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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#174; CRL-11147&#8482;</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&#39;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&#160;</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（<strong class="xf...

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

Research

JoVE Journal

Cancer Research

4.2K 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

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

科研

癌症研究

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

一种确定环境富集对小鼠结肠肿瘤模型结肠菌群生物多样性影响的方法

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

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 ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

分离近端液研究胰腺腺癌肿瘤微环境

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

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 ...

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 ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...