这项研究得到了"化学和辐射测量标准的制定"、授予编号为KRISS-202020-0003以及"生物材料和医学融合测量标准和技术的制定"的支持，赠款编号为KRISS-2020-GP2020-0004项目，由韩国标准与科学研究所资助。这项研究还得到了韩国科学和信息通信技术部（MSIT）、韩国国家研究基金会（NRF-2019M3A9F3065868）、卫生福利部（MOHW）、韩国卫生产业发展研究所（KHIDI、HI20C0558）、贸易、工业和能源部（MOTIE）和韩国工业技术评估研究所（KEIT，20009350）的支持。ORCID ID （何敏宇： 0000-0002-5951-2137：杜金康： 0000-0002-5924-9674：西尔·金： 0000-0003-3465-7118：李俊恩： 0000-0002-2495-1439：李吉娜： 0000-0002-3661-3701）。我们感谢张伍公园对实验的协助。

1. 细菌细胞培养和 无乳酸杆菌 无细胞超高分子（LCFS）的制备
注：步骤 1.2 - 1.9 在厌氧室中执行。
<ol><li>准备一个含有 L-西斯坦的人造黄油板和肉汤，并通过自动包装消毒。</li>
	<li>在H2 厌氧室中预孵育MRS agar板，维持在37°C，含20ppm氧气。</li>
	<li>解冻乳酸杆菌库存，并接种与细菌培养的阿加板（图1A（i）） 。</li>
	<li>在H2 厌氧室中以37°C和20ppm氧孵育细菌2-3天，直到获得单一细菌菌落。</li>
	<li>清洗和干燥洪高特型有氧培养管。在 121 °C 下将培养管高压灭菌 15 分钟。</li>
	<li>然后在H2 厌氧室中孵育管子，在37°C和20ppm氧中去除氧气。</li>
	<li>将 2 - 3 mL 的夫人肉汤放入管中。用丁基橡胶塞密封管子，拧紧盖子。</li>
	<li>获得一个带环的单个菌落，并将其放入 1.5 mL 培养管中，该培养管的 500 μL 为 1x PBS。（图1A（ 二））。</li>
	<li>使用 1 mL 注射器暂停殖民地 （图 1A （iii）。这样做，将 1 mL 注射器的针头插入管盖中心，吸气悬浮的菌落，然后将其重新插入 MRS 肉汤介质中。（图1A（四））。</li>
	<li>在摇床孵化器中孵育 MRS 肉汤介质 2 天（37 °C，5% CO 2，200 rpm）。</li>
	<li>使用光谱光度计测量光学密度 （OD）以监测细菌生长曲线，直到 OD620 的吸收率达到 2.0。</li>
	<li>将细菌颗粒和受条件介质分离，以 1，000 x g 离心 15 分钟进行离心。用 1x PBS 清洗收集的细菌颗粒，在 RPMI 1640 的 4 mL 中重新吸附，并辅以 10% 的胎儿牛血清。介质中不包括任何抗生素。</li>
	<li>在 RPMI 中保持细菌颗粒，并在 37 °C 的摇床孵化器中孵育 4 小时，以 100 rpm 的速度在 5% CO2 中孵育。</li>
	<li>对于益生菌超纳特的制备，在1000 x g的离心下通过离心去除细菌颗粒，在4°C下15分钟。 使用 0.22 μm 过滤器对已恢复的超纳特进行无菌过滤，并存储在 +80 °C 下，直到使用。</li>
</ol>2. 球形的生成
<ol><li>
准备结肠直肠癌细胞系
	<ol>
<li>将 DLD-1、HT-29 和 WiDr 细胞系作为单层培养，直到 70-80% 的汇合，并在 5% CO2 孵化器中以 37 °C 孵育板（生长介质：含有 10% 胎儿牛血清 （FBS） 和 1% 青霉素-链霉素的 RPMI）。</li>
		<li>对于生长在 100 mm Petri 盘中的细胞，用 4 mL 的 1x PBS 洗盘两次。加入 1 mL 的 0.25% 试用素-EDTA，在 5% CO 2 孵化器中在 37 °C 下孵育2 分钟，以分离细胞。</li>
		<li>孵化后，在显微镜下检查细胞分离，用5兆升的生长介质中和试丁二醇。</li>
		<li>将分离的细胞转移到15mL圆锥管和离心机3分钟在300×g。 </li>
		<li>丢弃超高分子，用3兆L的生长介质轻轻重新悬浮。</li>
		<li>用锥蓝计数细胞，使用血细胞计确定可行的细胞。（图1B （一） ）</li>
	</ol>
</li>
	<li>
球形形成
	<ol>
<li>在 15 mL 圆锥管中，将细胞从 2.1.5 中稀释，以获得 1 - 2 x 105个细胞/mL （图 1B （ii）</li>
		<li>将最终浓度为0.6%的甲基纤维素加入细胞悬浮物中，并将稀释后的细胞转移到无菌储液层中。 注：对于每个细胞系，所需的甲基纤维素量应进行点缀并相应确定。</li>
		<li>使用多通道移液器将 200 μL 的细胞分配到每个超低附件 96 井圆形底部微板的油井中。（图1B （三）</li>
		<li>在 5% CO2 孵化器中将板在 37 °C 下孵育，24 - 36 小时。</li>
		<li>24 -36小时后，在光显微镜下观察板块，以确保球形形成。</li>
	</ol>
</li>
</ol>3. 用LCFS治疗3D结肠直肠癌细胞
<ol><li>生成第 2 步和第 3 步中描述的球形。</li>
	<li>在进行LCFS治疗之前，在室温下解冻冷冻LCFS10 -20分钟。</li>
	<li>将LCFS股票解决方案接种到生长介质中。在生长介质中连续稀释至 25%、12.5% 和 6% （即 25% LCFS = 150 μL 的增长介质 = 50 μL 的 LCFS）。</li>
	<li>从孵化器中取出含有球体的细胞培养板，使用 200 μL 移液器从每口井中取出尽可能多的生长介质。</li>
	<li>在细胞上加入LCFS的生长介质，在37°C的CO2 孵化器中孵育24-48小时。 注：要使用的体积将取决于板大小如下：2 mL为6井细胞培养板：200 μL 用于 96 井细胞培养板。</li>
</ol>4. 球形细胞的生存能力
<ol><li>准备 8 - 10 LCFS 治疗的结肠直肠癌球体在不透明壁多井板（细胞生存能力检测执行 48 小时后 LCFS 治疗）。</li>
	<li>将细胞生存性试剂（参见 材料表）解冻，在 4 °C 下过夜。</li>
	<li>使用前将细胞生存性试剂与室温等距。</li>
	<li>在进行检测之前，从球体中取出 50% 的生长介质。</li>
	<li>在每个井中加入 100 μL 的细胞生存性试剂。 注：要使用的体积将取决于板大小如下：100 μL 的 96 井细胞培养板。</li>
	<li>将试剂大力混合5分钟，促进细胞裂解。</li>
	<li>孵育30分钟 = 2小时在37°C。</li>
	<li>记录发光。</li>
</ol>5. 球体的定量实时聚合酶链反应分析
<ol><li>对于每个情况，准备10-15球体在2 mL管和离心机3分钟在400×g。</li>
	<li>丢弃超高纳特，在1 mL的冰冷1xPBS中两次清洗球体。 注意：避免离心，让球体安定下来。</li>
	<li>尽可能多地吸气 1x PBS，并使用市售套件隔离 RNA。</li>
	<li>根据制造商的协议，使用市售套件从 1 微克 RNA 中合成 cDNA。</li>
	<li>准备一个主组合，以三重运行所有样品（参见表1和表2）。</li>
	<li>在模板主组合的 20 μL 中将放大到每个 qPCR 板中。</li>
	<li>混合反应良好，如有必要，旋转。</li>
	<li>根据仪器制造商的建议运行样品（表3）。</li>
</ol>6. 球体的西式印迹
注：在收集球体时，使用 200 μL 移液器并切割尖端，以避免干扰其结构。
<ol><li>对于每个条件，准备30 - 40球体在2mL管。</li>
	<li>将管子放在冰上，让球体沉降到2mL管的底部。</li>
	<li>丢弃超纳特，在 1 mL 冰冷 1x PBS 中两次清洗球体 注意：避免离心，让球体安定下来。</li>
	<li>尽可能多地吸气 1x PBS，并添加 RIPA 缓冲器与蛋白酶抑制剂鸡尾酒 （10 球体 = 30 μL RIPA 缓冲器）.</li>
	<li>通过上下管道来对细胞进行解剖，并进行30s的声波，30s在冰上休息10圈。</li>
	<li>将蛋白质以 15000 x g 为离心，在 4 °C 下 15 分钟。</li>
	<li>确定每个细胞解解剂的蛋白质浓度。</li>
	<li>在加载之前，将每个细胞在 100 °C 的样品缓冲中沸腾 10 分钟。</li>
	<li>将等量的蛋白质装载到 SDS-PAGE 凝胶的井中，并在 100 V 下将凝胶运行 1 - 2 小时。</li>
	<li>将蛋白质从凝胶转移到PVDF膜。</li>
	<li>转移后，使用阻塞缓冲器（5% 脱脂牛奶 + TBS，0.05% Tween-20）在室温下将膜阻隔 1 小时。</li>
	<li>在 1x TBST 中以 4 °C 的 5% BSA 缓冲器在 1x TBST 中用 1：1，000 稀释原发抗体 （表 4）来孵育膜。</li>
	<li>用结节水三次清洗膜，每次洗15分钟。</li>
	<li>在室温下在室温下在阻隔缓冲器中用1：2，500稀释二次抗体的膜孵化2小时（见 材料表）。</li>
	<li>用结节水三次清洗膜，每次洗15分钟。</li>
	<li>用化学发光基板为 HRP 检测准备膜。</li>
	<li>获取化学图像。</li>
</ol>7. 球体的碘化物（PI）染色
<ol><li>准备5-10球体，如第4.1步所述，并将球体放在37°C和5%CO2的孵化器中。</li>
	<li>在 1x PBS 中稀释 PI 1：100 的 1 毫克/mL 库存。</li>
	<li>从 96 井板的每口井中取出 50% 的介质。</li>
	<li>将 100 μL 的 PI 溶液添加到每口油井中，并将油井置于 37 °C 和 5% CO2 的孵化器中，10 - 15 分钟。</li>
	<li>用 1x PBS 洗掉 PI 溶液。</li>
	<li>加入200微升的生长介质，使用荧光显微镜拍摄图像。使用图像 J 分析荧光强度，以获得球体的生存能力计数。</li>
</ol>8. 球形的FACS分析
<ol><li>生成如前所述的球形。</li>
	<li>对于每个条件，准备30 - 40球体在FACS管和离心机3分钟在400 x g 和RT。</li>
	<li>吸气超纳特，在 1x PBS 的 3 mL 中清洗球体，然后以 400 x g离心机在 4 °C 下 3 分钟洗涤球体。</li>
	<li>吸气超纳坦，并添加200微升0.25%的Trypsin-EDTA，然后在RT孵育2-3分钟。 注：孵化时间取决于球体大小和细胞类型。</li>
	<li>添加 1 mL 的 FACS 缓冲器，并使用 200 μL 移液器轻轻分离球体。</li>
	<li>将分离的细胞在 400 x g和 4 °C 下离心 3 分钟。 注： FACS 缓冲器 = 1x PBS = 2.5% FBS，使用 0.22 μm 顶部过滤器进行过滤。</li>
	<li>丢弃超高纳特，加入 7-AAD/附件 V 试剂 （7AAD （5 μL）、附件 V （5 μL）/样品） 。</li>
	<li>轻轻地旋转细胞，在黑暗中的RT孵育13-30分钟。</li>
	<li>加入 500 μL 的 FACS 缓冲器，使用锥形聚苯乙烯试管过滤细胞，以去除聚合细胞。</li>
	<li>离心机在 400 x g和 4 °C 为 3 分钟。</li>
	<li>在每个管子中加入 500 μL 的附件 V 绑定缓冲器并重新悬浮。</li>
	<li>使用流细胞仪进行分析。</li>
</ol>

<ol>
	<li>Bron, P. A., Van Baarlen, P., Kleerebezem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+molecular+insights+into+the+interaction+between+probiotics+and+the+host+intestinal+mucosa.">Emerging molecular insights into the interaction between probiotics and the host intestinal mucosa.</a> Nature Reviews Microbiology. 10 (1), 66-78 (2012).</li><li>Ruiz, L., Delgado, S., Ruas-Madiedo, P., S&#225;nchez, B., Margolles, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifidobacteria+and+their+molecular+communication+with+the+immune+system.">Bifidobacteria and their molecular communication with the immune system.</a> Frontiers in Microbiology. 8, 1-9 (2017).</li><li>Sanders, M. E., Merenstein, D. J., Reid, G., Gibson, G. R., Rastall, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics+and+prebiotics+in+intestinal+health+and+disease:+from+biology+to+the+clinic.">Probiotics and prebiotics in intestinal health and disease: from biology to the clinic.</a> Nature Reviews Gastroenterology and Hepatology. 16 (10), 605-616 (2019).</li><li>Pandey, K. R., Naik, S. R., Vakil, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics,+prebiotics+and+synbiotics-+a+review.">Probiotics, prebiotics and synbiotics- a review.</a> Journal of Food Science and Technology. 52 (12), 7577-7587 (2015).</li><li>Harish, K., Varghese, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics+in+humans-evidence+based+review.">Probiotics in humans-evidence based review.</a> Calicut Medical Journal. 4 (4), 3 (2006).</li><li>Routy, 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=The+gut+microbiota+influences+anticancer+immunosurveillance+and+general+health.">The gut microbiota influences anticancer immunosurveillance and general health.</a> Nature Reviews Clinical Oncology. 15 (6), 382-396 (2018).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+of+the+cell-based+data-harvesting+efforts+that+drive+the+integration+of+cell+biology.">Most of the cell-based data-harvesting efforts that drive the integration of cell biology.</a> Nature Reviews Molecular Cell Biology. 8 (10), 839-845 (2007).</li><li>Shamir, E. R., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organotypic+culture:+Experimental+models+of+mammalian+biology+and+disease.">Three-dimensional organotypic culture: Experimental models of mammalian biology and disease.</a> Nature Reviews Molecular Cell Biology. 15 (10), 647-664 (2014).</li><li>Jong, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+culture+models+in+cancer+biology.">Three-dimensional tissue culture models in cancer biology.</a> Seminars in Cancer Biology. 15 (5), 365-377 (2005).</li><li>Zanoni, 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=3D+tumor+spheroid+models+for+in+vitro+therapeutic+screening:+a+systematic+approach+to+enhance+the+biological+relevance+of+data+obtained.">3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained.</a> Scientific Reports. 6, 19103 (2016).</li><li>Anton, D., Burckel, H., Josset, E., Noel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+A+breakthrough+in+vivo.">Three-dimensional cell culture: A breakthrough in vivo.</a> International Journal of Molecular Sciences. 16 (3), 5517-5527 (2015).</li><li>Lee, J. E., 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=Characterization+of+the+Anti-Cancer+Activity+of+the+Probiotic+Bacterium+Lactobacillus+fermentum+Using+2D+vs.+3D+Culture+in+Colorectal+Cancer+Cells.">Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus fermentum Using 2D vs. 3D Culture in Colorectal Cancer Cells.</a> Biomolecules. 9 (10), 557 (2019).</li><li>Koledova, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+cell+culture:+An+introduction.">3D cell culture: An introduction.</a> Methods in Molecular Biology. 1612, (2017).</li><li>Kapa&#322;czy&#324;ska, 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=2D+and+3D+cell+cultures+-+a+comparison+of+different+types+of+cancer+cell+cultures.">2D and 3D cell cultures - a comparison of different types of cancer cell cultures.</a> Archives of Medical Science. 14 (4), 910-919 (2018).</li><li>Langhans, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+cell+culture+models+in+drug+discovery+and+drug+repositioning.">Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.</a> Frontiers in Pharmacology. 9, 1-14 (2018).</li><li>Breslin, S., O'Driscoll, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+The+missing+link+in+drug+discovery.">Three-dimensional cell culture: The missing link in drug discovery.</a> Drug Discovery Today. 18 (5-6), 240-249 (2013).</li><li>Mazzocchi, A. R., Rajan, S. A. P., Votanopoulos, K. I., Hall, A. R., Skardal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+patient-derived+3D+mesothelioma+tumor+organoids+facilitate+patient-centric+therapeutic+screening.">In vitro patient-derived 3D mesothelioma tumor organoids facilitate patient-centric therapeutic screening.</a> Scientific Reports. 8, 2886 (2018).</li><li>Lv, D., Hu, Z., Lu, L., Lu, H., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+A+powerful+tool+in+tumor+research+and+drug+discovery.">Three-dimensional cell culture: A powerful tool in tumor research and drug discovery.</a> Oncology Letters. 14 (6), 6999-7010 (2017).</li><li>Thirumala, S., Gimble, J., Devireddy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+Based+Thermally+Reversible+Hydrogel+System+for+Tissue+Engineering+Applications.">Methylcellulose Based Thermally Reversible Hydrogel System for Tissue Engineering Applications.</a> Cells. 2 (3), 460-475 (2013).</li><li>Chandrashekran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+as+a+scaffold+in+the+culture+of+liver-organoids+for+the+potential+of+treating+acute+liver+failure.">Methylcellulose as a scaffold in the culture of liver-organoids for the potential of treating acute liver failure.</a> Cell and Gene Therapy Insights. 4 (11), 1087-1103 (2018).</li><li>Lee, W., Park, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+patterned+stem+cell+differentiation+using+thermo-responsive+methylcellulose+hydrogel+molds.">3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds.</a> Scientific Reports. 6, 1-11 (2016).</li><li>Fan, H., Demirci, U., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+organoid+models:+Leaping+forward+in+cancer+research.">Emerging organoid models: Leaping forward in cancer research.</a> Journal of Hematology and Oncology. 12 (1), 1-10 (2019).</li><li>Drost, J., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+in+cancer+research.">Organoids in cancer research.</a> Nature Reviews Cancer. 18 (7), 407-418 (2018).</li><li>Liou, C. 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=A+Metabolic+Pathway+for+Activation+of+Dietary+Glucosinolates+by+a+Human+Gut+Symbiont.">A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont.</a> Cell. 180 (4), 717-728 (2020).</li><li>Sherwin, E., Bordenstein, S. R., Quinn, J. L., Dinan, T. G., Cryan, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+and+the+social+brain.">Microbiota and the social brain.</a> Science. 366 (6465), 2016 (2019).</li><li>Honda, K., Littman, D. 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+microbiota+in+adaptive+immune+homeostasis+and+disease.">The microbiota in adaptive immune homeostasis and disease.</a> Nature. 535 (7610), 75-84 (2016).</li><li>B&#225;rcena, 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=Healthspan+and+lifespan+extension+by+fecal+microbiota+transplantation+into+progeroid+mice.">Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice.</a> Nature Medicine. 25 (8), 1234-1242 (2019).</li><li>Michalovich, 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=Obesity+and+disease+severity+magnify+disturbed+microbiome-immune+interactions+in+asthma+patients.">Obesity and disease severity magnify disturbed microbiome-immune interactions in asthma patients.</a> Nature Communications. 10, 5711 (2019).</li><li>Ansaldo, E., 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=Akkermansia+muciniphila+induces+intestinal+adaptive+immune+responses+during+homeostasis.">Akkermansia muciniphila induces intestinal adaptive immune responses during homeostasis.</a> Science. 364 (6446), 1179-1184 (2019).</li></ol>

作者没有相关的财务披露。

组织微环境，包括相邻细胞和细胞外基质（ECM），是组织生成的基础，在控制细胞生长和组织发育方面至关重要。然而，2D培养物有几个缺点，如细胞相互作用的中断，以及细胞形态的改变，细胞外环境，以及第14分区的方法。3D细胞培养系统经过严格研究，以更好地在体内繁殖效果，并已被证明是更精确的体外癌症检测系统15，16。<sup class="xre...

益生菌是肠道中最有利的微生物，可改善免疫平衡和宿主能量代谢1。乳酸杆菌和双歧杆菌的益生菌是肠2、3中同类中最先进的。先前的研究表明，乳酸杆菌对包括结肠直肠癌在内的几种癌症有抑制和反促进作用。此外，益生菌可预防炎症性肠病、克罗恩病和溃疡性结肠炎5、6。然而，大多数益生菌研究都是在生长在固体表面的二维（2D）单层体中进行的。
人工培养系统缺乏环境特征，这对癌细胞来说是不自然的。为了克服这一局限性，开发了3维（3D）文化系统7、8。3D中的癌细胞在基本生物机制方面表现出改善，如细胞生存能力、增殖、形态、细胞细胞通信、药物敏感性和体内相关性9、10。此外，球体由多细胞类型的结肠直肠癌制成，并依赖于细胞-细胞相互作用和细胞外基质（ECM）11。我们先前的研究已经报告说，使用乳酸杆菌发酵产生的益生菌无细胞超纳坦（CFS）对结肠直肠癌（CRC）细胞12的3D培养物具有抗癌作用。我们建议CFS是测试3D球体12的益生菌效应的合适替代策略。
在这里，我们提出了一种方法，可以容纳多细胞类型的3D结肠直肠癌，用于分析益生菌无细胞超高分子（CFS）对几个3D结肠直肠癌模仿系统的治疗效果。这种方法为体外分析相关的益生菌和抗癌效果提供了手段。

<table>
	<tbody>
		<tr>
			<td> 
			10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 &#181;l</td>
			<td>Biorad</td>
			<td>4561036</td>
			<td>Pkg of 10</td>
		</tr>
		<tr>
			<td>Applied Biosystems MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>100 covers</td>
		</tr>
		<tr>
			<td>10x transfer buffer</td>
			<td>Intron</td>
			<td>IBS-BT031A</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>10X Tris-Glycine (W/SDS)</td>
			<td>Intron</td>
			<td>IBS-BT014</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case</td>
			<td>Corning</td>
			<td>SCT-200-C</td>
			<td>500 Tubes/Pack, 10 Packs/Case</td>
		</tr>
		<tr>
			<td>BD Difco Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>BD Difco Lactobacilli MRS Broth</td>
			<td>BD</td>
			<td>DF0881-17-5</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell viability assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td>100&#956;l/assay in 96-well plates</td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
			<td>vial of 20 tablets</td>
		</tr>
		<tr>
			<td>Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 &#177; 0.1</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>EMD Millipore Immobilon-P PVDF Transfer Membranes</td>
			<td>fisher Scientific</td>
			<td>IPVH00010</td>
			<td>26.5cm x 3.75m roll; Pore Size: 0.45um</td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>25/Pack, 500/Case</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, US origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit, 25 x 20 &#181;l rxns #1708890</td>
			<td>Biorad</td>
			<td>1708890</td>
			<td>25 x 20 &#181;L rxns</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Biorad</td>
			<td>1725121</td>
			<td>5 x 1 mL</td>
		</tr>
		<tr>
			<td>Lactobacillus fermentum</td>
			<td>Korean Collection for Type Cultures</td>
			<td>KCTC 3112</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C6852-25G</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Methyl Cellulose (3500-5600mPa&#183;s, 2% in Water at 20&#176;C)</td>
			<td>TCI</td>
			<td>M0185</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td>20 plates</td>
		</tr>
		<tr>
			<td>Millex-GS Syringe Filter Unit, 0.22 &#181;m, mixed cellulose esters, 33 mm, ethylene oxide sterilized</td>
			<td>Millipore</td>
			<td>SLGS033SB</td>
			<td>250</td>
		</tr>
		<tr>
			<td>PE Annexin V Apoptosis Detection Kit with 7-AAD</td>
			<td>Biolegend</td>
			<td>640934</td>
			<td>100 tests</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Introgen</td>
			<td>P1304MP</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>89901</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit (250)</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td>250</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Name of Materials/Equipment/Software</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>anti - p-I&#954;B&#945; (B-9)</td>
			<td>Santa cruze</td>
			<td>sc-8404</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-BclxL (H-5)</td>
			<td>Santa cruze</td>
			<td>sc-8392</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-PARP 1 (C2-10)</td>
			<td>Santa cruze</td>
			<td>sc-53643</td>
			<td>50 &#181;l ascites</td>
		</tr>
		<tr>
			<td>anti-&#946;-actin (C4)</td>
			<td>Santa cruze</td>
			<td>sc-47778</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>BD FACSVerse</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>Synergy HTX Multi-Mode Microplate Reader</td>
			<td>BioT</td>
			<td>S1LFA</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo fisher</td>
			<td>HERAcell 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 15 ml</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 50 ml</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile</td>
			<td>Costar</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermofisher</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EnSpire Multimode Reader</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Enspire 2300</td>
		</tr>
		<tr>
			<td>Eppendorf Research Plus Multi Channel Pipette, 8-channel</td>
			<td>Eppendorf</td>
			<td>3122000051</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>TreeStar</td>
			<td></td>
			<td>Ashland, OR, USA</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>115-035-062</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>111-035-144</td>
			<td>2.0 mL</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Inc., San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000 mini</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Incubated shaker</td>
			<td>Lab companion</td>
			<td>SIF-6000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi Gauge Ver. 3.0,</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Optical density (OD)LAMBDA UV/Vis Spectrophotometers</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Phase-contrast microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>SPL microcentrifuge tube 1.5mL</td>
			<td>SPL</td>
			<td>60015</td>
			<td></td>
		</tr>
		<tr>
			<td>SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile</td>
			<td>SPL</td>
			<td>21012</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Vibra-Cell Ultrasonic Liquid Processors</td>
			<td>SONICS-vibra cell</td>
			<td>VC 505</td>
			<td>500 Watt ultrasonic processor</td>
		</tr>
		<tr>
			<td>Vinyl Anaerobic Chamber</td>
			<td>COY LAB PRODUCTS</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating cell death using cell-free supernatant of probiotics in three-dimensional spheroid cultures of colorectal cancer cells

这份手稿描述了一个协议，评估癌细胞死亡的三维（3D）球形多细胞类型的癌细胞使用超分子从 乳酸杆菌发酵 细胞培养， 被认为是益生菌培养物。使用 3D 培养物来测试 乳酸杆菌 无细胞超高分子 （LCFS） 比在 2D 单层测试更好，特别是因为 L. 发酵 可以在肠道内产生抗癌效果。 L. 发酵 超高分子被鉴定在3D培养条件下对几种结肠直肠癌（CRC）细胞具有增加的抗增殖作用。有趣的是，这些效应与培养模型密切相关，显示了 L.发酵 诱导癌细胞死亡的显著能力。稳定的球体来自不同的CRC（结肠直肠癌细胞），使用下面提出的协议。生成 3D 球体的这种协议节省时间且具有成本效益。该系统的开发是为了轻松研究LCFS在多种CRC球体中的抗癌作用。不出所料，在实验中用LCFS治疗的CRC球体强烈诱导细胞死亡，并表达由qRT-PCR、西侧印迹和FACS分析分析的特定凋亡分子标记。因此，该方法对于探索细胞生存能力和评估抗癌药物的疗效具有重要的价值。

我们描述了从不同的结肠直肠癌细胞系中获取球状体的程序。需要补充甲基纤维素才能产生球状体。我们还介绍了LCFS制备方法，并提出了一个模型来研究益生菌与结肠直肠癌之间的相关性。球形形成和LCFS制备协议在图1A，B中示意图说明。如图2A所示，甲基纤维素浓度为0.6%，将癌细胞转化为紧凑的球体。这一结果表明，球状体可以通过使用...

Watch this Scientific Journal Video about Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells at JoVE.com

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

这里介绍了了解 乳酸杆菌 无细胞超高分子（LCFS）的抗癌作用的方法。结肠直肠癌细胞系显示在3D培养物中用LCFS治疗时细胞死亡。生成球体的过程可以根据脚手架进行优化，并且所呈现的分析方法可用于评估所涉及的信号通路。

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

evaluating-cell-death-using-cell-free-supernatant-probiotics-three

Research

JoVE Journal

Cancer Research

5.4K 次观看.  Korea Research Institute of Standards and Science (KRISS). 这里介绍了了解 乳酸杆菌 无细胞超高分子（LCFS）的抗癌作用的方法。结肠直肠癌细胞系显示在3D培养物中用LCFS治疗时细胞死亡。生成球体的过程可以根据脚手架进行优化，并且所呈现的分析方法可用于评估所涉及的信号通路。

视频: Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

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

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

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

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

向上移动: 一个简单而灵活的体外三维入侵检测协议

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

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

三维流状入侵检测中的全人肿瘤基基质

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

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. ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

从3维球体培养中分离出类似干细胞的细胞

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

癌症患者血浆样本中无细胞DNA检测

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

研究结肠癌干细胞的球体三维模型

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

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 ...