Name: modeling oral-esophageal squamous cell carcinoma in 3d organoids
Uploaded: 2022-12-23T14:30:00
Description: Watch this Scientific Journal Video about Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids at JoVE.com

我们感谢哥伦比亚大学赫伯特欧文综合癌症中心的共享资源（流式细胞术，分子病理学和共聚焦和专业显微镜）的技术支持。我们感谢Alan Diehl博士，Adam J. Bass博士和Kwok-Kin Wong博士（NCI P01食管癌发生机制）以及Rustgi和Nakagawa实验室的成员进行了有益的讨论。这项研究得到了以下NIH资助的支持：P01CA098101（H.N.和A.K.R.），R01DK114436（H.N.），R01AA026297（H.N.），L30CA264714（SF），DE031112-01（F.M.H.），KL2TR001874（F.M.H.），3R01CA255298-01S1（J.G.），2L30DK126621-02
（J.G.）R01CA266978 （C.L.）， R01DK132251 （C.L.）， R01DE031873 （C.L.）， P30DK132710 （C.M. 和 H.N.）， 和 P30CA013696 （AKR）。H.N.和C.L.是哥伦比亚大学赫伯特欧文综合癌症中心多PI飞行员奖的获得者。H.N.是范可尼贫血研究基金奖的获得者。F.M.H.是马克癌症研究基金会奖（20-60-51-MOME）和美国癌症研究协会奖的获得者。J.G.是美国胃肠病学协会（AGA）奖的获得者。

小鼠实验是根据法规和动物协议#AABB1502计划和进行的，由哥伦比亚大学机构动物护理和使用委员会审查和批准。小鼠被饲养在适当的动物护理设施中，以确保对小鼠的人道待遇，并为小鼠提供适当的兽医护理，并为实验室人员提供实验室安全培训。
1.用4NQO治疗小鼠诱导食管IEN和ESCC病变（时间考虑：长达28周）
注意：为了产生代表肿瘤性食管病...

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此处描述的协议中，生成和分析MEO有几个关键步骤和注意事项。为了确保MEO实验的可重复性和严谨性，生物学和技术重复都很重要。对于生物学重复，每个实验条件通常有两到三只携带ESCC的独立小鼠就足够了。然而，适当的生物学重复次数可能会有所不同，具体取决于单个研究中要测试的参数。例如，目前尚不清楚从没有肉眼可见病变或组织学IEN和ESCC病变的4NQO处理小鼠中检测到肿瘤类器官的早?...

食管鳞状细胞癌 （ESCC） 是人类鳞状细胞癌中最致命的，因为它诊断较晚、治疗耐药和转移1，2。ESCC 起源于分层鳞状上皮，其排列在食管的管腔表面。鳞状上皮由增殖性基底细胞和基底上细胞层内的分化细胞组成。在生理条件下，基底细胞表达标记物，如p63，Sox2和细胞角蛋白K5和K14，而分化的细胞表达K4，K13和IVL。基底细胞本身是异质的，包括由K153 和CD734等标记物定义的推定干细胞。在体内平衡中，基底细胞在基底上细胞层内经历有丝分裂后终末分化，而分化的细胞迁移并脱屑到腔内以完成上皮更新。让人联想到它们的起源细胞，ESCC在不同程度上显示出鳞状细胞分化。ESCC 通常伴有多灶性组织学前体病变，称为上皮内瘤变 （IEN） 或异型增生，包括非典型基底细胞。除了上皮变化外，ESCC还显示上皮下隔室内的组织重塑，其中发生癌症相关成纤维细胞（CAFs）的活化和免疫/炎症细胞的募集以促进肿瘤的微环境。
ESCC的发病机制涉及遗传改变和环境危险因素暴露。关键的遗传病变包括肿瘤抑制基因TP53和CDKN2A（p16INK4A）的失活以及CCND1（细胞周期蛋白D1）和EGFR癌基因的活化，最终导致细胞周期检查点功能受损，异常增殖以及在与暴露于环境致癌物相关的遗传毒性应激下存活。事实上，遗传变化与行为和环境风险因素密切相关，最常见的是烟草和酒精的使用。烟草烟雾中含有乙醛等人类致癌物质，乙醛也是酒精的主要代谢产物。乙醛诱导DNA加合物和链间DNA交联，导致DNA损伤和DNA突变和染色体不稳定的积累。鉴于过量的有丝分裂刺激和癌基因激活引起的异常增殖，应对遗传毒性应激的机制促进了食管上皮细胞的恶性转化，包括抗氧化剂的激活、自噬和上皮-间充质转化（EMT）。有趣的是，这些细胞保护功能通常在以高CD44（CD44H）表达为特征的ESCC癌症干细胞（CSC）中被激活，并具有肿瘤起始，侵袭，转移和治疗耐药的能力5，6，7。
ESCC已在细胞培养和啮齿动物模型8，9中建模。在过去的三十年中，已经开发出强大的ESCC基因工程小鼠模型。这些包括CCND1和EGFR转基因小鼠10，11和p53和p120Ctn敲除小鼠12，13。然而，单个基因改变通常不会导致快速发作的ESCC。通过使用食道致癌物克服了这一挑战，这些致癌物很好地概括了ESCC14中的人类遗传病变。例如，4-硝基喹啉-1-氧化物（4NQO）加速CCND1转基因小鼠ESCC的发育15。近年来，在细胞谱系可追溯小鼠模型3，4中研究了假定的食管上皮干细胞，祖细胞及其各自的命运。此外，这些细胞谱系可追溯的小鼠已被用于探索ESCC的起源细胞以及这些细胞如何通过常规组织学和基于组学的分子表征产生CD44HCSCs7。
与这些小鼠模型相关的一个新兴领域是细胞培养技术在三维（3D）类器官系统中分析活ESCC和前体细胞的新应用，其中原始组织的结构在体外重现7，8，9。这些 3D 类器官从从小鼠组织中分离的单细胞悬液中快速生长，包括原发性和转移性肿瘤（例如淋巴结、肺和肝脏病变）。将细胞包埋在基底膜提取物（BME）中，并用明确的无血清细胞培养基进料。3D类器官在7-10天内生长，所得的球形结构适合传代培养，冷冻保存和测定，用于分析各种细胞特性和功能，包括CSC标志物，EMT，自噬，增殖，分化和凋亡细胞死亡。
这些方法可广泛应用于从任何分层鳞状上皮组织建立的3D类器官培养物，例如头颈部粘膜（口腔，舌头，咽部和喉部）甚至前胃。头颈部黏膜与食管相邻，两种组织具有相似的组织组织、功能和对疾病的易感性。头颈部鳞状细胞癌（HNSCC）和ESCC都有遗传病变和生活方式相关的环境危险因素，如烟草和酒精暴露。强调这种相似性，用烟草烟雾模拟4NQO处理的小鼠很容易发展HNSCC和ESCC。鉴于下面描述的方案可以很容易地应用于HNSCC建模，我们提供了从这些病变建立3D类器官培养物的具体说明。
在本文中，我们提供了用于生成小鼠食管3D类器官（MEO）的详细方案，这些类器官代表在用4NQO治疗的小鼠中发展的正常，癌前和ESCC病变。可以使用各种小鼠品系，包括常见的实验室品系，如C57BL / 6和细胞谱系可追溯以及其他基因工程衍生物。我们强调关键步骤，包括分离正常或患病的小鼠食管上皮，制备单细胞悬浮液，培养和监测生长中的3D类器官，传代培养，冷冻保存以及后续分析的处理，包括形态学和其他应用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>25-300-120</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>25-200-114</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4% Trypan Blue</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL tuberculin syringe without needle</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell strainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L wide bore micropipette tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>212361A</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needles</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-556-006</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Nitroquinoline-1-oxide (4NQO)</td>
			<td>Tokyo Chemical Industry</td>
			<td>NO250</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-565-270</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>12556004</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>99.9% ethylene propylene glycol</td>
			<td>SK picglobal</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>15290018</td>
			<td>Stock concentration 250 &#181;g/mL, final concentration 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td>Stock concentration 100x, final concentration 1x</td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td>Stock concentration 50x, final concentration 1x</td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator, e.g.Heracell 150i</td>
			<td>Thermo Fisher Scientific</td>
			<td>51026406</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMQAX1000</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Thermo Fisher Scientific</td>
			<td>03-337-7D</td>
			<td></td>
		</tr>
		<tr>
			<td>DietGel 76A</td>
			<td>Clear H2O</td>
			<td>72-07-5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>MilliporeSigma</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Corning</td>
			<td>354235</td>
			<td>Stock concentration 50 U/mL, final concentration 2.5&#8211;5 U/mL</td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>VWR</td>
			<td>25870-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190250</td>
			<td>Stock concentration 1x</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>VWR</td>
			<td>82027-386</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing container</td>
			<td>Corning</td>
			<td>432002</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Thermo Fisher Scientific</td>
			<td>G7-500</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Stock concentration 100x, final concentration 1x</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td>Stock concentration 1 M, final concentration 10 mM</td>
		</tr>
		<tr>
			<td>Hot plate/stirrer</td>
			<td>Corning</td>
			<td>PC-420D</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Lab Armor bead bath (or water bath)</td>
			<td>VWR</td>
			<td>89409-222</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Laboratory balance</td>
			<td>Ohaus</td>
			<td>71142841</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Matrigel basement membrane extract (BME)</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Minispin</td>
			<td>Eppendorf</td>
			<td>22620100</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube rack</td>
			<td>Southern Labware</td>
			<td>0061</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502048</td>
			<td>Stock concentration 100x, final concentration 1x</td>
		</tr>
		<tr>
			<td>N-acetylcysteine (NAC)</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>Stock concentration 0.5 M, final concentration 1 mM</td>
		</tr>
		<tr>
			<td>Parafilm M wrap</td>
			<td>Thermo Fisher Scientific</td>
			<td>S37440</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>MilliporeSigma</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272416</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase-contrast microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Recombinant mouse epidermal growth factor (mEGF)</td>
			<td>Peprotech</td>
			<td>315-09-1mg</td>
			<td>Stock concentration 500 ng/&#181;L, final concentration 100 ng/mL</td>
		</tr>
		<tr>
			<td>RN cell-conditioned medium expressing R-Spondin1 and Noggin (RN CM)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Available through the Organoid and Cell Culture Core upon request, final concentration 2%</td>
		</tr>
		<tr>
			<td>Sorval ST 16R centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004380</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor (STI)</td>
			<td>MilliporeSigma</td>
			<td>T9128</td>
			<td>Stock concentration 250 &#181;g/mL</td>
		</tr>
		<tr>
			<td>ThermoMixer C</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-285-562 PM</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleck Chemicals</td>
			<td>S1049</td>
			<td>Stock concentration 10 mM, final concentration 10 &#181;M</td>
		</tr>
	</tbody>
</table>

modeling oral-esophageal squamous cell carcinoma in 3d organoids

食管鳞状细胞癌（ESCC）在世界范围内普遍存在，每年占所有食管癌病例的90%，是所有人类鳞状细胞癌中最致命的。尽管最近在确定伴随ESCC启动和发展的分子变化方面取得了进展，但患者的预后仍然很差。这些分子变化的功能注释是必要的下一步，并且需要既能捕获ESCC的分子特征，又可以轻松且廉价地操作功能注释的模型。用烟草烟雾模拟物4-硝基喹啉1-氧化物（4NQO）处理的小鼠可预测地形成ESCC和食管癌前发育。值得注意的是，4NQO病变也出现在口腔中，最常见于舌头以及前胃，它们都共享分层鳞状上皮。然而，这些小鼠不能简单地操纵进行功能假设检验，因为生成同基因小鼠模型是时间和资源密集型的。在这里，我们通过从用4NQO处理的小鼠中生成单细胞衍生的三维（3D）类器官来克服这一限制，以表征小鼠ESCC或体 外肿瘤前细胞。这些类器官捕获了ESCC和食管癌前增生的显着特征，可以廉价快速地用于形成等基因模型，并可用于同源移植实验。我们演示了如何从正常，肿瘤前和SCC鼠食管组织中生成3D类器官，并维持和冷冻保存这些类器官。这些多功能类器官的应用非常广泛，包括利用基因工程小鼠和通过流式细胞术或免疫组织化学进一步表征，使用CRISPR技术生成同基因类器官系，以及药物筛选或同源移植。我们相信，广泛采用该议定书中展示的技术将加速该领域的进展，以应对ESCC的严重负担。

该协议描述了根据特定的治疗方案从正常食管组织或来自4NQO处理的小鼠的ESCC肿瘤组织产生小鼠食管类器官（MEO）的过程，该方案包括在饮用水中施用16周的4NQO，然后是10周至12周的观察期（图1）。然后将小鼠安乐死以解剖舌头或食道组织（图2 和 图3）。我们描述了一种从完整食道中分离上皮层的方法（图4?...

Watch this Scientific Journal Video about Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids at JoVE.com

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

该协议描述了生成和表征鼠口腔 - 食管3D类器官的关键步骤，这些类器官代表通过化学致癌引起的正常，癌前和鳞状细胞癌病变。

在 3D 类器官中模拟口腔-食管鳞状细胞癌

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

食管鳞状细胞癌（ESCC）在世界范围内是致命的和普遍的。三维类器官可用于加速该领域的进展，以应对ESCC的严重负担。来自用4NQO处理的小鼠的单细胞衍生的3D类器官可以很容易地和廉价地操作，以对伴随ESCC启动和发育的分子变化进行功能注释。该模型捕获了诱变原诱导的肿瘤的遗传异质性。因此，这些类器官提供了一个生理相关的平台来测试新的治疗策略或识别驱动肿瘤发生的显着遗传变化。帮助展示动物解剖和器官收集的是工作人员Yasuto Tomita，工作人员助理，演示用于石蜡包埋程序的类器官的制备将由我实验室的博士后研究员Norihiro Matsuura。首先，通过捏中腹皮毛并使用手术剪刀打开安乐死小鼠的皮肤，从下腹部到下巴做一个颅尾腹中线切口。从中线切口开始，进行径向切口延伸到小鼠两侧的四肢，使皮瓣膨胀。要暴露颈气管，请使用解剖剪刀将唾液腺分开中线。要暴露胸气管，请切除胸骨。用镊子轻轻捏住并提起腹膜，然后用剪刀将腹膜颅尾分开。并沿肋骨横向。轻轻地将肝脏从横膈膜的尾部表面收回，并使用剪刀在胸骨切口处的横膈膜上做一个小切口，特别是在剑突的背表面。一旦肋骨与胸内容物分离，将剪刀插入横膈膜的切口，并解剖紧贴胸骨背表面的颈带，以避免损伤下面的器官。用剪刀剪断胸骨两侧的肋骨并移除胸骨。要暴露腹部食道，请用镊子握住胃窦，轻轻地向前提起胃。用剪刀从胃和食道解剖脾脏、胰腺和肠系膜。要暴露胸食管，请立即轻轻抬起气管尾部至甲状腺软骨，并使用虹膜剪刀解剖气管背侧的食管。用虹膜剪刀将甲状腺软骨处的气管分开。通过小心地在尾部方向解剖气管，从食道的其余部分剥离气管。用气管完全切除肺、心脏和胸腺。用剪刀将幽门处的胃分开。用镊子夹住胃窦并解剖颅骨，将食道与椎骨分开。将食道分开到甲状腺软骨的水平，并完全收获食道和胃。通过在贲门处分离食道来分离胃和食道，并解剖食道外表面的任何筋膜。要保留用于组织学的样本，请用剪刀纵向分开。将剩余的完整食道和冷的PBS放在冰上。沿着较大的曲率打开胃，并用PBS充分清洗。分离前胃并用冷PBS清洗。要收获舌头，请从鼻子上取下针头，然后用镊子拔出舌头。尽可能长时间地切开舌头，并将其放在冰上的冷PBS中。将解离的组织转移到培养皿中后，使用镊子小心地从上皮上去除肌肉层。将上皮细胞转移到含有500微升0.25%胰蛋白酶的微量离心管中，并在37摄氏度和800rpm的温度混合器中孵育10分钟。短暂离心后，通过保持在50毫升锥形管上的100微米细胞过滤器转移细胞悬液，使用圆周运动进行宽口径尖端，然后加入3毫升大豆胰蛋白酶抑制剂或STI，通过过滤器使用圆周运动洗涤，然后用1毫升结核菌素注射器柱塞的底部擦洗过滤器以推动细胞通过。用3毫升PBS清洗过滤器3至5次，在洗涤之间用注射器底部擦洗过滤器。通过离心沉淀细胞后，除去上清液，在管中留下1毫升溶液进行重悬。一旦沉淀被重新悬浮，通过70微米细胞过滤器将细胞悬液转移到一个新的50毫升锥形管中。再次离心管后，将沉淀重悬于100微升小鼠类器官培养基或MOM中，并进行自动细胞计数。在75%基底膜提取物或BME和MOM中板5，000个活细胞，每孔总体积为50微升。使用200微升宽口径尖端，在每个孔的中心缓慢添加50微升液滴，避免尖端与孔底部或侧面接触。让BME在37摄氏度的培养箱中凝固30分钟，5%二氧化碳和95%相对湿度。孵育后，每孔小心地加入500微升MOM。在第 3 天或第 4 天更换 MOM，然后每 2 到 3 天更换一次，直到准备好通过。使用前将解冻的 BME 放在冰上，预热 MOM、0.05% 胰蛋白酶和 STI 至 37 摄氏度。使用宽口径微量移液器吸头，将 BME 圆顶中的类器官与上清液一起收集，并通过上下移液破坏 BME。通过短暂离心获得类器官沉淀后，一旦将其移开，将其重悬于500微升0.05%胰蛋白酶中。将管在37摄氏度和800 rpm的热混合器中孵育10分钟。用600微升STI灭活胰蛋白酶。离心并弃去上清液后，将细胞沉淀重悬于100微升MOM中。通过台盼蓝排除进行自动细胞计数。要固定类器官，轻轻地移开沉淀并在 300 微升 4% 多聚甲醛中重悬 4 摄氏度过夜。接下来，通过倒置微量离心管架并用天花板薄膜覆盖表面来准备包埋表面，然后用相应的类器官ID标记。通过在管的侧面添加50微升琼脂，小心地覆盖在PBS洗涤的类器官沉淀中去除的多聚甲醛。重复此步骤。在不干扰沉淀的情况下，将琼脂凝胶液滴中的沉淀转移到包埋表面上的天花板膜上，以便在 4 摄氏度下固化 45 分钟。使用镊子，小心地将含有固化类器官颗粒的液滴转移到标记的病理盒中。通过明场成像和组织学染色对正常鼠食管舌和前胃类器官进行形态分析。来自4NQO治疗小鼠的食管鳞状细胞癌显示核异型性和突然角化。通过测定传代培养时类器官形成率评估的类器官自我更新能力表明，形成速率随时间下降，反映了成熟类器官中增殖基础细胞的减少。类器官的生长动力学通过它们在不同时间点的形态来揭示。类器官内核的角质化在第10天变得突出。增殖性基底细胞保留在最外层的细胞层中，即使在第14天和第21天的时间点也是如此。由4NQO未处理小鼠产生的正常小鼠舌类器官的群体倍增曲线表明它们在多次传代和长期培养中稳定生长。类器官是用于高通量筛选以识别新治疗靶点的平台，基于CRISPR的编辑用于询问特定基因功能或共培养以定义肿瘤微环境中的哪些相互作用影响转化。这项技术使我们能够研究部分EMT和HPV感染等现象，以及它们与呼吸消化道鳞状细胞癌生物学中免疫系统的相互作用。

Research

JoVE Journal

Cancer Research

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

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

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

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

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 ...

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

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

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

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

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 ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

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

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

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 ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...