作者对国家自然科学基金 (31371390)、国家高新技术发展项目 (2014AA020702) 和广东省科技计划 (2016B030231001) 的研究资助给予了认可。

所有动物工作都是按照《动物福利法》、《机构指南》和中山大学动物保育和使用委员会批准的。
1. 制备 TEM
<ol><li>用不育的手术剪刀和镊子来执行小鼠颈椎脱位并去除舌头。</li>
	<li>浸泡在75% 乙醇3分钟的舌头, 然后把每一个舌头到1.5 毫升离心 (EP) 管与1毫升的10毫米无菌磷酸盐缓冲溶液 (PBS)。 注意: PBS 在以下所有步骤中的浓度与此步骤中的浓度相同。</li>
	<li>冷冻解冻细胞裂解: 将 EP 管中的舌头冻成-80 摄氏度, 1 小时, 然后在室温下将舌头解冻45分钟3圈。</li>
	<li>用手术针将每条舌头装入手术缝合线上, 用一小块不育的锡纸包裹缝合的末端。在不育条件下, 在含有75% 乙醇的3.5 厘米或6厘米培养皿中进行操作。 注: 每个手术缝合的适当长度约为20厘米, 每块锡纸的适当尺寸约为0.3 厘米2 (1 厘米 x 0.3 厘米)。舌头应该装在锡纸附近。</li>
	<li>三十年代用3.5 厘米或6厘米培养皿冲洗每舌3毫升的无菌 PBS. 在无菌条件下执行此操作。</li>
	<li>用超纯水冲洗舌头: 将 ampicilin 放入一个宽口瓶中, 250 毫升的无菌超纯水, 最终浓度为90µg/毫升。把舌头放进装有搅拌棒的瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。 注: 可将多达5种舌头放入同一瓶中, 考虑缠绕缝线。锡纸在瓶子的缝合端, 以防止舌头滑脱。舌头应该被放置 2 cm 高从瓶子的底部, 调整长度的缝合留在瓶子内。此注释还用于步骤1.8、1.10、1.12 和1.16。</li>
	<li>把瓶子放在磁力搅拌器上12小时。</li>
	<li>用氯化钠冲洗舌头: 将 ampicilin 添加到一个宽口瓶中, 250 毫升无菌1米氯化钠, 最终浓度为90µg/毫升。把舌头和搅拌棒放进瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。</li>
	<li>把瓶子放在磁力搅拌器上24小时。</li>
	<li>细胞裂解的海卫 X-100: 添加 ampicilin 到最后浓度的90µg/毫升成一个宽口瓶与250毫升无菌2% 海卫 X-100 在 PBS。把舌头和搅拌棒放进瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。</li>
	<li>把瓶子放在磁力搅拌器上48小时。</li>
	<li>洗舌头与 CaCl2/氯化镁2: 添加 ampicilin 到一个宽口瓶与250毫升无菌5毫米 CaCl2/氯化镁2到最后浓度90µg/毫升。把舌头和搅拌棒放进瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。</li>
	<li>把瓶子放在磁力搅拌器上24小时。</li>
	<li>DNase 消化: 添加1毫升的汉克平衡盐溶液 (HBSS) 到每个 EP 管。将 DNase 分别添加到 HBSS 中, 最终浓度为300µM. 将每一舌移入每个 EP 管, 并将部分缝合到管外。在无菌条件下执行此操作。 注: 在本步骤中, 确保保留在瓶子内的缝合部分也留在 ep 管内, 并确保在前一步骤中保持在瓶外的缝合部分也保持在本步骤的 ep 管之外。</li>
	<li>在37摄氏度的 EP 管中孵育舌头24小时。</li>
	<li>用 pbs 冲洗舌头: 将 ampicilin 加入250毫升无菌 pbs 的宽口瓶中, 最终浓度为90µg/毫升。把舌头和搅拌棒放进瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。</li>
	<li>把瓶子放在磁力搅拌器上24小时。</li>
	<li>将所制备的 TEM 在无菌 PBS 中贮存4摄氏度, 直至使用。</li>
</ol>2. 三维 (3D) TSCC 的重建
<ol><li>
静态 TSCC 模型构造
	<ol>
<li>种子 1.0 x 106单 TSCC 细胞 (Cal27) 成3.5 厘米培养皿。添加3毫升的 Dulbecco 的改良鹰的 medium/F12 (DF12), 含有10% 胎牛血清 (血清), 90 µg/毫升 ampicilin, 90 µg/毫升卡那霉素。</li>
		<li>培养的 Cal27 细胞在37°c 2 至3天。确保细胞覆盖至少60% 区域的盘子底部。</li>
		<li>将透射电镜加载到培养皿中的 Cal27 单层上。</li>
		<li>在37摄氏度的28天内, 将盘子放入 CO2孵化器中。</li>
		<li>在细胞培养过程中每天刷新培养基。在孵化器中的 CO2浓度是5%。</li>
	</ol>
</li>
	<li>
搅拌 TSCC 模型施工
	<ol>
<li>
自制搅拌 minibioreactor 的研制
		<ol>
<li>从10毫升注射器中取出柱塞。</li>
			<li>在杆的下端附近挖一个孔 (直径1厘米), 并在孔中装入搅拌杆。</li>
			<li>在塑料宽口瓶的瓶盖中央挖一个孔 (直径0.5 厘米), 并将活塞杆穿过瓶盖。</li>
			<li>切割50毫升离心管的一半, 并将其焊接在瓶盖的外侧。</li>
			<li>通过将拉杆与与鱼钩相连的钓线包裹起来, 将鱼钩连接到拉杆上。 注: 最多可连接4鱼钩杆。</li>
			<li>在使用前先将自制的复合釜压蒸。 注: 请勿蒸压塑料宽口瓶。在培养细胞时使用新的无菌塑料宽口瓶。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
动态细胞培养
	<ol>
<li>种子 1.0 x 106单 Cal27 细胞在自制的 minibioreactor。添加150毫升的 DF12 培养基, 其中含有10% 的血清, 90 µg/毫升 ampicilin, 90 µg/毫升卡那霉素。</li>
		<li>使用连接到杆上的鱼钩将 TEM 加载到 minibioreactor 上。</li>
		<li>拧紧瓶盖, 将 minibioreactor 放在磁力搅拌器上。激活 minibioreactor 在 200 rpm 在 CO2孵化器在37°c 7 到14天。 注: co2孵化器中 co2的浓度为5%。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

一个完善的瓣膜 ecm 制造协议应保留本机 ecm 成分, 同时去除组织中的细胞成分几乎完全21。尽管目前报告的去细胞协议需要通过血管灌流来去除蜂窝材料通过对流运输, 这里采用了机械搅拌, 称为传统的简单和廉价的方法22,23,24,25,26. 此外, 由于舌头富含 lingualis, 且有少量体积庞大的血管, 本议定书比上文所述的其他协议更适合舌组织。
此外, 该 TEM 生产的协议进行适当的适度强度去细胞, 避免破坏或解散可能是由高强度去细胞, 如月桂酸钠的基膜 (SDS) 治疗3。此外, 该议定书还有效地在鼠和猪的舌头 (没有显示的数据), 表明该方法可以普遍使用的舌头从不同物种3。
值得注意的是, 该协议中有一些关键步骤或细节, 这可能直接影响到结果。一个重要的步骤是通过 DNase 消化舌细胞。如果消化时间不够, 或者 DNase 工作不有效, 舌头的去细胞就很难实现。另一件值得注意的是, 考虑到 TEM 的损坏, 生物反应器的转速不应该太快。此外, 这种操作的无菌环境在协议中非常重要。
尽管上面有严格的规定, 但在一定程度上可以调整 TEM 准备的协议。用超纯水或 PBS 清洗舌头比议定书建议的时间多几个小时, 不会明显影响 TEM 的制作。然而, 由于该议定书需要将近一周的时间来准备 tem, 它无法满足对 tem 的即时要求。
透射电镜在 TSCC 模型施工中具有重要的价值。与自制的 minibioreactor, 悬浮 Cal27 细胞可以附着在 TEM 中, 形成类似人类 TSCC 病理组织学3的相似浸润结构。这可能是一个理想的模型, 监测和调查 TSCC 的侵袭和转移的体外。该模型还有利于 TSCC 药物试验的工作。考虑到这一切, 这里提出的协议在 TSCC 研究中可能具有很大的潜力。

舌有多种重要功能, 如吞咽、发音和味觉。因此, 舌功能的损害对患者的生活质量有很大的影响1。口腔内最常见的恶性肿瘤是舌鳞状细胞癌 (TSCC), 通常发生在饮酒或吸烟2的人身上。
近年来, TSCC 的基础研究取得了很小的进展。缺乏有效的体外研究模式仍然是最大的问题之一。因此, 细胞外基质 (ECM) 结果是一个潜在的解决方案。由于 ecm 是由高度组织的矩阵元件组成的复杂网络框架, 因此具有 ecm 类结构和成分的脚手架材料将胜任癌症研究。瓣膜 ecm 能够完美地为细胞从同一来源的体外提供利基, 这是 ecm 最显著的优势。
ECM 可以通过去细胞使用洗涤剂和酶将细胞成分从组织中去除。各种 ECM 组件, 包括胶原蛋白、纤连蛋白和瓣膜基质层粘连蛋白, 为培养细胞提供了一种类似于组织的微环境, 促进了细胞的存活、增殖和分化3。此外, 在 ECM 中缺乏细胞成分的情况下, 移植的免疫原性可降至最低水平。
到目前为止, 瓣膜 ECM 的制备方法已经尝试了不同的组织和器官, 如心脏4,5,6,7, 肝8,9,10 ,11, 肺12,13,14,15,16,17, 和肾脏18,19,20. 然而, 在我们最了解的情况下, 没有就舌头上的类似工作找到相关的研究。
在本研究中, 瓣膜舌外基质 (TEM) 是通过一系列物理、化学和酶处理, 高效、廉价地制备出来的。然后用透射电镜对体外TSCC 进行了重述, 对 TSCC 的行为和发育进行了适当的模拟。tem 具有良好的生物相容性以及引导细胞到组织特异性的能力, 这表明 tem 在 TSCC 研究3中可能具有很大的潜力。此处显示的协议为研究 TSCC 的发病机制或临床治疗的研究者提供了选择。

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			<td height="42" style="height:42px;width:216px;">C57-BL/6J mice</td>
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			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
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			<td style="width:119px;">A501218</td>
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			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A100395</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dibasic Sodium Phosphate</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">BE14-GR-500G</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Monobasic&#160;</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501211</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">1.5 mL EP tube</td>
			<td style="width:292px;">Axygen</td>
			<td style="width:119px;">MCT-150-A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-low temperature freezer&#160;</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3.5 cm cell culture dish</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">153066</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 cm cell culture dish</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">V900502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">449164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium sulphate</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A601988</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">158968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">PHR1487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical suture</td>
			<td style="width:292px;">Shanghai Jinhuan</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 mL wide-mouth bottle</td>
			<td style="width:292px;">SHUNIU</td>
			<td style="width:119px;">1407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic stirrer</td>
			<td style="width:292px;">AS ONE</td>
			<td style="width:119px;">1-4602-32</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">CO2 incubator</td>
			<td style="width:292px;">SHEL LAB</td>
			<td style="width:119px;">SCO5A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL syringe</td>
			<td style="width:292px;">Hunan Pingan</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL centrifuge tube</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">227270</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cal27 cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Tongue squamous cell carcinoma cell line</td>
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			<td height="42" style="height:42px;width:216px;">U2OS cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Human osteosarcoma cell line</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes free acid</td>
			<td style="width:292px;">BBI</td>
			<td style="width:119px;">A600264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:292px;">Hyclone</td>
			<td style="width:119px;">SH30084.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4 &#176;C fridge</td>
			<td style="width:292px;">Haier</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water purifier</td>
			<td style="width:292px;">ELGA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:292px;">BLAU</td>
			<td style="width:119px;">717805</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro

为了构建一种有效的、逼真的舌鳞癌 (TSCC) 模型, 建立了瓣膜舌细胞外基质 (TEM), 为 TSCC 的构建提供了功能性支架。TEM 为细胞生长、分化和细胞迁移提供了体外小生境。本机细胞外基质 (ECM) 的显微结构和瓣膜基质中保留的生化成分为锚固细胞提供组织特异的龛位。经脱氧核糖核酸酶 (DNase) 消化, 同时具有严重的有机或无机预处理, 可实现透射电镜的制备。该协议易于操作, 并确保了去细胞的高效率。透射电镜显示, 在静态或搅拌培养条件下, TSCC 细胞具有良好的细胞相容性, 使 TSCC 模型得以构建。自制的生物反应器也用于细胞培养的持续搅拌条件。应用 TEM 重建 TSCC 显示了类似临床 TSCC 组织病理学的特点和性质, 提示 TSCC 研究的潜力。

本议定书的编写 TEM 证明是有效和适当的。与母语组织相比, 透射电镜显示出完美的去细胞。去细胞的疗效经苏木精-伊红染色 (图 1A B) 证实。he 染色结果显示透射电镜核染色完全消失 (图 1B)。此外, 从先前的工作中 dna 含量的量化表明, dna 几乎完全从 TEM3中去除。该协议在去除细胞成分的同时也显示了对组织完整性的罕见损害 (图 1B)。
3D. 采用 TEM 和自制 minibioreactor (图 2a B) 进行 TSCC 重建, 取得了令人满意的效果。he 染色表明, TEM 中的 Cal27 细胞呈现典型的 TSCC 病理特征 (图 2C D)。搅拌培养条件下的细胞在不同病变区域呈现单细胞迁移 (图 2C) 或集体迁移 (图 2D)。在静态培养条件下, Cal27 细胞在 TEM 中也形成了侵入性结构, 但花了较长的时间 (图 2E)。此外, 人类骨肉瘤细胞系 U2OS 也被引入到相同的搅拌培养系统。虽然 U2OS 细胞可以生活在培养基中, 但在 TEM 中找不到它们 (图 2F)。透射电镜对不同类型的癌细胞具有不同的生物相容性, 表明不同的肿瘤细胞可能需要不同的微环境。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57235/57235fig1.jpg"> 图 1: 制备 TEM.(一) 他用老鼠的母语染色。刻度条 = 100 µm (B) 他从小鼠身上染色瓣膜透射电镜。刻度条 = 100 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57235/57235fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57235/57235fig2.jpg"> 图 2: 经 TEM 重建 TSCC.(a) 自行制作的 minibioreactor 的概览。(B) 自行制作的 minibioreactor 的电镜加载位置的观点。(三) 用 TEM 对14天搅拌培养 TSCC 进行染色。单细胞侵入现象由黑色箭头表示。刻度条 = 50 µm (D) he 染色14天搅拌培养 TSCC 与 TEM。集体入侵现象由黑箭指示。刻度条 = 50 µm (E) he 染色28天静态培养 TSCC 与 TEM。单细胞侵入现象由黑色箭头表示。刻度条 = 100 µm (F) 他用 TEM 染色14天搅拌培养的 U2OS 细胞。刻度条 = 50 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57235/57235fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>

Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

舌细胞外基质的制备与舌鳞癌体外重建

给出了一种有效去细胞的舌胞外基质 (TEM) 的制备方法。透射电镜可作为功能性支架, 用于在静态或搅拌培养条件下重建舌鳞癌 (TSCC) 模型。

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

fabrication-tongue-extracellular-matrix-reconstitution-tongue

Nanyang Technological University

Research

JoVE Journal

Cancer Research

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

6.2K 次观看.  Sun Yat-sen University. 给出了一种有效去细胞的舌胞外基质 (TEM) 的制备方法。透射电镜可作为功能性支架, 用于在静态或搅拌培养条件下重建舌鳞癌 (TSCC) 模型。

视频: Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

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, ...

科研

癌症研究

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

研究使用细胞外基体水凝胶的正常组织辐射效应

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

M1巨噬细胞-4T1小鼠乳腺癌细胞在共培养中噬菌体活性的时移2D成像

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

比较在小鼠肾脏和鸡胆膜上建立的转移性透明细胞肾细胞癌模型

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

多维共同培养系统模拟肺鳞癌进展

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...