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步, 稍后将化验纳入我们的三级护理中心的临床通路。今后, 它可用于 (i) 评估个别肿瘤易感性的共同第一或二线/第三线治疗协议 (化学试剂, 辐射) 和实验治疗协议, (ii) 进一步表征肿瘤细胞从新鲜标本并将分子表面或细胞质形态与治疗反应相关联。球体从来自不同肿瘤定位的标本,如例如, 原发性和淋巴结转移, 是可想而知的。
在整个协议和方法的建立过程中, 我们使用了不同的已知细胞线, 几十年来已经建立了一些。仅仅是细胞系的年龄就引起了他们对目前实际人类肿瘤细胞的连接和可比性的怀疑, 据称已经失去了几个性质和标志性分子标记。体外实验的结果很难在人体或体内试验27中重现。因此, 我们专注于最近在我们的设施中由人类主要肿瘤细胞产生的细胞线, 其特征是16。有了这个细胞线的异食癖, 我们能够进行更大规模的治疗实验, 并确认目前的治疗预期。这支持的理论, 异食癖细胞反映实际人类肿瘤行为优于其他, 更老的和传代细胞系, 接受了许多通道。由于长期的体外条件而可能接受非生理选择的细胞可能会使化疗敏感性偏倚。在最近的实验中, 我们证实了与主细胞有关的可靠球体形成的可行性;然而, 必须进行进一步和更大的实验, 才能将该化验结果实施到临床常规中。
假设球体和三维细胞培养一般与常规的 2 d-培养相比, 在接触化疗药物或辐射28时反应不同。球体的结构导致了从外表面到核心的氧气和营养的梯度, 最近的研究表明, 对三维细胞簇的不同部位的药物传递也不均匀29。此外, 球体培养中的细胞似乎保留了某些基因表达模式, 与动物模型中的耐药性有关30。我们建议, 也所谓的辐射抵抗, 这一特点已经与头颈癌的许多患者, 是由于细胞相互作用和明显的微环境导致变化的氧气供应和代谢活动。由于体内功能增加, 这种现象可能在球体中被模仿。总之, 使用主细胞和生成球体可以使我们尽可能多地保留体内肿瘤生长的可能特征, 并结合成本效益的方法研究个体治疗反应。
这一做法当然有局限性。到目前为止, 尚不清楚不同患者的细胞如何在接受肿瘤治疗的实验中表现出来, 以及如何预测该化验与后来治疗结果的相关性。必须进行过多的研究, 才能简单比较体外与临床课程的性能。其中, 两个因素导致了这种不确定性: 肿瘤的异质性和缺乏与基质和免疫细胞的共培养。肿瘤异质性是一种不完全理解的现象, 存在于主要的不同子站点之间, 以及主要和转移的定位12之间。自从最近的研究, 有一个第三个站点, 非常独特的和专门的肿瘤细胞可以存在和盛行的利基: 血液和骨髓可以住房循环和传播肿瘤细胞9,10。细胞培养中缺乏其他细胞类型如基质细胞和 T 细胞 (作为免疫系统的代表) 并不局限于化验, 而是大多数体外测定肿瘤治疗敏感性的有效缺点。鉴于最近的药物批准和正在进行的研究, 影响 T 细胞反应, 未来的药物发现实验必须结合不同的化验和方法进行。

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 &amp;micro;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&amp;micro;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 &amp;micro;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, 板处理, 随后损失的球体与沉重的振动, 和更多的可重现的球体增长的一致性 (图 2)。在 HD 方法中, 每瓶多球体的发生更常见。我们检查了一个更明显的球体形态的特征。图 3显示了 Ki67 的免疫化学染色, 它是从异食细胞中球体的增殖标记。正如文献中所描述的, 球体是一个不扩散的核心与增殖外层, 模仿不同地区的固体 HNSCC 增殖的人。体外作为体内, 这大概是由细胞培养基所提供的养分和氧气梯度引起的, 这导致从外围到肿瘤中心的减少。
然后, 我们试图表明, 该检测能够检测到球形大小的变化后, 孵化与化疗药物或暴露于电离辐射, 这里代表两个共同标准治疗方案的 HNSCC。在可能将化验纳入治疗前的例行程序之前, 验证检测的敏感性是先决条件。有两个参数可用作试验的终点: 球体大小 (面积; 步骤5.1 中的参数 1) 和扩散 (见步骤5.4 中的参数 2), 是治疗后细胞活力和生长潜能的替代标记。治疗研究的结果与共同化疗和化学辐射协议显示在图 4中。球体与顺铂或5FU 的接触和孵化导致了随着时间的推移, 与未经治疗的对照组相比 (p< 0.001) 显著降低了增长速度。2Gy 的辐射能够显著地降低生长速度 (p< 0.01)。存活率测定 (WST-8) 能够检测到与顺铂相比仅辐射 (p = 0.017) 的化疗放疗的额外的、显著的价值, 这是在对球体大小的评估中先前未被发现的差异。这强调它的重要性, 作为一个整体的化验, 增加其敏感性小变化治疗反应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57012/57012fig1.jpg"> 图 1: 对未来个性化治疗方式的模型的概念.患者有粘膜 HPV 感染的背景和/或吸烟和酗酒的历史, 目前我们的三级转诊中心的疑似诊断头颈癌。在麻醉下, 对可疑粘膜区或宏观肿瘤肿块进行活检, 以确保诊断。此外, 我们还进行肿瘤活检, 在实验室中生成三维球形细胞培养物。在基于原细胞的球体的充分生长后, 我们将其暴露于目前的标准或实验治疗方案中, 以测试个别治疗反应。在分析治疗结果后, 可以增加对遗传或分子变异的进一步分析。治疗结果体外可以考虑为病人规划治疗概念的过程。这个数字是从哈格曼, J.等等转载的。26, 并具有作者和日记本授予的权限。<a href="/files/ftp_upload/57012/57012fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57012/57012fig2.jpg"> 图 2: 所有单元格线都可以生成可靠的球体; 读取的大小和时间的差异根据图 1.在初步实验中, 主细胞没有形成可重现的球体在 HD, 但 ULA 板 (左下)。还将进行进一步的研究, 以评估主要细胞的球状生长特征。右侧的单元格数是放置在瓶子中形成球体的初始单元格数。这个数字是从哈格曼, J.等等转载的。26, 并具有作者和日记本授予的权限。<a href="/files/ftp_upload/57012/57012fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57012/57012fig3.jpg"> 图 3: Ki67 免疫化学染色球形切片 (异食癖) 。周围的文化显示比中央细胞更高的增殖率, 模仿营养分布在实体黏膜肿瘤往往显示坏死的核心。这个数字是从哈格曼, J.等等转载的。26, 并具有作者和日记本授予的权限。<a href="/files/ftp_upload/57012/57012fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57012/57012fig4.jpg"> 图 4: 治疗研究.(A)在 ULA 板中培养出了用于生成球体 (每组 n = 12) 的异位癖细胞。细胞被刺激根据协议以 PBS 作为控制, 顺铂在5µM 或5FU 在30µM. 根据标准协议, 在6、10和16天确定了球体的大小。顺铂和5FU 处理导致尺寸显著减小 (p-值在左下角给出 ( B)与 (a) (n = 12) 相同的实验设计, 但附加辐射2Gy。(C)绘图显示最小到最大值。根据 (C) 中给出的p值, 以前的2Gy 辐射导致对照组的球体大小显著下降, 模仿放射治疗, 并显示异位癖细胞的辐射敏感性。与顺铂治疗相结合的辐射并没有导致球体 (p> 0.05) 的大小进一步显著下降。图显示最小值为 max。(C)计算p-从实验 A 和 B. (D) WST8 化验的2路方差分析的值, 作为平面 (区域) 分析球体的替代品。WST8 分析发现, 化疗后的活细胞明显减少 (2Gy 辐射和顺铂), 仅与顺铂相比 (p = 0.017), 因此显示了结合平面分析和 WST8 的优势。测定时的色度测定。图显示最小到最大值。这个数字是从哈格曼, J.等等转载的。26, 并具有作者和日记本授予的权限。<a href="/files/ftp_upload/57012/57012fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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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 ...

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Nanyang Technological University

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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

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

科研

癌症研究

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

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

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

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. ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

医学

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

胶质母细胞瘤的3D球形模型

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

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

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

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

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

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

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

生成用于药物评估研究的 3D 肿瘤微球

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

功能性 3D 球状体细胞培养物的半自动表型分析

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...