该项目由慕尼黑大学 (FöFoLe 项目-no: 789-781) 拨款资助。

本手稿中所显示的所有研究, 即人类肿瘤标本的使用, 均受《美因茨大学医学》/慕尼黑大学医学中心伦理委员会的事先决定的保护。根据国家法律准则, 病人同意科学使用在其治疗过程中获得的过量生物材料, 因此, 患者已给予知情许可。已按照所有机构、国家和国际人类福利准则进行研究。
1. 头部和见鬼鳞状细胞癌的肿瘤活检
<ol><li>执行一般 (异丙酚和/或七氟醚, 肌肉放松剂) 或局部浸润 (2% ultracaine, 肾上腺素)/表面 (利多卡因) 麻醉在手术室或耳鼻喉检查椅。在口腔/咽/喉/上消化道和呼吸道的其他部分用标准操作仪器可视化肿瘤肿块, 如有需要, 在显微镜下观察。</li>
	<li>用钝器或切割器械在癌性病变的周围进行新的肿瘤活检。避免因本区大量坏死导致病灶中心。将所获得的组织放在一个无菌容器中, 采用等渗氯化钠溶液。</li>
	<li>活检后, 进行必要的止血, e. g, 与双极或单凝血装置, 除了使用缩血管物质。</li>
	<li>将用于细胞培养实验的肿瘤活检直接应用于相邻的实验道。像往常一样向病理学家发送其他肿瘤活检以排除癌症。</li>
	<li>确保实验室技术员可以直接处理样品。</li>
</ol>2. 处理肿瘤标本
<ol><li>把肿瘤标本放在一个合适的和不育的表面上, 用一把无菌的单用手术刀彻底切开, 尽可能少的片断。 注意: 确保感染性和血液传播的疾病的状况有充分的记录, 技术员注意到机构的标准协议, 以防止针棒或切割伤害与潜在的生物危害患者材料和可能受伤后的后续议定书。</li>
	<li>在有足够的机械分离的主要组织后, 将组织放入含有胶原酶 I/II 的瓶子中, 并在37摄氏度孵育1小时。筛过70µm 猎鹰细胞过滤器, 并用汉克斯平衡盐溶液 (HBSS) 冲洗悬浮液。</li>
	<li>
成功分离和随后清洗后, 将包含 1-2 x 106单元格的悬浮液放置到 T75 细胞培养烧瓶 (75 厘米2) 中, 以在 5% CO2和温度为37摄氏度的情况下增加到子融合。此步骤可能需要10天。
	<ol>
<li>使用特殊角质细胞培养培养基, 包括: 125 毫升 Dulbecco 的修饰鹰培养基 (DMEM), 250 毫升角质细胞补充培养基 (补充培养基, 包括500毫升角质细胞 SF 培养基, 15 毫克牛垂体提取物 (BPE), 2.5 毫升青霉素/链霉素, 150 ng 重组人上皮生长因子 (EGF), 516 µL 300 毫米 CaCl2, 预先准备的库存混合), 125 毫升 F12 营养混合物, 10 毫克 BPE (0.75 毫升), 75 ng 重组人 EGF (2 µL), 3.75 毫升200毫米 l-丙-Glutamin 二肽 (材料表)。</li>
	</ol>
</li>
</ol>3. 将细胞播种到超低黏附细胞培养板中
<ol><li>在显微镜下确认肿瘤细胞的生长。将培养基中的细胞 (主要或细胞培养) 和种子5000主要肿瘤细胞或 1,000-2, 000 细胞的中间细胞线/其他细胞系在200-300 µL 的介质中 (步骤 3.2) 成一个超低粘附板, 凹, 圆底 (96-井)。</li>
	<li>
培养细胞在 5% CO2在37°c 和在相等的部分 DMEM 和气道上皮细胞培养基 (BEGM), 10% 胎牛血清, 1% 青霉素/链霉素, 1% 钠丙酮酸, 1% 非必需氨基酸, 1% l-谷氨酰胺 (步 2.3.)。如果使用细胞线, DMEM 的基础上的介质就足够了。
	<ol>
<li>每隔一天执行一次媒体更改。注意不要在介质变化过程中用吸管吸入球体。文化球体直到增长水平作为 descibed 在3.3 被到达 (大约7-10 天)。</li>
	</ol>
</li>
	<li>通过寻找三维球形细胞企业集团, 在显微镜下确认自发球体的形成。排除不规则和/或多球体形成的井进一步调查。 注: 球体应该是肉眼可见, 也有助于进一步处理和媒体的变化, 如下所述。</li>
</ol>4. 将球体暴露于多模标准或实验性肿瘤治疗中
<ol><li>选择一个理想的治疗方案。设计足够大的控制组, 允许比较治疗球体或球体只接受部分治疗方案, e. g。仅辐射。控制组的大小取决于实验组的设计, 不能普遍定义。</li>
	<li>用添加剂将介质与介质交换, 即在所需浓度下化疗和/或单克隆抗体的培养基。添加顺铂浓度为 2.5/5/10 µM 或 5-fluoruracil (5FU) 在30µM。 注: 对于高通量实验或大组大小/大量组, 我们可以使用自动吹打机器人, 因为我们正在进一步建立在实验中。</li>
	<li>或者, 用合适的辐射设施在2球体的细胞培养板上辐射。 注意: 尊重该机构关于防止雇员有害辐射暴露的议定书。根据辐射防护指导方针, 只与指定和训练有素的技术人员合作。如有需要, 添加化疗, 如下所述4.2。之后。</li>
	<li>在先前描述的培养条件 (37 °c, 步骤 3.2) 孵育细胞24小时。</li>
	<li>孵化后, 每隔一天, 继续进行细胞培养至少6天的介质变化。</li>
</ol>5. 评估读取的球体大小和细胞增殖的程度
<ol><li>用图形软件 (参数 1) 在6天 (10 天, 16 天) 的数字照片文档中测量球体大小。</li>
	<li>离心后在 520 x g 2.5 分钟的板, 取出上清。用足够数量的 1x PBS 冲洗细胞, 再将盘子重新离心, 然后将其移除上清 (PBS)。</li>
	<li>添加100µL 酶细胞脱离溶液, 以使球体溶解。在37摄氏度孵育8分钟的盘子。</li>
	<li>检查显微镜下球体是否成功溶解。添加100µL 的 DMEM。离心板在 520 x g 2.5 分钟. 取出上清液并将细胞悬浮在100µL 的 DMEM 中。</li>
	<li>根据制造商的说明25, 执行一项商业上可获得的比色扩散试验, 例如, 在每个瓶子上 WST-8 化验。阅读酶联免疫吸附试验 (ELISA) 阅读器 (参数 2) 中的检测。</li>
</ol>

<ol>
	<li>Hunter, K. D., Parkinson, E. K., Harrison, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+early+head+and+neck+cancer.">Profiling early head and neck cancer.</a> Nat Rev Cancer. 5 (2), 127-135 (2005).</li><li>Jerjes, W., 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=The+effect+of+tobacco+and+alcohol+and+their+reduction/cessation+on+mortality+in+oral+cancer+patients:+short+communication.">The effect of tobacco and alcohol and their reduction/cessation on mortality in oral cancer patients: short communication.</a> Head Neck Oncol. 4, 6 (2012).</li><li>Chen, H. H. W., Kuo, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+radiotherapy+in+cancer+treatment:+Promises+and+challenges.">Improving radiotherapy in cancer treatment: Promises and challenges.</a> Oncotarget. 8 (37), 62742-62758 (2017).</li><li>Boeckx, C., 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=Anti-epidermal+growth+factor+receptor+therapy+in+head+and+neck+squamous+cell+carcinoma:+focus+on+potential+molecular+mechanisms+of+drug+resistance.">Anti-epidermal growth factor receptor therapy in head and neck squamous cell carcinoma: focus on potential molecular mechanisms of drug resistance.</a> Oncologist. 18 (7), 850-864 (2013).</li><li>Brand, T. M., Iida, M., Wheeler, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+resistance+to+the+EGFR+monoclonal+antibody+cetuximab.">Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab.</a> Cancer Biol Ther. 11 (9), 777-792 (2011).</li><li>Seidl, D., Schild, S. E., Wollenberg, B., Hakim, S. G., Rades, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+Factors+in+Patients+Irradiated+for+Recurrent+Head-and-Neck+Cancer.">Prognostic Factors in Patients Irradiated for Recurrent Head-and-Neck Cancer.</a> Anticancer Res. 36 (12), 6547-6550 (2016).</li><li>Shirai, K., 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=Clinical+Outcomes+of+Definitive+and+Postoperative+Radiotherapy+for+Stage+I-IVB+Hypopharyngeal+Cancer.">Clinical Outcomes of Definitive and Postoperative Radiotherapy for Stage I-IVB Hypopharyngeal Cancer.</a> Anticancer Res. 36 (12), 6571-6578 (2016).</li><li>Theile, D., 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=Evaluation+of+drug+transporters'+significance+for+multidrug+resistance+in+head+and+neck+squamous+cell+carcinoma.">Evaluation of drug transporters' significance for multidrug resistance in head and neck squamous cell carcinoma.</a> Head Neck. 33 (7), 959-968 (2011).</li><li>Slade, M. J., 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=Comparison+of+bone+marrow,+disseminated+tumour+cells+and+blood-circulating+tumour+cells+in+breast+cancer+patients+after+primary+treatment.">Comparison of bone marrow, disseminated tumour cells and blood-circulating tumour cells in breast cancer patients after primary treatment.</a> Brit J Cancer. 100 (1), 160-166 (2009).</li><li>Mockelmann, N., Laban, S., Pantel, K., Knecht, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+in+head+and+neck+cancer:+clinical+impact+in+diagnosis+and+follow-up.">Circulating tumor cells in head and neck cancer: clinical impact in diagnosis and follow-up.</a> Eur Arch Otorhinolaryngol. 271 (1), 15-21 (2014).</li><li>Gerlinger, M., 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=Intratumor+heterogeneity+and+branched+evolution+revealed+by+multiregion+sequencing.">Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.</a> N Engl J Med. 366 (10), 883-892 (2012).</li><li>Ledgerwood, L. 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=The+degree+of+intratumor+mutational+heterogeneity+varies+by+primary+tumor+sub-site.">The degree of intratumor mutational heterogeneity varies by primary tumor sub-site.</a> Oncotarget. 7 (19), 27185-27198 (2016).</li><li>Loyo, M., 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=Lessons+learned+from+next-generation+sequencing+in+head+and+neck+cancer.">Lessons learned from next-generation sequencing in head and neck cancer.</a> Head Neck. 35 (3), 454-463 (2013).</li><li>Morris, L. 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=The+Molecular+Landscape+of+Recurrent+and+Metastatic+Head+and+Neck+Cancers:+Insights+From+a+Precision+Oncology+Sequencing+Platform.">The Molecular Landscape of Recurrent and Metastatic Head and Neck Cancers: Insights From a Precision Oncology Sequencing Platform.</a> JAMA Oncol. , (2016).</li><li>Bauml, J. M., Cohen, R. B., Aggarwal, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunotherapy+for+head+and+neck+cancer:+latest+developments+and+clinical+potential.">Immunotherapy for head and neck cancer: latest developments and clinical potential.</a> Ther Adv Med Oncol. 8 (3), 168-175 (2016).</li><li>Mack, B., 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=Rapid+and+non-enzymatic+in+vitro+retrieval+of+tumour+cells+from+surgical+specimens.">Rapid and non-enzymatic in vitro retrieval of tumour cells from surgical specimens.</a> PLoS One. 8 (1), e55540 (2013).</li><li>Ham, S. L., Joshi, R., Thakuri, P. S., Tavana, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid-based+three-dimensional+tumor+models+for+cancer+research+and+drug+discovery.">Liquid-based three-dimensional tumor models for cancer research and drug discovery.</a> Exp Biol Med (Maywood). 241 (9), 939-954 (2016).</li><li>Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+generation+of+homogeneous+multicellular+tumor+spheroids+applicable+to+a+wide+variety+of+cell+types.">Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.</a> Biotechnol Bioeng. 83 (2), 173-180 (2003).</li><li>Kunz-Schughart, L. A., Freyer, J. P., Hofstaedter, F., Ebner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+3-D+cultures+for+high-throughput+screening:+the+multicellular+spheroid+model.">The use of 3-D cultures for high-throughput screening: the multicellular spheroid model.</a> J Biomol Screen. 9 (4), 273-285 (2004).</li><li>Lin, R. Z., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+three-dimensional+multicellular+spheroid+culture+for+biomedical+research.">Recent advances in three-dimensional multicellular spheroid culture for biomedical research.</a> Biotechnol J. 3 (9-10), 1172-1184 (2008).</li><li>Weiswald, L. B., Bellet, D., Dangles-Marie, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spherical+cancer+models+in+tumor+biology.">Spherical cancer models in tumor biology.</a> Neoplasia. 17 (1), 1-15 (2015).</li><li>Cancer Genome Atlas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+genomic+characterization+of+head+and+neck+squamous+cell+carcinomas.">Comprehensive genomic characterization of head and neck squamous cell carcinomas.</a> Nature. 517 (7536), 576-582 (2015).</li><li>Duarte, S., 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=Isolation+of+head+and+neck+squamous+carcinoma+cancer+stem-like+cells+in+a+syngeneic+mouse+model+and+analysis+of+hypoxia+effect.">Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect.</a> Oncol Rep. 28 (3), 1057-1062 (2012).</li><li>Reid, P. A., Wilson, P., Li, Y., Marcu, L. G., Bezak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+understanding+of+cancer+stem+cells:+Review+of+their+radiobiology+and+role+in+head+and+neck+cancers.">Current understanding of cancer stem cells: Review of their radiobiology and role in head and neck cancers.</a> Head Neck. 39 (9), 1920-1932 (2017).</li><li>Cossu, F., 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=Structural+Insight+into+Inhibitor+of+Apoptosis+Proteins+Recognition+by+a+Potent+Divalent+Smac-Mimetic.">Structural Insight into Inhibitor of Apoptosis Proteins Recognition by a Potent Divalent Smac-Mimetic.</a> PLoS ONE. 7 (11), e49527 (2012).</li><li>Hagemann, J., 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=Spheroid-based+3D+Cell+Cultures+Enable+Personalized+Therapy+Testing+and+Drug+Discovery+in+Head+and+Neck+Cancer.">Spheroid-based 3D Cell Cultures Enable Personalized Therapy Testing and Drug Discovery in Head and Neck Cancer.</a> Anticancer Res. 37 (5), 2201-2210 (2017).</li><li>Worp, H. B., 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=Can+animal+models+of+disease+reliably+inform+human+studies?.">Can animal models of disease reliably inform human studies?.</a> PLoS Med. 7 (3), e1000245 (2010).</li><li>Wilding, J. L., Bodmer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+lines+for+drug+discovery+and+development.">Cancer cell lines for drug discovery and development.</a> Cancer Res. 74 (9), 2377-2384 (2014).</li><li>Chitcholtan, K., Asselin, E., Parent, S., Sykes, P. H., Evans, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+growth+properties+of+endometrial+cancer+in+three+dimensional+(3D)+culture+and+2D+cell+monolayer.">Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer.</a> Exp Cell Res. 319 (1), 75-87 (2013).</li><li>Longati, 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+pancreatic+carcinoma+spheroids+induce+a+matrix-rich,+chemoresistant+phenotype+offering+a+better+model+for+drug+testing.">3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing.</a> BMC Cancer. 13, 95 (2013).</li></ol>

作者没有什么可透露的。

我们能够建立一个协议, 以产生可再生的球体从细胞悬浮, 对细胞系和在初步实验中, 原发性人类肿瘤细胞。我们首先评估了两种以前描述的方法, 并确定了 ULA 方法, 这种方法是使用超低粘附表面的培养板, 是一个更安全, 更可靠的三维球体生成。通过结合两种不同的方法来测定读出 (大小/面积和细胞的生存能力), 这种多模式检测是足够敏感, 以确定小的差异组之间。这是迈向技术可行性证明的第一?...

头颈鳞状细胞癌 (HNSCC) 是世界上第六最常见的癌症, 其发病率上升, 粘膜人乳突病毒 (HPV) 感染相关的发病机制, 在大多数情况下, 过量的尼古丁和酒精引起消耗量1,2。虽然较小的肿瘤和前侵入阶段通常是很好的治疗与手术切除, 通常结合宫颈淋巴结清扫, 晚期和复发 HNSCC 治疗仍然是挑战, 由于侵袭性肿瘤侵袭转移扩散和抗辐射和化疗协议3,4,5,6,7,8。最近的研究表明细胞表型的变异很高, 循环和弥散肿瘤细胞的亚型特征刚刚开始9,10。早期对固体、均匀肿瘤质量的信念必须根据最近几年的研究进行修订:11,12,13,14。目前的肿瘤鉴定和关键突变鉴定方法可以识别出一些与治疗耐药性相关的基因, 但仍然是一种成本密集型的方法。而且, 基因型的知识并不一定能对表型及其治疗反应作出可靠的预测。
在改善晚期和复发性疾病的总体和无疾病生存方面, 进展甚少。对于尼古丁-以及与病毒相关的癌症, 目前的治疗方案除了手术外, 还附上了积极的辐射和铂基化学疗法。对 HPV 阴性和阳性癌的反应速率有不同的影响;然而, 这还没有导致一般治疗指南的改变。抗辐射和化疗是在所有肿瘤阶段普遍存在的现象, 并存在于铂基化疗以及靶向治疗 (抗 EGFR;表皮生长因子受体)和最近出现的检查点抑制15。无效的放疗和化疗在吞咽困难、粘膜炎、干口和肾脏或心脏功能下降的风险等方面有很高的病人发病率。预测治疗反应之前, 对每个病人的一般治疗概念的决定似乎是关键的目标, 防止不必要的治疗概念, 副作用和成本。
我们试图建立一个模型, 以测试病人的治疗敏感性目前标准的化疗-辐射, 可纳入常规和质量控制的肿瘤治疗算法从一个技术的站点。远的目标是使用该模型, 而不使用严重改变和老化细胞系, 因为他们不太代表实际的人肿瘤细胞没有他们的变异性和异质性, 因为我们现在知道, 而建立的协议是在不同的细胞系进行。为了独立于商业上可用的细胞线, 我们最近成功地从人类肿瘤标本的原始 HNSCC 细胞中生成了一种称为 "异食癖" 的中间细胞线, 其表面和有限通道上有保守的细胞标记16. 这个异食细胞系应作为一个准备, 以发展的道路上, 然后以后的试验与新鲜的人类癌细胞从肿瘤活检。已经表明, 三维细胞培养细胞在体内的反应不同, 并且比在单分子膜17,18,19,20 中生长的肿瘤药物的治疗更像是癌症药品的管理.21, 主要是由于某些单元格子集22 、23、24的迁移和分异属性的保护。在这里, 我们描述了从中间细胞线和原发性人鳞状细胞癌细胞的球体为基础的三维模型的协议, 以及如何将这种模型整合到头颈外科医生和肿瘤学家的癌症治疗中 (图 1)。

<table>
	<tbody>
		<tr>
			<td>Dulbeccos modified Eagles medium (DMEM)</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>F 0425</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco Life Technologies, Paisley, UK</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>A2212</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L0473</td>
			<td></td>
		</tr>
		<tr>
			<td>non-essential amino acids</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>K0293</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>K0293</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche Life Sciences, Basel, Switzerland</td>
			<td>5401127001</td>
			<td></td>
		</tr>
		<tr>
			<td>GravityPLUS 3D Culture and Assay Platform</td>
			<td>InSphero, Schlieren, Switzerland</td>
			<td>PB-CS 06-001</td>
			<td></td>
		</tr>
		<tr>
			<td>GravityTRAP plate</td>
			<td>InSphero, Schlieren, Switzerland</td>
			<td>PB-CS-01-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low attachment (ULA) culture plates</td>
			<td>Corning, Corning, NY, USA</td>
			<td>4520</td>
			<td></td>
		</tr>
		<tr>
			<td>airway epithelial cell growth medium</td>
			<td>Promocell, Heidelberg, Germany</td>
			<td>C-21060</td>
			<td></td>
		</tr>
		<tr>
			<td>amphotericin B</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>A 2612</td>
			<td></td>
		</tr>
		<tr>
			<td>airway epithelial cell growth medium supplement mix</td>
			<td>Promocell, Heidelberg, Germany</td>
			<td>C39165</td>
			<td></td>
		</tr>
		<tr>
			<td>WST-8 test</td>
			<td>Promocell, Heidelberg, Germany</td>
			<td>PC PK-CA705-CK04</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocyte SFMedium + L-Glutamine 500mL</td>
			<td>Invitrogen</td>
			<td>#17005-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Pituitary Extract (BPE), 25mg</td>
			<td>Invitrogen</td>
			<td>#37000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human Epithelial Growth Factor 2.5 &#181;g</td>
			<td>Invitrogen</td>
			<td>#37000015</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM High Glucose</td>
			<td>Invitrogen</td>
			<td>#21068-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin 10000U/mL Penicillin/ 10000&#181;g/mL Streptomycin</td>
			<td>Invitrogen</td>
			<td>#15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>F12 Nutrient Mix</td>
			<td>Invitrogen</td>
			<td>#21765-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax (200 mM L-Alanyl-L-Glutamin-Dipeptide in NaCl)</td>
			<td>Invitrogen</td>
			<td>#35050087</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (Ca, Mg)</td>
			<td>Life Technologies</td>
			<td>#14025-092</td>
			<td>(no phenol red)</td>
		</tr>
		<tr>
			<td>1x TrypLE Expres Enzyme</td>
			<td>Invitrogen</td>
			<td>#12604-013</td>
			<td>(no phenol red)</td>
		</tr>
		<tr>
			<td>Accutase (enzymatic cell detachment solution)</td>
			<td>Innovative cell technologies</td>
			<td>Cat# AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Falcon cell strainer</td>
			<td>BD Biosciences, USA</td>
			<td>#352350</td>
			<td></td>
		</tr>
	</tbody>
</table>

therapy testing in a spheroid-based 3d cell culture model for head and neck squamous cell carcinoma

目前的治疗方案的晚期和复发头颈部鳞状细胞癌 (HNSCC) 包围辐射和化疗的方法与或没有手术。虽然铂基化疗方案目前代表的是金标准的功效, 并在绝大多数情况下, 新的化疗方案, 即免疫疗法正在出现。然而, 无论是化疗方案的反应率和治疗耐药性机制都难以预测, 而且仍未得到充分了解。目前已知的化疗和耐辐射机制的广泛变化。本研究介绍了一种标准化的高通量体外检测方法, 以评估 HNSCC 细胞系对各种治疗方案的反应, 希望能将个体患者的原细胞作为个性化肿瘤的未来工具。治疗。本试验的目的是将 HNSCC 患者的质量控制标准算法纳入我们的三级护理中心;然而, 这将是未来研究的主题。从实际患者的肿瘤活检中, 技术可行性看起来很有希望。标本然后转移到实验室。活检是机械分离后, 酶消化。细胞, 然后培养在超低黏附细胞培养瓶, 促进可再生, 标准化和自发形成三维球形细胞企业集团。球体然后准备在必要时暴露在化学辐射协议和免疫治疗协议中。最终细胞生存能力和球体大小是治疗敏感性的指标, 因此可以考虑在未来评估患者的可能的治疗反应。这一模式可能是一个宝贵的, 经济高效的工具, 以个性化治疗头颈癌。

我们能够重现性产生球体从单细胞悬浮, 首先从不同的细胞系, 包括专有的异种细胞系, 后来从最初的人类癌细胞的新肿瘤活检中所描述的哈格曼et al.26. 我们评估了两种已建立的球体生成方法;两者是所谓的吊滴 (HD) 方法和超低粘附 (ULA) 方法, 后者是更有效和安全的一个。我们能够突出的优势, ULA 方法相比, HD, 包括更快的球体增长, 更少的问题, 板处?...

Watch this Scientific Journal Video about Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma at JoVE.com

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

我们描述了一个基于球体的三维体外模型的进化, 它使我们能够测试细胞系头颈部鳞状细胞癌实验治疗方案的现行标准, 旨在评估治疗未来人体标本对原代细胞的敏感性和耐药性。

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 ...

therapy-testing-spheroid-based-3d-cell-culture-model-for-head-neck

Research

JoVE Journal

Cancer Research

9.7K 次观看.  Johannes-Gutenberg University Medical Center. 我们描述了一个基于球体的三维体外模型的进化, 它使我们能够测试细胞系头颈部鳞状细胞癌实验治疗方案的现行标准, 旨在评估治疗未来人体标本对原代细胞的敏感性和耐药性。

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

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

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 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 ...

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 ...

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 ...

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

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

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

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

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

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 ...

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, ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

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 ...