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>

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</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。模型系统需要更准确地预测对化疗的个性化反应。
3D脚手架是为组织工程开发的。它作为ECM的代孕损失，代表肿瘤细胞的可用空间。此外，脚手架为细胞粘附和增殖提供物理相互作用，并导致细胞形成适当的空间分布和细胞-ECM或细胞-细胞相互作用18。甲基纤维素（MC）聚合物经过不断研究，以确定其是否适合在3D细胞培养工程19、20中应用基于MC的水凝胶系统。然而，这些水凝胶完好无损地融入生物材料，如3D细胞网络，在技术上仍然具有挑战性。因此，此处提出的球形形成协议建议将 MC 浓度的点状与每个 CRC 细胞线的不同时间点进行优化。细胞系特异性特征，如细胞聚集、生存能力和死亡，可以显著影响我们测试的每一种情况。这种方法可以提供一种产生均匀球体的方法，用于在癌细胞上测试LCFS。
益生菌是有益细菌，产生活性代谢物，可能模仿抗癌效果。因此，我们的研究旨在分离乳酸细菌（LAB），并测试其代谢提取物从无细胞超分子（CFS）的抗癌效果。我们的研究提供了一种方法来观察L.发酵无细胞超纳特的影响，这种超分子在3D系统中诱导结肠直肠癌细胞凋亡。在 LCFS 暴露在 3D 条件下后，参与凋亡通路的凋亡标记物的 mRNA 水平会显著诱导。此外，与图4 C、D、E的控制相比，LCFS处理的3D球体控制中PARP1和BCL-XL水平有所下降。NF-éB 活化抑制也是在 LCFS 治疗后在 3D 培养物中观察到的。综合起来，在3D中培养细胞的优势包括增加细胞细胞相互作用和对信号分子的反应，以更好地模仿体内系统。使用球体的西式印迹可以导致对各种信号分子状态的定量洞察。
细胞系作为癌症研究的临床前模型具有一定的局限性。最近，癌症器官被用于个性化抗癌疗法22，23的建模。LCFS在器官中使用益生菌的治疗有望被用作测试抗癌效果的强大平台。此外，我们只在癌症模型中的各种益生菌中测试了一种乳酸杆菌。各种益生菌也正在测试预防代谢综合征，免疫学和神经系统疾病24，25，26。来自双歧杆菌、糖核酸菌、链球菌、肠球菌和阿克曼西亚物种的LCFS是通过各种疾病模型27、28、29测试健康益处的潜在候选者。
基于这项研究，可以得出结论，了解球体中的信号和对3D模型LCFS治疗的各种反应可能有助于使用我们建议的方法测试抗癌效果。此外，3D 癌症模型可以提供传统 2D 单层机型无法实现的几个优势。

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>&lt;br/&gt; 10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 &amp;micro;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&amp;mu;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 &amp;plusmn; 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 &amp;micro;l rxns #1708890</td><td>Biorad</td><td>1708890</td><td>25 x 20 &amp;micro;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&amp;middot;s, 2% in Water at 20&amp;deg;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 &amp;micro;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>&lt;strong&gt;Name of Materials/Equipment/Software&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>anti - p-I&amp;kappa;B&amp;alpha; (B-9)</td><td>Santa cruze</td><td>sc-8404</td><td>200 &amp;micro;g/mL</td></tr><tr><td>anti-BclxL (H-5)</td><td>Santa cruze</td><td>sc-8392</td><td>200 &amp;micro;g/mL</td></tr><tr><td>anti-PARP 1 (C2-10)</td><td>Santa cruze</td><td>sc-53643</td><td>50 &amp;micro;l ascites</td></tr><tr><td>anti-&amp;beta;-actin (C4)</td><td>Santa cruze</td><td>sc-47778</td><td>200 &amp;micro;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我们的甲基纤维素协议从几种类型的结肠直肠癌中产生。接下来，用25%的LCFS治疗球体，48小时后使用光显微镜研究形态。如图3A所示，使用LCFS治疗的群体的球体表面被破坏。为了以适当的浓度研究LCFS的抗癌效果，用LCFS的各种剂量治疗了48小时:0（对照）、6%、12.5%和25%。在用25%LCFS治疗的球形中观察到球形形态的中断，如图3B所示。此外，球体在24小时和48小时用25%的LCFS治疗，在48小时治疗后观察到球体形态的中断。球形的微观图像显示在图3C中。
48小时后，用细胞生存能力检测对样本进行评估，用LCFS以剂量依赖的方式观察结肠直肠癌细胞死亡，观察到的细胞死亡（图3D）的LCFS含量更高。然后，我们用碘化物（PI）染色样品，以观察凋亡。不出所料，凋亡的诱导取决于LCFS剂量（图4A、B、C）。RT-PCR是为了检测凋亡的分子标记的变化，即BAX，BAK和NOXA（图5A，B）。最后，通过FACS（图6A、B、C、D）使用西式印迹和附件V/7AAD对凋亡标记进行了研究。这些观察表明，LCFS有效地诱导了3D模型中的凋亡。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61285/61285fig01.jpg"> 图1:球形形成和LCFS制备的示意图表示。 （A） LCFS 生成协议的示意图表示图像标记为 （i-iv）（B）甲基纤维素介质介质球体形成的示意图标记为 （i-iii）。 <a href="https://www.jove.com/files/ftp_upload/61285/61285fig01large.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/61285/61285fig02.jpg"> 图2:甲基纤维素介质球体形成。 （A） HT-29、DLD1 和 WiDr 的甲基纤维素介质球体形成的代表图像。细胞被播种在超低附件96井圆底板与甲基纤维素浓度0.1-1.2%为48小时。缩放条 10 μm（每个实验 3 个）。 <a href="https://www.jove.com/files/ftp_upload/61285/61285fig02large.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/61285/61285fig03.jpg"> 图3:LCFS浓度和球形形态评估。 （A） HT-29 的代表图像，用 LCFS 处理 48 小时。比例尺 100 μm。（B） HT-29， DLD1 和 WiDr 球体治疗与增加剂量的 LCFS 48 小时.所有球体都以 12.5- 25% LCFS 的速度中断了边缘。规模条 20 μm （每个实验 n=3）（C）HT-29、DLD1 和 WiDr 球体的球形形态，在 24 小时和 48 小时使用 25% LCFS 处理。比例杆 20 μm （n = 3）（D）测量细胞生存能力，显示为平均± SEM. ***，P < 0.05（n = 3 每个实验）。 <a href="https://www.jove.com/files/ftp_upload/61285/61285fig03large.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/61285/61285fig04.jpg"> 图4:球体的碘化物污渍。 在（A） HT-29（B） DLD1 和（C） 48 小时 LCFS 治疗后的 PI 染色代表图像。图像是使用荧光显微镜获得的，PI 强度的增加是使用图像 J. 刻度条 10 μm 测量的。显示 SEM ±平均值。， P < 0.05 （n = 每个实验 3） 。 <a href="https://www.jove.com/files/ftp_upload/61285/61285fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61285/61285fig05.jpg"> 图5:使用 qRT-PCR 识别凋亡标记。 对百色、BAK 和 NOXA 等凋亡标记进行了量化。mRNA 定量表示为（A） β-actin 和（B） 18s rRNA 的相对表达。显示 SEM ±平均值。，P < 0.05 （每个实验 n=3）。 <a href="https://www.jove.com/files/ftp_upload/61285/61285fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61285/61285fig06.jpg"> 图6:通过西式印迹和FACS对球体的分析确定凋亡标记。 图中显示的是LCFS治疗后的（A）HT-29、（B） DLD1和（C）WiDr细胞的西式污点。检测到了 PARP1、BCL-XL 和 p-IéB+ 。β作用素被用作内部控制。（D） FACS 分析 HT-29、DLD1 和 LCFS 孵育的 WiDr 球体中的凋亡。附件素V-FITC荧光强度的增加检测出凋细胞。<a href="https://www.jove.com/files/ftp_upload/61285/61285fig06large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>反应</td>
			<td>每单 20 μL 的音量</td>
		</tr>
<tr>
<td>2X qPCR 组合</td>
			<td>10 微升</td>
		</tr>
<tr>
<td>前进引物 （10 pmols/μL）</td>
			<td>1 微升</td>
		</tr>
<tr>
<td>反向引物 （10 pmols/μL）</td>
			<td>1 微升</td>
		</tr>
<tr>
<td>cDNA （50 ng/μL）</td>
			<td>1 微升</td>
		</tr>
<tr>
<td>PCR 级水</td>
			<td>7 μL</td>
		</tr>
</tbody></table>表1:PCR反应混合物。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>底漆</td>
			<td>序列</td>
		</tr>
<tr>
<td>BAX 向前</td>
			<td>切加加特克特克特克加格</td>
		</tr>
<tr>
<td>BAX 反向</td>
			<td>卡奇卡特加特克特加特加特</td>
		</tr>
<tr>
<td>烘焙前进</td>
			<td>阿特克塔克塔克特克察</td>
		</tr>
<tr>
<td>烘焙反向</td>
			<td>特卡塔格特克特加特格特克</td>
		</tr>
<tr>
<td>NOXA-转发</td>
			<td>阿卡格特加特格加特</td>
		</tr>
<tr>
<td>诺萨反向</td>
			<td>阿克特克塔克特克特克特克特克格</td>
		</tr>
<tr>
<td>18s rRNA-转发</td>
			<td>加特格格格加塔格</td>
		</tr>
<tr>
<td>18s rRNA 反向</td>
			<td>格格特加特塔塔塔塔特</td>
		</tr>
<tr>
<td>β-行动-前进</td>
			<td>特克特格特克卡特卡卡特卡卡特</td>
		</tr>
<tr>
<td>β-行动-反向</td>
			<td>加格卡特格特加特</td>
		</tr>
</tbody></table>表2:qRT-PCR 分析中使用的引物序列。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>阶段</td>
			<td>温度 （oC）</td>
			<td>时间</td>
		</tr>
<tr>
<td>初始变性</td>
			<td>95</td>
			<td>10 分钟</td>
		</tr>
<tr>
<td colspan="3">40 周期:</td>
		</tr>
<tr>
<td>第 1 步</td>
			<td>95</td>
			<td>15 秒</td>
		</tr>
<tr>
<td>第 2 步</td>
			<td>60</td>
			<td>60秒</td>
		</tr>
<tr>
<td rowspan="3">熔化曲线阶段</td>
			<td>95</td>
			<td>15 秒</td>
		</tr>
<tr>
<td>60</td>
			<td>60秒</td>
		</tr>
<tr>
<td>95</td>
			<td>15 秒</td>
		</tr>
</tbody></table>表3:qRT-PCR条件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>抗体</td>
			<td>稀释</td>
		</tr>
<tr>
<td>PARP 1 （C2-10）</td>
			<td>1:1000</td>
		</tr>
<tr>
<td>BCL-XL （H-5）</td>
			<td>1:1000</td>
		</tr>
<tr>
<td>p-伊布埃 （B-9）</td>
			<td>1:1000</td>
		</tr>
<tr>
<td>β-行动素 （C4）</td>
			<td>1:1000</td>
		</tr>
<tr>
<td>山羊抗鼠标IgG （H+L）</td>
			<td>1:2500</td>
		</tr>
<tr>
<td>山羊反兔伊格 （H+L）</td>
			<td>1:2500</td>
		</tr>
</tbody></table>表4:用于西方斑点分析的抗体。

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

Universidad Científica del Sur

Research

JoVE Journal

Cancer Research

6.5K 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

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 ...

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. ...

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

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

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

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 ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

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 ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

量化响应细胞毒性扰动巴根的人结肠样细胞中的细胞死亡

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-&#945; and IFN-&#947;, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

作者聚焦:使用人类类器官了解细胞因子诱导的肠上皮细胞和癌细胞死亡

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

使用实时荧光显微镜定量人结肠类器官中细胞因子诱导的细胞死亡

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ...

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

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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

一种用汽车 t 细胞进行的切杀检测

评估癌细胞3D球形培养中的细胞活力和死亡

监测三D培养中的癌细胞入侵和T细胞毒性

研究结肠癌干细胞的球体三维模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

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

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

使用实时荧光显微镜定量人结肠类器官中细胞因子诱导的细胞死亡

使用实时荧光显微镜定量人结肠类器官中细胞因子诱导的细胞死亡

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

摘要

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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

一种用汽车 t 细胞进行的切杀检测

评估癌细胞3D球形培养中的细胞活力和死亡

监测三D培养中的癌细胞入侵和T细胞毒性

研究结肠癌干细胞的球体三维模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

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

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

使用实时荧光显微镜定量人结肠类器官中细胞因子诱导的细胞死亡

使用实时荧光显微镜定量人结肠类器官中细胞因子诱导的细胞死亡

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型