确定癌细胞和成纤维细胞的总数。确认活细胞的比例为 >80%。</li>
	<li>确认有足够数量的黑色素瘤细胞和成纤维细胞用于所有计划的注射，假设在注射过程中样品损失至少20%。</li>
	<li>将所需数量的细胞转移到新的锥形管中，并通过离心（180 x g 5分钟）沉淀细胞。</li>
	<li>用 10 mL 移液管取出上清液。</li>
	<li>将沉淀重悬于所需浓度的适量美国药典（USP）级PBS（皮内注射每个肿瘤50μL，皮下注射每个肿瘤100μL）。</li>
</ol>6.将癌细胞和成纤维细胞注射到小鼠体内
注意:如果得到机构动物护理委员会的批准，将两个肿瘤注射到每只小鼠中，每侧一个。随机化哪只小鼠将在左右两侧接受哪种注射。根据要注射的小鼠数量，麻醉小鼠并将癌细胞和成纤维细胞分批注射到小鼠体内。
<ol><li>用于癌细胞的皮下注射<ol><li>如第4.1节所述麻醉小鼠。</li>
		<li>用酒精棉签擦拭剃光的皮肤。</li>
		<li>用所需体积的细胞或细胞混合物填充无菌注射器，并用27号针头盖住注射器。 注意:确保不存在气泡。根据需要轻弹注射器以去除气泡。将细胞引入注射器后，通过连续倒置注射器以防止结块，使注射器内的细胞充分混合。</li>
		<li>将针头插入小鼠侧腹的皮下区域，然后将细胞轻轻注射到小鼠中。皮下注射时，每个肿瘤使用100μL细胞悬液（不建议每次注射使用超过100μL的体积）。</li>
		<li>监测小鼠，直到它们从麻醉中恢复。如果需要，为小鼠提供额外的绝缘材料，如小屋和/或加热垫，以帮助小鼠完全康复。</li>
	</ol></li>
	<li>用于癌细胞的皮内注射<ol><li>如第4.1节所述麻醉小鼠。</li>
		<li>用酒精棉签擦拭剃光的皮肤。</li>
		<li>用所需体积的细胞或细胞混合物填充无菌注射器，并用27号针头盖住注射器。 注意:确保不存在气泡。根据需要轻弹注射器以去除气泡。将细胞引入注射器后，通过连续倒置注射器以防止结块，使注射器内的细胞充分混合。</li>
		<li>将27号针插入小鼠侧面皮肤的皮内区域，然后将细胞轻轻注射到小鼠的皮内区域。每个肿瘤使用50μL细胞悬液进行皮内注射（不建议每次注射使用超过50μL的体积）。</li>
		<li>监测小鼠，直到它们从麻醉中恢复。如果有迹象表明小鼠处于困境，请咨询兽医。如果需要，为小鼠提供额外的绝缘材料，如小屋和/或加热垫，以帮助小鼠完全康复。</li>
	</ol></li>
</ol>7.监测小鼠肿瘤生长过程中
<ol><li>每天监测小鼠是否有任何疼痛或痛苦的迹象（驼背姿势、扭动、发声、沿笼子伸展、将腹部压在笼底或不愿在笼子周围移动）或脓肿形成。如果有迹象表明小鼠处于困境，请咨询兽医。</li>
	<li>一旦肿瘤形成，大约每两到三天测量一次肿瘤体积。用卡尺测量肿瘤长度（最长边）和宽度（垂直于长度）。 注意:最佳做法是让同一个人在整个实验中执行这些测量，以减少数据的变异性。对于测量，如第4.1节所述麻醉小鼠</li>
	<li>用公式计算肿瘤体积 体积= 0.5 x（长 x 宽2）。</li>
</ol>8.收获肿瘤并测量肿瘤重量
<ol><li>当肿瘤生长达到动物护理委员会建立的实验终点时，对小鼠实施安乐死。将小鼠置于腔室中，并在停止呼吸后用压缩CO2 缓慢（每分钟20-30%）置换腔室空气一分钟，对它们实施安乐死。</li>
	<li>对小鼠进行颈椎脱位。将啮齿动物约束在坚固、平坦的表面上。将坚固的钢笔或其他物体放在颅底的颈部后部。快速向前和向下推动，物体约束头部，同时用握住尾巴根部的手向后拉。触诊确认脊髓被切断。 注意:颈椎脱位应由经验丰富的实验室人员进行。</li>
	<li>对于实验中的每只小鼠，通过检查没有呼吸，没有心跳和对脚趾捏的反应来确认小鼠不再活着。</li>
	<li>使用无菌手术刀和手术剪刀，从小鼠身上切除整个肿瘤。用手术剪刀从肿瘤中修剪非肿瘤组织。</li>
	<li>在分析天平上称量肿瘤并记录肿瘤重量。</li>
</ol>9.肿瘤体积和肿瘤权重的统计分析
<ol><li>通过重复测量方差分析（ANOVA）比较每组肿瘤体积随时间的变化。如果每只小鼠注射两个肿瘤，则进行配对分析。</li>
	<li>使用双尾检验比较方差分析组之间的肿瘤权重。然后，Tukey的事后测试可用于确定哪些组彼此显着不同。如果每只小鼠注射两个肿瘤，则进行配对分析。</li>
</ol>

作者没有什么可透露的。

在图1的实验中，与引入人A2058黑色素瘤细胞相比，与引入黑色素瘤细胞而不共同注射成纤维细胞时，导致更大的肿瘤。这种差异可以很容易地根据肿瘤体积和肿瘤重量检测到。结果与癌症相关成纤维细胞可以促进肿瘤生长的多个报道一致5，6，7，8。除了这里讨论的终点，如肿瘤体积和肿瘤重量，还可以监测其他终点。还可以进行其他分析。例如，发育中的肿瘤可以收集到福尔马林中进行固定，石蜡包埋并用苏木精和伊红进行分析以进行组织学分析。或者，可以将发育的肿瘤收集到最佳切割温度化合物（OCT）中，以便通过免疫荧光对特定蛋白质进行进一步分析。
这些实验的一个重要限制是，与癌细胞共同注射的成纤维细胞可能在几次细胞分裂后衰老或死亡。或者，共注射的成纤维细胞可能远离肿瘤，在这种情况下，它们可能不会影响肿瘤生长。如果成纤维细胞衰老，死亡或远离肿瘤，那么随着时间的推移，它们将变得不那么丰富，并且共同引入的成纤维细胞的作用对肿瘤生长将变得不那么重要。事实上，引入的癌细胞有望从宿主中招募更多的成纤维细胞。对于在引入的成纤维细胞中敲低或敲除特定基因的实验，随着时间的推移，表达编码蛋白的宿主成纤维细胞的募集有望减少可能观察到的任何表型。为了监测引入的成纤维细胞的存在，可以对成纤维细胞进行基因工程改造以表达荧光蛋白，例如GFP26，27。如果成纤维细胞表达GFP，则可以使用流式细胞术来监测肿瘤内在引入后不同日期存在的成纤维细胞的数量。作为替代方案，荧光素酶可以引入成纤维细胞中，生物发光成像可用于随着时间的推移监测小鼠中成纤维细胞的数量。如果成纤维细胞从肿瘤微环境中迅速消除，Cre-lox基因工程小鼠模型将解决这个问题并补充所描述的研究。
如果通过将人类癌细胞和成纤维细胞引入裸鼠中进行实验，那么有限的免疫系统预计将对结果产生重大影响。无胸腺裸鼠（NU / J）缺乏T细胞，因此缺乏细胞介导的免疫力。它们在B细胞发育中也有部分缺陷。细胞介导的免疫，即通过活化的CD8 + T细胞杀死细胞，可以在抑制肿瘤生长中发挥重要作用28，29。在最近的研究中，癌症相关成纤维细胞和CD8 + T细胞之间的相互作用已被报道30，31，32。将小鼠癌细胞和小鼠成纤维细胞引入具有免疫系统的小鼠可以被认为是解决此问题的替代方法。
另一种替代方法是使用基因工程小鼠模型，其中Cre-lox系统用于调节宿主成纤维细胞中特定蛋白质的水平或活性。其中一种方法是使用富含成纤维细胞的 Cre-lox 模型和驱动程序，例如 FSP1、Col1a1、Col1a2 或 PDGFRa 33，34，35。使用Cre / lox重组酶模型，不会担心注射的成纤维细胞会死亡或从肿瘤迁移。
协议中提供的几个步骤对其成功至关重要。成纤维细胞的生长是协议的重要组成部分，可能对结果有重大贡献。原代成纤维细胞容易衰老，必须仔细监测传代或倍增，以确保成纤维细胞处于指数生长期并且没有衰老。衰老的成纤维细胞有望分泌高水平的细胞因子，这些细胞因子可以对肿瘤生长产生显着影响36，37，38。因此，仔细记录传代数并且注射的成纤维细胞不会衰老非常重要，除非协议要求分析衰老的成纤维细胞。
当成纤维细胞汇合时，可以预期它们会发生大量变化，其中一些变化可能会在它们重新进入细胞周期39，40，41，42，43，44时逆转。将成纤维细胞保持在增殖状态并在它们90%汇合时分裂它们对于确保研究尽可能一致非常重要。在组织培养板上培养成纤维细胞也可能影响其行为，应考虑3D胶原蛋白基质等替代品23，24，25。在3D基质中培养的成纤维细胞显示出与在2D表面上生长的成纤维细胞截然不同的迁移模式45，46，47，48，49，这可能会影响它们在引入肿瘤时的肿瘤促进能力，其中成纤维细胞将在建立肿瘤细胞外基质微环境中发挥积极作用50。
通过皮内注射将黑色素瘤细胞注射到小鼠中可以产生比皮下注射更可重复的肿瘤生长。这是一个棘手的步骤，因为皮内区域的空间很小。小心定位针头很重要。如果进行皮内注射，则仅注射50μL溶液。即使对于经验丰富的研究人员，这些注射也可能泄漏。重要的是要注意每次注射是否泄漏，以便如果没有肿瘤形成，则可以从最终分析中消除它。
准备注射器是另一个关键步骤。从注射器中去除所有气泡很重要，这可能需要轻弹注射器以消除气泡。同样重要的是，细胞不会结块。反复倒置注射器以防止结块。
这些实验的另一个棘手方面是测量肿瘤体积可能因人而异。为了限制肿瘤体积测量的可变性，分配单个实验室成员负责在整个实验过程中的所有小鼠和所有时间点进行所有肿瘤体积测量是有帮助的。
最好尽可能向小鼠注射与宿主小鼠相同性别的癌细胞。在方差分析模型中，可以使用两种性别并将性别作为变量，以确定性别是否随着时间的推移影响肿瘤大小。
了解肿瘤微环境对于深入了解肿瘤发生至关重要。该协议可以为理解成纤维细胞，不同类型的成纤维细胞以及成纤维细胞内可能影响肿瘤生长的不同途径的作用提供基础。这些实验的结果可能会确定新的治疗靶点，以防止成纤维细胞促进肿瘤生长18。

在肿瘤微环境中，最突出的细胞类型之一是癌症相关成纤维细胞（CAF）1。这些癌相关成纤维细胞可发挥肿瘤抑制作用2，3。例如，表达S100A的成纤维细胞分泌胶原蛋白，可以包封致癌物并防止癌的形成4。此外，胰腺癌中α平滑肌肌动蛋白（SMA）阳性肌成纤维细胞的耗竭会导致免疫抑制并加速胰腺癌的进展2。CAF还可以与癌细胞共同进化并促进肿瘤进展5，6，7，8。成纤维细胞可以合成和分泌细胞外基质蛋白，从而产生促进肿瘤的环境8。这些细胞外基质蛋白可引起组织的机械僵硬，这与肿瘤进展有关9，10。沉积的细胞外基质可以作为抑制免疫浸润的物理屏障11。CAF的基质沉积也与肿瘤浸润有关，因为CAF产生的纤连蛋白已被证明可以促进肿瘤浸润12。CAF通过分泌转化生长因子-β（TGF-β），血管内皮生长因子（VEGF），白细胞介素-6（IL-6）和CXC-趋化因子配体12（CXCL12）13，14，15，促进血管生成并将免疫抑制细胞募集到肿瘤微环境中。由于其在促进肿瘤生长中的核心作用，癌症相关成纤维细胞是抗癌治疗的新兴靶标6，16，17，18。
下面的协议描述了一种在成熟且广泛使用的肿瘤生长小鼠模型中测试成纤维细胞如何影响肿瘤生长的方法。为了了解成纤维细胞在肿瘤微环境中的重要性，修改了将癌细胞引入小鼠以监测其生长的标准方案，以包括癌细胞引入的成纤维细胞。癌细胞可以皮下或皮内引入。皮内引入会导致由皮肤本身引起的肿瘤。将癌细胞和成纤维细胞共同注射到小鼠体内的异种移植物代表了剖析成纤维细胞，成纤维细胞亚群和蛋白质因子在促进癌症生长能力中的作用的重要方法工具19，20，21。提供了将癌细胞和成纤维细胞共同注射到小鼠中的详细方案。该方法可用于比较成纤维细胞的存在与否，比较来自不同来源的成纤维细胞20，或比较具有和没有特定蛋白质表达的成纤维细胞19。引入癌细胞和成纤维细胞后，可以随着时间的推移监测肿瘤大小。在实验结束时，可以解剖和称重肿瘤。通过监测肿瘤随时间的生长，可以剖析不同因素的重要性。
有可能的替代方法来研究成纤维细胞在肿瘤生长中的作用。例如，有基于Cre-loxed的模型，这些模型提供了组织特异性基因敲除，其驱动因素优先在成纤维细胞中表达。这些方法也为研究成纤维细胞中特定基因和途径对肿瘤进展的作用提供了机会。与基于Cre-lox的方法相比，所提供的方案将代表一种明显更快速的方法来监测成纤维细胞的作用，因为肿瘤生长将在短短几周内被监测。所提供的方法也便宜得多，因为它不需要产生和容纳基因工程小鼠的菌落。所提供的协议可用于使用shRNA快速测试不同基因的敲低效果，而无需开发小鼠集落。所提供的方法也更加灵活，因为它允许比较不同数量的成纤维细胞，不同比例的癌细胞和成纤维细胞，敲低不同的基因，甚至比较来自不同组织部位或物种的成纤维细胞。Cre-lox方法的优点是成纤维细胞在更生理的背景下存在于小鼠体内。
这里报道的方案对于寻求快速且经济高效地监测成纤维细胞对肿瘤生长影响的科学家来说很有价值。该协议对于研究来自不同来源的成纤维细胞或成纤维细胞的不同亚群对肿瘤生长的肿瘤生长特别有价值。如果肿瘤起始发生在生理背景下很重要，那么应考虑基因工程小鼠模型。
有几种可能的方法可以执行这些实验。免疫能力小鼠可用作宿主，这将允许研究成纤维细胞 - 免疫细胞相互作用。对于免疫能力小鼠模型，必须注射小鼠癌细胞和小鼠胚胎成纤维细胞（MEF）。MEF的使用还允许研究人员利用广泛的敲除小鼠品系来测试目的基因的存在与否。或者，免疫缺陷小鼠可用于测试人成纤维细胞在促进源自人类癌细胞的小鼠肿瘤生长中的作用。癌细胞的引入可以皮下或正位进行。对于黑色素瘤，如下所述，可以皮内注射肿瘤-成纤维细胞混合物以进行原位注射，从而更密切地模拟皮肤内黑色素瘤发展的位置。

<table><tbody><tr><td>26G Needles</td><td>Fisher Scientific</td><td>14-826-10</td><td></td></tr><tr><td>Alcohol swabs</td><td>Fisher Scientific</td><td>326895</td><td></td></tr><tr><td>Animal clipper miniARCO with surgical blade #40</td><td>WAHL Professional</td><td>8787-450A</td><td></td></tr><tr><td>Athymic nude mice (NU/J)</td><td>The Jackson labs</td><td>002019</td><td>These mice are immunocompromised and can be used for experiments in which human cells are introduced. Immunocompetent mice can also be used if mouse cancer cells and fibroblasts will be introduced.</td></tr><tr><td>Cancer cells</td><td>ATCC</td><td>ATCC&amp;reg; CRL-11147&amp;trade;</td><td>This is the catalog number for a primary human melanoma cell line. Other cancer cell types can also be used.</td></tr><tr><td>Cell Culture Multi Flasks</td><td>Fisher Scientific</td><td>14-826-95</td><td></td></tr><tr><td>Centrifuge for conical tubes capable of reaching 180 x g</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Countess Cell Counting Chamber</td><td>Fisher Scientific</td><td>C10228</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Fisher Scientific</td><td>11965-118</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Fisher Scientific</td><td>MT35010CV</td><td></td></tr><tr><td>Fibroblasts&amp;nbsp;</td><td>ATCC</td><td>PCS-201-012</td><td>We isolate fibroblasts from skin in our lab. This is a catalog number for an adult primary human dermal fibroblast cell line. MEFs and fibroblasts derived from other sites can also be used.</td></tr><tr><td>Isoflurane</td><td>Henry Schein Animal Health</td><td>NDC 11695-6776-2</td><td></td></tr><tr><td>PBS USP grade for injection into mice</td><td>Fisher Scientific</td><td>50-751-7476</td><td></td></tr><tr><td>Sterile 10 ml serological pipet</td><td>Celltreat</td><td>667210B</td><td></td></tr><tr><td>Sterile 5 ml serological pipet</td><td>Celltreat</td><td>229005B</td><td></td></tr><tr><td>Sterile 50 ml centrifuge tubes</td><td>Genesee Scientific</td><td>28-108</td><td></td></tr><tr><td>Sterile Syringe Filters pore size 0.2 microns</td><td>Fisher Scientific</td><td>09-740-61A</td><td></td></tr><tr><td>Sterile tissue culture-grade Trypsin-EDTA</td><td>Fisher Scientific</td><td>15400054</td><td></td></tr><tr><td>Sterile tissue-culture grade PBS</td><td>Fisher Scientific</td><td>50-751-7476</td><td></td></tr><tr><td>Sterle 25 ml serological pipet</td><td>Celltreat</td><td>667225B</td><td></td></tr><tr><td>TC treated 100 x 20 mm dishes</td><td>Genesee Scientific</td><td>25-202</td><td></td></tr><tr><td>TC treated 150 x 20 mm dishes</td><td>Genesee Scientific</td><td>25-203</td><td></td></tr><tr><td>TC treated 60 x 15 mm dishes</td><td>Genesee Scientific</td><td>25-260</td><td></td></tr><tr><td>Trypan blue</td><td>Fisher Scientific</td><td>C10228</td><td></td></tr></tbody></table>

a mouse model to investigate the role of cancer-associated fibroblasts in tumor growth

癌症相关成纤维细胞（CAF）可以通过创造促进肿瘤的微环境在肿瘤生长中发挥重要作用。研究CAF在肿瘤微环境中作用的模型有助于理解成纤维细胞，来自不同组织的成纤维细胞以及成纤维细胞中特定遗传因子的功能重要性。小鼠模型对于了解体内肿瘤生长和进展的贡献因素至关重要。在这里，提供了一种方案，其中癌细胞与成纤维细胞混合并引入小鼠以发展肿瘤。确定肿瘤大小随时间的变化和最终的肿瘤权重并在组间进行比较。所描述的方案可以更深入地了解CAF在肿瘤生长和进展中的功能作用。

A2058人黑色素瘤细胞和原代人真皮成纤维细胞在无菌条件下培养。收集细胞并用PBS洗涤三次。免疫缺陷小鼠（NU / J - Foxn1裸体菌株）在一侧皮下注射，仅用25万个A2058黑色素瘤细胞。在另一侧，小鼠注射25万个A2058黑色素瘤细胞和75万个成纤维细胞的混合物。将细胞注射到12只免疫缺陷小鼠中。向左右侧注射是随机的。在注射后第12、14、16、19和21天监测肿瘤体积（图1A）。安乐死后，肿瘤被切除，成像（图1B）和称重（图1C）。成纤维细胞的存在导致肿瘤明显变大。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61883/61883fig01.jpg"> 图1:存在和不存在共注射成纤维细胞时黑色素瘤生长的比较。 将黑色素瘤细胞引入有或没有原代皮肤成纤维细胞的裸鼠中。（A）有和没有共同注射成纤维细胞的黑色素瘤随时间推移的肿瘤体积图。体积计算为0.5*长*宽2。绘制平均体积和均值标准误差 （SEM）。进行方差分析以确定显著性。（B）切除肿瘤的图像。（C）第21天实验结束时肿瘤的最终重量。绘制平均权重和 SEM。使用未配对的双尾t检验来比较最终权重。* 表示 p < 0.05，*** 表示 p < 0.001。 <a href="https://www.jove.com/files/ftp_upload/61883/61883fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth at JoVE.com

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

提供了共同注射癌细胞和成纤维细胞并随着时间的推移监测肿瘤生长的方案。该协议可用于了解成纤维细胞作为肿瘤生长调节因子的作用的分子基础。

研究癌症相关成纤维细胞在肿瘤生长中的作用的小鼠模型

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

a-mouse-model-to-investigate-role-cancer-associated-fibroblasts-tumor

Nanyang Technological University

Research

JoVE Journal

Cancer Research

4.3K 次观看.  University of California, Los Angeles. 提供了共同注射癌细胞和成纤维细胞并随着时间的推移监测肿瘤生长的方案。该协议可用于了解成纤维细胞作为肿瘤生长调节因子的作用的分子基础。

视频: A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

科研

癌症研究

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

发育生物学

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

医学

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

将原发性癌症相关纤维细胞与乳腺癌的合成素 Murine 模型分离，用于靶向纳米粒子的研究

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

评估从3D多细胞肺肿瘤球体中分离的癌症相关成纤维细胞的线粒体健康

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...