作者感谢霍华德-克劳福德博士 (密歇根大学) 实验室的技术支持, 为这项研究提供材料和设备, 艾伦凯莱赫技术支持。
这项工作由密歇根大学 Rogel 癌症中心核心赠款 CA046592-26S3、nih K08 CA201335-01A1 (进工党)、BCAN YIA (进工党)、nih R01 CA17483601A1 (DMS) 资助。

1. 生长中的癌症球体
<ol><li>
从细胞线生长
	<ol>
<li>培养人膀胱癌细胞在常规黏附细胞培养情况下和维护在37°c 孵化器供应与 5% CO2。保持细胞在 < 90% 融合。 注: 所使用的培养基是 Dulbecco 的改良最小必需培养基 (DMEM), 含有4.5 克/升 d-葡萄糖, l-谷氨酰胺, 110 毫克/升丙酮钠, 并在本议定书中提供10% 胎牛血清 (血清)。</li>
		<li>在实验开始前的一天, 胰蛋白酶处理 (通过使用0.25% 胰蛋白酶-EDTA) 细胞, 量化细胞浓度, 并在3毫升培养基中分布 1 x 106细胞, 每一个井中都有一高的超低附着板。</li>
		<li>在低附着条件下孵化细胞在37°c ≥16 h。这应该允许足够的时间让细胞聚集成球体的大多数膀胱癌细胞株。 注: 球体的形成可以通过倒亮场显微镜观察。球体的最佳大小和数量可能取决于个别实验, 但我们发现, 直径从50µm 到150µm 的球体通常适用于使用20X 物镜的时间推移成像。</li>
		<li>要将其他细胞类型 (如成纤维细胞或免疫细胞) 纳入球体, 在超低附着板中混合所需的细胞和癌细胞, 并在37摄氏度内孵育≥16 h 以允许形成混合细胞类型球体。</li>
	</ol>
</li>
	<li>
从原发肿瘤生长 注: 肿瘤球体也可从原发膀胱肿瘤来源, 如由致癌性膀胱肿瘤模型开发的肿瘤8。例如, 用 n-丁基 n (4-丁基) 中亚硝胺 (BBN) 喂养的小鼠产生的膀胱肿瘤, 可以用无菌手术器械收割和碎成小块 (约0.5 毫米3)。本系统还可以研究消化性膀胱癌的主要标本。<ol>
<li>收集和洗涤0.5 毫米3肿瘤片在5毫升冰冷磷酸盐缓冲生理盐水 (PBS)。离心在 200 x g 5 分钟在4摄氏度。重复这个洗涤步骤一次。</li>
		<li>以 200 x g为10分钟, 以4摄氏度的离心法收集肿瘤片。取出上清液, 加入5毫升的 DMEM, 含10% 的血清。将肿瘤球体/介质混合物转移到6井超低附着板上。在嵌入胶原基质之前, 在37摄氏度的≥16中孵化 (见步骤 2.3)。</li>
	</ol>
</li>
</ol>2. 准备3维文化室
<ol><li>
稀释 type-1 胶原蛋白 (来源于鼠尾) 在 DMEM 含有10% 的血清, 使2毫克/毫升混合物根据制造商的指示。
	<ol>
<li>简而言之, 根据制造商的指示, 以达到生理 pH 值, 将胶原蛋白与适量的 1 N 氢氧化钠溶液和 DMEM 混合在一起。</li>
		<li>在混合了胶原和 DMEM 后, 快速地涂上腔板的水井 (对于大多数应用来说, 在凝固前用胶原-DMEM 混合物 (200 µL/井), 带1.5 盖玻璃的会议厅幻灯片是最佳的)。允许涂布室滑动在室温下固定15分钟。 注: 根据实验的目的, 通过添加其他细胞外基质成分, 如纤维粘连蛋白或层粘连蛋白, 可以对胶原基基质的组成进行修改。</li>
	</ol>
</li>
	<li>轻轻吸管500µL 的媒体包含球体 (或肿瘤片断, 如果主要肿瘤样本使用) 从6井超低附着板成空1.5 毫升离心管。等待2分钟让球体安定到管子的底部。</li>
	<li>小心地从试管中取出上清液。准备另一管 type-1 胶原蛋白 (2 毫克/毫升) 混合 DMEM 含有10% 的血清, 并迅速添加500µL 的这种混合物球体。用慢吹打轻轻地混合球体和胶原蛋白。</li>
	<li>添加250µL 的球体/胶原蛋白混合物的一个良好的胶原预涂膜室幻灯片。允许含有球体的胶原基质完全凝固 (约30分钟37摄氏度)。</li>
	<li>胶原基质固化后, 添加1毫升的 DMEM, 其中每一个井含10% 个血清 (图 1B)。孵化的房间幻灯片在37摄氏度孵化器提供 5% CO2 , 直到准备成像。</li>
</ol>3. 活细胞时间推移成像
<ol><li>根据制造商的指示打开共焦显微镜, 确保气候室达到37摄氏度, 并提供 5% CO2。 注: 我们的显微镜和腔室通常需要1小时才能达到系统平衡。</li>
	<li>小心地将会议厅滑动到附着在显微镜上的滑动适配器。</li>
	<li>使用低功耗目标 (如5X) 定位感兴趣的球体, 并以更高的功率目标开始成像 (在当前协议中, 20X 目标用于大多数活细胞成像, 以提高图像质量)。 注: 对于原发性膀胱肿瘤标本, 可能需要5X 和10X 的目标, 以覆盖较大的成像区域。对于我们的时间推移成像, 成像间隔设置为30分钟, Z 堆栈用于图像的整个球体。通常, z 切片之间的距离设置为20X 目标的4µm, 而 z 轴的总距离约为200µm. 图像可以使用 DIC 和荧光如果细胞/组织海港荧光蛋白标记。</li>
	<li>执行 24–72 h 的时间推移成像。 注意: 持续时间由细胞类型和实验需要决定。</li>
</ol>4. 用于冰冻组织切片的肿瘤球体样品块的制备
<ol><li>用小镊子小心地将胶原凝胶块和球体从腔内滑出。将胶原凝胶块置于塑料组织学模型中。</li>
	<li>简单地用 1x pbs 冲洗胶原凝胶, 然后在室温下用4% 多聚甲醛 (粉煤灰) 在 pbs 中固定30分钟。 注意: 粉煤灰是潜在的致癌物, 应该小心处理。</li>
	<li>用1.5 毫升的 1x pbs 在振动筛上洗15分钟的胶原凝胶. 更换 PBS 并重复上一个洗涤步骤3x。</li>
	<li>应用一个薄层 (约3毫米) 的最佳切削温度 (OCT) 化合物覆盖底部的一个新的塑料组织学模具。将固定的和水洗的胶原凝胶放在 oct 化合物的顶部, 然后用 oct 化合物填充霉菌, 并避免在 oct 中形成任何气泡, 仔细地嵌入整个凝胶。</li>
	<li>将模具保持在4摄氏度, 1 小时。</li>
	<li>将含有样品的霉菌和 OCT 化合物放在漂浮在液氮上的100毫米培养皿上。允许样品完全闪光冷冻。</li>
	<li>将冷冻样品块存放在-80 摄氏度, 供将来使用。</li>
</ol>5. 冰冻切片肿瘤球体免疫荧光成像
<ol><li>将冰冻样品块从-80 °c 冷藏库转移到恒温器的-20 °c 室。</li>
	<li>
通过将节间隔设置为7µm 来执行常规的冻结切片。让切片样品附着在没有皱纹或被困空气的玻璃滑梯上。
	<ol>
<li>将幻灯片存放在-80 摄氏度前, 再进行染色。 注意: 不同的间隔时间可以用来适应实验需要。</li>
	</ol>
</li>
	<li>空气干燥的幻灯片在室温下1小时。</li>
	<li>Permeabilize 样品与 PBS 包含0.5% 海卫 X-100 15 分钟。</li>
	<li>洗涤样品3x 与 PBS 为10分钟每洗涤。</li>
	<li>用疏水性屏障笔将样品用疏水屏障包围在滑梯上。</li>
	<li>用阻断溶液 (1x PBS 含5% 牛血清白蛋白) 治疗样品, 室温1小时。</li>
	<li>应用40µL 的原发性抗体溶液 (1x PBS, 含 5% BSA, 1:100 稀释原抗体), 每张幻灯片上的样品。</li>
	<li>以37°c 为1小时, 或在室温下, 在一夜之间孵化出初级抗体的幻灯片 (潜伏期和条件因原抗体而异)。</li>
	<li>用 PBS 清洗样品 3x (每次清洗) 15 分钟。</li>
	<li>应用40µL 的二次抗体含液 (1x PBS 含有5% 的 BSA 1:300 次抗体稀释) 的每一个样本在幻灯片上。</li>
	<li>用 PBS 清洗样品 3x (每次清洗) 15 分钟。</li>
	<li>将样品与赫斯特33342溶液 (1 µg/毫升, 在 PBS 稀释) 在室温下染色10分钟。然后用 PBS 冲洗样品5分钟。</li>
	<li>装上安装介质的样品并盖上适当大小的盖板。在室温下将幻灯片保持在24小时的黑暗中, 然后进行共焦显微术。</li>
</ol>

<ol>
	<li>American Cancer Society. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Society. , (2018).</li><li>Knowles, M. A., Hurst, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+bladder+cancer:+new+insights+into+pathogenesis+and+clinical+diversity.">Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity.</a> Nature Reviews Cancer. 15 (1), 25-41 (2015).</li><li>DeGeorge, K. C., Holt, H. R., Hodges, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+Cancer:+Diagnosis+and+Treatment.+American+Family+Physician.">Bladder Cancer: Diagnosis and Treatment. American Family Physician.</a> American Family Physician. 96 (8), 507-514 (2017).</li><li>Repesh, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro.+assay+for+quantitating+tumor+cell+invasion.">A new in vitro. assay for quantitating tumor cell invasion.</a> Invasion Metastasis. 9 (3), 192-208 (1989).</li><li>Doillon, C. J., Gagnon, E., Paradis, R., Koutsilieris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+system+as+a+model+for+studying+cancer+cell+invasion+capacity+and+anticancer+drug+sensitivity.">Three-dimensional culture system as a model for studying cancer cell invasion capacity and anticancer drug sensitivity.</a> Anticancer Research. 24 (4), 2169-2177 (2004).</li><li>Duong, H. S., Le, A. D., Zhang, Q., Messadi, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3-dimensional+culture+system+as+an+in+vitro.+model+for+studying+oral+cancer+cell+invasion.">A novel 3-dimensional culture system as an in vitro. model for studying oral cancer cell invasion.</a> International Journal of Expermintal Pathology. 86 (6), 365-374 (2005).</li><li>Rebelo, S. P., 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=3D-3-culture:+A+tool+to+unveil+macrophage+plasticity+in+the+tumour+microenvironment.">3D-3-culture: A tool to unveil macrophage plasticity in the tumour microenvironment.</a> Biomaterials. , 185-197 (2018).</li><li>Vasconcelos-Nobrega, C., Colaco, A., Lopes, C., Oliveira, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+BBN+as+an+urothelial+carcinogen.">Review: BBN as an urothelial carcinogen.</a> In Vivo. 26 (4), 727-739 (2012).</li><li>Palmbos, P. L., 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=ATDC/TRIM29+Drives+Invasive+Bladder+Cancer+Formation+through+miRNA-Mediated+and+Epigenetic+Mechanisms.">ATDC/TRIM29 Drives Invasive Bladder Cancer Formation through miRNA-Mediated and Epigenetic Mechanisms.</a> Cancer Research. 75 (23), 5155-5166 (2015).</li><li>Wang, L., 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=ATDC+induces+an+invasive+switch+in+KRAS-induced+pancreatic+tumorigenesis.">ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis.</a> Genes &amp; Development. 29 (2), 171-183 (2015).</li><li>Fife, C. M., McCarroll, J. A., Kavallaris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movers+and+shakers:+cell+cytoskeleton+in+cancer+metastasis.">Movers and shakers: cell cytoskeleton in cancer metastasis.</a> British Journal of Pharmacology. 171 (24), 5507-5523 (2014).</li><li>Akhmanova, A., Steinmetz, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+microtubule+organization+and+dynamics:+two+ends+in+the+limelight.">Control of microtubule organization and dynamics: two ends in the limelight.</a> Nature Reviews Molecular Cell Biology. 16 (12), 711-726 (2015).</li><li>Cheung, K. J., Gabrielson, E., Werb, Z., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+invasion+in+breast+cancer+requires+a+conserved+basal+epithelial+program.">Collective invasion in breast cancer requires a conserved basal epithelial program.</a> Cell. 155 (7), 1639-1651 (2013).</li><li>Kidd, M. E., Shumaker, D. K., Ridge, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+vimentin+intermediate+filaments+in+the+progression+of+lung+cancer.">The role of vimentin intermediate filaments in the progression of lung cancer.</a> American Journal of Respiratory Cell and Molecular Biology. 50 (1), 1-6 (2014).</li><li>Lowery, J., Kuczmarski, E. R., Herrmann, H., Goldman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate+Filaments+Play+a+Pivotal+Role+in+Regulating+Cell+Architecture+and+Function.">Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function.</a> Journal of Biological Chemistry. 290 (28), 17145-17153 (2015).</li><li>Papafotiou, 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=KRT14+marks+a+subpopulation+of+bladder+basal+cells+with+pivotal+role+in+regeneration+and+tumorigenesis.">KRT14 marks a subpopulation of bladder basal cells with pivotal role in regeneration and tumorigenesis.</a> Nature Communications. 7, 11914 (2016).</li><li>Sun, W., Lim, C. T., Kurniawan, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+adaptability+of+cancer+cells+strongly+affects+anti-migratory+drug+efficacy.">Mechanistic adaptability of cancer cells strongly affects anti-migratory drug efficacy.</a> Journal of the Royal Society Interface. 11 (99), (2014).</li><li>Goddette, D. W., Frieden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+polymerization.+The+mechanism+of+action+of+cytochalasin+D.">Actin polymerization. The mechanism of action of cytochalasin D.</a> Journal of Biological Chemistry. 261 (34), 15974-15980 (1986).</li><li>Berrier, A. L., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-matrix+adhesion.">Cell-matrix adhesion.</a> Journal of Cellular Physiology. 213 (3), 565-573 (2007).</li><li>Lee, J. H., 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=Collagen+gel+three-dimensional+matrices+combined+with+adhesive+proteins+stimulate+neuronal+differentiation+of+mesenchymal+stem+cells.">Collagen gel three-dimensional matrices combined with adhesive proteins stimulate neuronal differentiation of mesenchymal stem cells.</a> Journal of the Royal Society Interface. 8 (60), 998-1010 (2011).</li><li>LeBleu, V. S., Macdonald, B., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+basement+membranes.">Structure and function of basement membranes.</a> Experimental Biology and Medicine. 232 (9), 1121-1129 (2007).</li><li>Rakha, E. 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=Invasion+in+breast+lesions:+the+role+of+the+epithelial-stroma+barrier.">Invasion in breast lesions: the role of the epithelial-stroma barrier.</a> Histopathology. , (2017).</li><li>Erler, J. T., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+context+regulation+of+metastasis.">Three-dimensional context regulation of metastasis.</a> Clinical &amp; Experimental Metastasis. 26 (1), 35-49 (2009).</li></ol>

作者声明没有竞争的财政利益。

在这里, 我们描述了一个3维的肿瘤球体模型, 允许实时观察膀胱癌侵袭, 这是至关重要的癌症进展和转移。该系统可以将各种基质和细胞成分结合在一起, 使研究者能够更好地复述发生膀胱癌侵袭的组织微环境。膀胱癌球体可以从各种来源, 如细胞系 (包括基因修饰细胞线, 用于检查影响侵袭过程的信号通路) 和小鼠原发性肿瘤 (这里描述)。它也有可能适应人类原发性肿瘤的分析。根据所使用的成像系统, 荧光可以实时监测或通过固定和免疫荧光染色在不同的时间点。该系统还可用于测试潜在的入侵抑制剂。这使得该系统成为评估膀胱癌生物学的多功能和强有力的工具。
具有温度控制、湿度控制和 CO2供应的气候室共焦显微系统是构建本协议所描述模型的关键设备。
据广泛报道, 3 维文化提供了专门的微环境, 更好地模仿本地组织的细胞生物学和癌症研究19,20。许多研究依靠渗透膜插入化验, 不反映在体内发生侵袭的条件。此外, 这些常规研究既不便于实时监测入侵过程, 也不允许在入侵期间或之后对样本进行详细分析。在此, 我们描述了一个系统, 这是一个有用的实时和端点审问的侵袭性癌症生物学。
胶原蛋白是额外细胞基质 (ECM) 的重要组成部分, 存在多种形式。Type-1 胶原是纤维胶原蛋白的主要形式, 而 Type-4 胶原蛋白是构成基底膜21的 nonfibrillar 胶原蛋白。癌细胞必须穿透基底膜, 并通过 type-1 胶原在非侵袭性肿瘤的进展22。由于其在 ECM 中无处不在, type-1 胶原已被用作构建3维文化的基质, 它比二维培养皿5、6、23更能模仿 ECM。虽然这里概述的系统是基于一个 type-1 胶原基质, 它可以被修改, 包括其他基质成分的基础上的实验问题和需要。
我们用来产生膀胱癌球体细胞系的方法涉及低附着条件下的细胞培养和细胞聚集。并非所有细胞都能耐受这些培养条件, 有些人可能会在胶原嵌入过程中发生凋亡或松散聚集的形成。在悬浮液中改变培养时间、细胞数或合并其他细胞类型可以改善结果。为这种化验选择的细胞系取决于实验的目的。我们成功地使用了这种技术与7/7 独特的人膀胱癌细胞系。然而, 可能有些细胞线可能不适合这个实验设置。此外, 一些细胞系具有很大的侵入性表型, 导致早期解除的球体, 癌细胞移出观察区的时间推移实验。为了解细胞系的一般行为, 我们强烈建议进行试验, 以确定研究的最佳时间框架。表 1列出了球体生成和成像常见问题的疑难解答。
本系统适用于限制癌细胞侵袭的药理学化合物的有限筛选。在此, 我们使用细胞松弛素 D, 一个细胞渗透性的肌动蛋白聚合抑制剂, 以说明这种潜在的应用18。如上图所示, 0.2 µM 的细胞松弛素有效地抑制了 UM-UC9 和 UM-UC18 球体的侵袭。利用多腔片和全自动显微分期控制, 多种化合物对肿瘤球体侵袭的影响是可行的。
虽然这些化验是最适合定性地描述侵入行为, 量化的迁移和入侵的程度也是可行的。在实验开始和不同时间点获取球体的图像, 以及随后的图像分析, 以测量 X、Y 或 Z 方向的入侵最远的线性距离, 可以提供入侵迁移行为的量化。更高级的图像分析使用荧光标记的细胞可以用来定义和定量个别细胞或细胞类型行为取决于实验需要或问题。

侵袭是癌症进展的一个关键步骤, 这是转移的需要, 并与较低的生存和预后较差的患者。在人类膀胱癌中, 泌尿道最常见的恶性肿瘤每年导致大约16.5万人死亡, 癌症分期、治疗和预后直接关系到入侵1的存在或缺失。大约75% 的膀胱癌病例为非肌肉侵入, 并以局部切除术进行管理。相反, 肌肉侵袭性膀胱癌 (约25% 的所有病例) 是积极的肿瘤, 转移率高, 并以积极的综合治疗2,3治疗。因此, 了解触发入侵的分子通路, 对于更好地表征侵入性进展的危险和发展能够预防侵入性进展的治疗干预是至关重要的。
肿瘤侵袭性进展发生在复杂的三维 (3) 环境中, 与其他肿瘤细胞、基质、基底膜和其他类型的细胞 (包括免疫细胞、成纤维细胞、肌肉细胞和血管) 相互作用。内皮细胞。渗透性支持 (例如Transwell) 检测系统通常用于定量癌细胞入侵4, 但这些系统是有限的, 因为它们不允许对入侵过程进行实时的显微监测和对样品进行进一步染色和分子分析的检索具有挑战性。研制一种3维膀胱肿瘤球体系统来研究入侵是可取的, 因为它允许在体外系统的方便的情况下纳入已定义的微环境成分。
在本议定书中, 我们描述了一个系统, 以检查人体膀胱癌细胞的侵袭过程, 使用3维球形入侵检测, 包括胶原基凝胶基质和共焦显微镜, 以允许调查人员监测细胞活力和实时入侵 (图 1A)。该系统是多才多艺的, 可以修改, 以询问各种基质/肿瘤的设置。它可以纳入大多数膀胱癌细胞系或原发性膀胱肿瘤和其他基质细胞, 如癌症相关的成纤维细胞和免疫单元5,6,7。该协议描述了由 type-1 胶原组成的基质, 但可以被修改以加入其他分子, 如纤维粘连蛋白, 层粘连蛋白, 或其他胶原蛋白。侵入过程可以遵循72小时或更长的时间取决于显微镜和系统使用的能力。3维基质中嵌入的肿瘤的固定和免疫荧光染色允许对侵入细胞中的蛋白质上调进行审问, 从而提供通常不存在或难以收集的关键信息。使用其他3维文化模型。该系统还可用于屏蔽入侵的化合物, 并描绘受此类化合物影响的信号通路。

<table border="0" cellpadding="0" cellspacing="0" style="width:1511px;" width="1511">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Human bladder cancer cell lines UM-UC9, UM-UC13, UM-UC14, UM-UC18, 253J</td>
			<td style="width:203px;"></td>
			<td style="width:169px;"></td>
			<td style="width:612px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">DMEM cell culture medium</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">11995065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-Antimycotic (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:169px;">A3803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS), pH 7.4&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar Ultral-low attachment 6-well cluster&#160;</td>
			<td>Corning</td>
			<td align="right" style="width:169px;">3471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional inverted microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;">491206-0001-000</td>
			<td>General use for cell culture and checking spheroids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen type 1 from rat tail, high concentration&#160;</td>
			<td>Corning</td>
			<td align="right" style="width:169px;">354249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab-Tek II Chambered Coverglass</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">155382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;">LSM800</td>
			<td>A confocal miscoscope with climate chamber, multi-location imaging, and Z-stack scanning function&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat micromtome</td>
			<td>Leica Biosystems</td>
			<td style="width:169px;">CM3050 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zen 2 Image processing software&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde solution</td>
			<td>Electron Microscopy Sciences</td>
			<td align="right" style="width:169px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImmEdge Hydrophobic Barrier PAP Pen</td>
			<td>Vector Laboratories&#160;</td>
			<td style="width:169px;">H4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">O.C.T compound&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">23730571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 solution&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">62249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-ATDC (Trim29) antibody</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:169px;">HPA020053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-Cytokeratin 14 antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:169px;">ab7800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-Vimentin antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:169px;">ab24525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond&#160;</td>
			<td style="width:203px;"></td>
			<td style="width:169px;"></td>
			<td>Mounting medium</td>
		</tr>
	</tbody>
</table>

3-d cell culture system for studying invasion and evaluating therapeutics in bladder cancer

膀胱癌是一个重要的健康问题。据估计, 今年美国将有超过1.6万人死于膀胱癌。虽然75% 的膀胱癌是无创性的, 而且不太可能转移, 但有25% 的进展是侵入型生长模式。多达半数的侵袭性癌症患者将会产生致命的转移性复发。因此, 了解膀胱癌侵袭性进展的机制对于预测患者预后和预防致死性转移是至关重要的。在本文中, 我们提出了一个三维的癌症入侵模型, 允许合并肿瘤细胞和基质成分, 模仿在体内的条件发生在膀胱肿瘤微环境。该模型提供了利用时间推移成像实时观察侵入过程的机会, 并利用共焦荧光成像和筛查化合物, 以阻断入侵的可能性, 对所涉及的分子通路进行审问。虽然本议定书的重点是膀胱癌, 但也有可能采用类似的方法来检查其他肿瘤类型的侵袭和运动。

成功地创建浸润性膀胱癌肿瘤球体需要形成适当大小的肿瘤球体从细胞系或原发肿瘤。图 2A显示适当大小的球体开发从四人膀胱癌细胞线 (UM-UC9, UM-UC13, UM-UC14, 253J 和 UM-UC18)。图 2B显示一个肿瘤球体的 BBN 产生的小鼠膀胱肿瘤嵌入胶原基质。这些球体被嵌入如上文所述, 有代表性的图像被捕获使用一个显微镜装备的活体细胞成像。20X 物镜用于成像 UM-UC9、UM-UC13、UM-UC14 和 UM-UC18 球体, 而小鼠膀胱肿瘤球体用5X 和10X 物镜进行检查。
人膀胱细胞系球体 24–72 h (图 2A) 和小鼠 BBN 原发性膀胱肿瘤球体 (图 2B) 的代表性图像显示膀胱癌移植到胶原基质中。253J, 非侵入性细胞系, 在很大程度上保持完好, 很少或根本没有迁移 (图 2a)。视频 1、 2和3分别演示了 UM-UC9、UM-UC13 和 UM-UC14 球体的时间推移入侵过程。视频 4显示了 UM-UC18 球体的入侵特性。视频 5显示了小鼠膀胱肿瘤的两个区域从66小时到 87 h 后胶原嵌入。
为了证明在侵袭性肿瘤球体中使用免疫荧光染色的可行性, 他们被固定和染色24或 72 h.图 3显示了共济失调-毛细血管扩张症组染色的代表性样本d-相关蛋白 (ATDC, 也称为 TRIM29), 在人膀胱和胰腺癌的发生和肿瘤进展中起重要作用的蛋白质9,10, tubulins (α和β), 形成微管和发挥关键作用, 塑造细胞和细胞运动11,12, 角蛋白 14 (KRT14), 一个基底上皮标记涉及入侵13和波形 (VIM), 一个间充质标记14,15, 16。这些图像表明, 可以使用这种方法进行细胞和亚胞结构的可视化。UM-UC14 的高放大度视图显示 ATDC 的丝状染色 (图 3A)。从 UM-UC18 球体传播的高度侵入细胞在24小时的入侵表达高水平的 VIM, 一个标志的上皮间骨髓转移 (急诊室,图 3B)。
将不同细胞类型 (癌症相关成纤维细胞) 纳入肿瘤球体是可行的, 并提供了一种检查异源细胞相互作用的方法 (图 4和视频 6)。在这个例子中, 我们使用了红色荧光蛋白 (RFP) 标记的人成纤维细胞和253J 膀胱癌细胞系转基因与一个载体表达绿色荧光蛋白 (GFP)。这些细胞混合形成肿瘤球体和嵌入胶原蛋白。通过共焦显微镜监测入侵过程。图 4显示了与成纤维细胞的相互作用可以调节 253 j 的侵袭行为。
图 5展示了检测系统对入侵抑制剂的检测效用。细胞松弛素 D 是一种已知的细胞迁移和侵袭抑制剂, 其作用是阻断肌动蛋白聚合17,18 , 并作为一个例子。细胞松弛素 D 治疗抑制 UM-UC9 球体的侵袭 (图 5A)。相同浓度的细胞松弛素 D (0.2 µM) 能够部分抑制 UM-UC18 球体的侵袭 (图 5B)。由于该系统可用于多井和肿瘤 organoids 的并行成像, 因此可以筛选出一组有限的药理制剂对侵袭性的影响。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58345/58345fig1.jpg"> 图 1: 建立3维入侵检测与活细胞成像.(A) 3 维活细胞成像和免疫荧光的示意图概述。(B) 本实验使用的胶原蛋白和培养基的盖玻片室。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/58345fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58345/58345fig2.jpg"> 图 2: 膀胱癌巨球形侵袭.(A) 植入胶原凝胶基质的人膀胱癌细胞系球体的例子。球体显示在嵌入时 (0 小时, 顶行) 或 72 h (UM-UC9, UM-UC13, UM-UC14, 253J) 或 24 h (UM-UC18)。刻度条 = 50 µm. (B) BBN 诱导的小鼠膀胱肿瘤侵入胶原基质的例子。箭指的是肿瘤球体的侵袭边缘。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/58345fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58345/58345fig3.jpg"> 图 3: 浸润性膀胱癌球体免疫荧光分析.(A) UM-UC14 72 小时后肿瘤球体侵入胶原蛋白。对 ATDC (绿色)、蛋白 (紫罗兰) 和细胞核 (赫斯特染色蓝) 进行常规免疫荧光检测, 对样品进行染色。白色正方形表示在底部行右两个面板中显示的放大视图较高的区域。缩放条 = 50 µm. (B) UM-UC18 肿瘤球体染色 KRT14 (绿色), VIM (红色), ATDC (紫罗兰) 和细胞核 (赫斯特染色蓝) 后24小时侵入胶原蛋白。缩放条 = 50 µm. 白色正方形表示在右下面板中显示的放大视图更高的区域。虚线粗略地表示原始肿瘤球体的边界。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/58345fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58345/58345fig4.jpg"> 图 4: 荧光标记的肿瘤球体合并成纤维细胞.对 GFP 标记的253J 膀胱癌细胞的侵袭 (下板) 或共培养与 RFP 标记成纤维细胞 (顶板) 监测24小时的比例酒吧 = 50 µm. 通过增加成纤维细胞到培养系统 (顶板), 侵入增强。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/58345fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58345/58345fig5.jpg"> 图 5: 小分子药物靶向性肌动蛋白聚合在侵袭中的作用.(A) 用细胞松弛素 d (0.2 µM) 或二甲基亚砜 (车辆控制) 治疗的 UM-UC9 肿瘤球体, 并监测 72 h (B) UM-UC18 肿瘤球体, 细胞松弛素 D 或亚砜治疗 24 h. 刻度条 = 50 µM. 虚线表示原始球体。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/58345fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>可能的问题</td>
			<td>建议的解决方案</td>
		</tr>
<tr>
<td>低数量的可行球体</td>
			<td>·增加悬浮培养细胞的数量 ·确保 > 90% 细胞在 typsinization 后可行 ·检查细胞是否有污染 ·在 typsinizing 前保持细胞的对数生长 ·优化细胞培养介质 ·改变悬浮条件下的培养时间 ·尝试其他单元格线</td>
		</tr>
<tr>
<td>球体太大或太小</td>
			<td>·调整添加到低附着板的单元格数 ·使用温和的吹打分解更大的细胞聚集物</td>
		</tr>
<tr>
<td>在想象中很难集中注意力</td>
			<td>·调整胶原凝胶的厚度。对于工作距离短的目标, 尽量使胶原凝胶变薄。 ·确保会议厅滑动或盖玻片 #1. 5 ·优化球体尺寸 (50–150µm 直径)</td>
		</tr>
<tr>
<td>入侵检测中细胞从成像区移出</td>
			<td>·减少球体的大小 ·如果需要, 每24小时重新居中成像区域 ·从事瓷砖成像技术 (如果适用) 覆盖较大的区域 ·考虑使用替代的细胞系</td>
		</tr>
<tr>
<td>会议厅滑动干燥</td>
			<td>·确保在气候室设置中没有渗漏 ·确保水分控制机制的功能</td>
		</tr>
</tbody></table>表 1: 对3维入侵模型进行故障排除。
<img alt="Movie 1" src="/files/ftp_upload/58345/58345movie1.jpg"> 视频 1: 显示从0到72小时胶原蛋白培养的 UM-UC9 球体的时间推移视频.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/Video_1_UC9-wt1_0-72hr.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Movie 2" src="/files/ftp_upload/58345/58345movie2.jpg"> 视频 2: 显示从0到72小时内胶原培养的 UM-UC13 球体集体迁移的时间推移视频.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/Video_2_UC13-wt1_0-72hr.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Movie 3" src="/files/ftp_upload/58345/58345movie3.jpg"> 视频 3: 从0到72小时的胶原蛋白培养的 UM-UC14 球体的时间推移视频.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/Video_3_UC14-wt1_0-72hr.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Movie 4" src="/files/ftp_upload/58345/58345movie4.jpg"> 视频 4: 从0到24小时的胶原蛋白培养的 UM-UC18 球体的时间推移视频.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/Video_4_UC18_0-24hr.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Movie 5" src="/files/ftp_upload/58345/58345movie5.jpg"> 视频 5: BBN 诱导的小鼠膀胱肿瘤的时间推移视频 66-84 h 后胶原嵌入.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/Video_5_mouse_tumor_66-84hrs_10X_composite.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Movie 6" src="/files/ftp_upload/58345/58345movie6.jpg"> 视频 6: 时间推移视频显示癌症球体形成从 GFP 标记的253J 细胞混合与 RFP 标签的人成纤维细胞 (左) 或 GFP 标记253J 细胞单独 (右)。球体植入胶原蛋白, 监测24小时.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58345/Video_6_253J-CAF_0-24hr.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>

Watch this Scientific Journal Video about 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer at JoVE.com

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

控制膀胱癌的过程代表了生物标志物和治疗发展的机会。在这里, 我们提出了一个膀胱癌入侵模型, 其中包括3维的肿瘤球体文化, 时间推移成像和共焦显微术。该技术可用于定义侵入过程的特征和筛选治疗药物。

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

3-d-cell-culture-system-for-studying-invasion-evaluating-therapeutics

Nanyang Technological University

Research

JoVE Journal

Cancer Research

8.8K 次观看.  University of Michigan Medical Center. 控制膀胱癌的过程代表了生物标志物和治疗发展的机会。在这里, 我们提出了一个膀胱癌入侵模型, 其中包括3维的肿瘤球体文化, 时间推移成像和共焦显微术。该技术可用于定义侵入过程的特征和筛选治疗药物。

视频: 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

在头颈部鳞状细胞癌的神经浸润模型

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

小鼠原位膀胱肿瘤模型和肿瘤检测系统

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living biobanks&#34; of patient derived cell lines. For many types of epithelial cells, various three dimensional (3D) culture approaches have been described that support an improved differentiated state. While CRCs retain their lineage commitment to the tissue from which they are isolated, they fail to express many of the differentiation markers associated with the tissue of origin when grown under normal two dimensional (2D) culture conditions. To enhance the application of patient-derived CRCs for prostate cancer research, a 3D culture format has been defined that enables a rapid (2 weeks total) luminal cell differentiation in both normal and tumor-derived prostate epithelial cells. Herein, a filter insert-based format is described for the culturing and differentiation of both normal and malignant prostate CRCs. A detailed description of the procedures required for cell collection and processing for immunohistochemical and immunofluorescent staining are provided. Collectively the 3D culture format described, combined with the primary CRC lines, provides an important medium- to high- throughput model system for biospecimen-based prostate research.

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

基于插入 - 滤池3D文化系统原发性前列腺细胞分化

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

利用有机体培养评价小鼠前列腺皮细胞的分化能力

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

小鼠膀胱肿瘤器官的培养、操作和正交移植

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...