细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测
1School of Life Sciences,The Chinese University of Hong Kong, 2State Key Laboratory of Agrobiotechnology,The Chinese University of Hong Kong, 3Key Laboratory for Regenerative Medicine, Ministry of Education,The Chinese University of Hong Kong, 4Centre for Novel Biomaterials,The Chinese University of Hong Kong
Summary
在这里, 我们提出了一个方案, 以分离凋亡乳腺癌细胞通过荧光激活细胞分类, 并进一步检测转变乳腺癌非干细胞到乳腺癌干细胞样细胞后, 细胞凋亡逆转流式细胞术。
Abstract
癌症复发长期以来一直由肿瘤学家进行研究, 而其潜在机制尚不清楚。最近, 我们和其他人发现, 一种叫做细胞凋亡逆转的现象会导致不同刺激下各种细胞模型的致瘤性增加。以前的研究重点是在体外和体内跟踪这一过程;然而, 真正的逆转细胞的分离尚未实现, 这限制了我们对细胞凋亡逆转后果的理解。在这里, 我们利用一种里海—-绿色检测染料, 在凋亡诱导后, 用活化的酪蛋白标记细胞。具有正信号的细胞通过荧光激活细胞分选 (facs) 进一步进行排序, 以进行恢复。共聚焦显微镜下的形态学检查有助于证实流式细胞仪前的凋亡状态。致瘤性的增加通常可归因于癌症干细胞 (csc) 样细胞的百分比升高。此外, 鉴于乳腺癌的异质性, 确定这些 cscc 样细胞的来源对癌症治疗至关重要。因此, 我们准备乳房非干细胞之前, 触发细胞凋亡, 分离卡斯帕塞激活细胞, 并执行细胞凋亡逆转程序。流式细胞术分析显示, 乳腺 csc 样细胞在逆转组重新出现, 表明在细胞凋亡逆转过程中, 乳腺 csc 样细胞从乳腺癌非干细胞中转移。总之, 该协议包括分离凋亡乳腺癌细胞和检测 csc 百分比的变化, 通过流式细胞术。
Introduction
癌症一直是导致死亡的主要原因, 给世界各国造成沉重负担1。乳腺癌在所有类型癌症1的女性患者中的发病率和死亡率都名列前茅。由于癌症的异质性, 药物的组合通常用于化疗, 以实现癌细胞死亡2,3,4。然而, 由于常见的化疗药物通常以 dna5,6、蛋白质合成7,8和微管动力学9 为目标,快速生长的细胞受影响最大。肿瘤干细胞等静止细胞受影响通常较小.因此, csc 在治疗后更有可能存活, 这后来导致耐药和癌症复发1 0、1 1.因此, 消除 csc 已成为癌症治疗的一个重要课题, 研究 csc 的起源是必要的。
近十年来, 对细胞凋亡逆转现象进行了更多的研究:12、13、14、15、16、17、18,19. 在这一概念出现之前, 人们普遍认为, 细胞在酪蛋白酶激活后将不可逆转地发生细胞凋亡。酪蛋白是一种蛋白质酶家族, 在细胞凋亡的启动和执行阶段发挥着关键作用, 包括凋亡复合体的形成和下游底物20的裂解。激活行刑细胞的 caspassase, 如酪蛋白酶3或酪蛋白酶7已被认为是细胞凋亡的“不归点” 21。然而, 研究人员最近观察到, 细胞凋亡逆转在体外和体内都会发生,在此过程中, 即使在 caspase 激活 12,13, 14 之后, 细胞也能从细胞凋亡中恢复,15,16,17,18,19. 此外, 在逆转的癌细胞15中检测到了对原始凋亡诱导剂的更高抵抗力和更高的侵入性等攻击性特征。因此, 有人提出, csc 样细胞的百分比将高于未治疗的细胞, 最终有助于更恶性的特征后, 细胞凋亡逆转 18。
此前, 已经做出了许多努力来跟踪细胞凋亡逆转的体外, 更重要的是, 在体内, 这大大有助于确认这一过程的普遍性 16,17,19。然而, 由于真正经历了细胞凋亡逆转的细胞隔离不能令人满意, 因此缺乏对逆转细胞后果的系统研究。有必要获得纯化的凋亡细胞并将其恢复, 以便进一步研究。因此, 我们使用传统上公认的行刑者酪蛋白酶活化标记作为细胞凋亡的 “不归点”21的标记, 并利用荧光激活细胞分选 (facs) 来鉴别被染色的酪蛋白酶活化细胞用里海———————————该染料共价地连接到一个短的氨基酸序列 devd, 它可以被活性的酪蛋白识别和裂解。裂解有助于释放染料, 它将从细胞溶胶转移到细胞核, 在那里它与 dna 结合并发出强烈的荧光。此过程避免使用大量细胞群, 其中一些细胞可能没有经历过细胞凋亡。
在许多实体肿瘤中, csc 或肿瘤引发细胞被发现, 使用的是单个或多个表面标记的组合, 这些细胞的数量很少足以在免疫缺陷的小鼠22,23形成肿瘤。 24,25,26,27。cd44 和 cd24 的组合已被广泛用于乳腺癌研究, cd44+/cd24–细胞被定义为乳房 csc26,27,28,29,30. 最近, 我们进行了一系列实验, 以证实细胞凋亡逆转与 csc 之间的关系, 并证明逆转乳腺癌细胞在体外和体内获得了更大的肿瘤形成能力。csc 标记18的细胞百分比升高。虽然我们不能排除乳房 csc 更好地存活, 从而在细胞凋亡逆转后得到丰富的可能性, 但重要的是, 当我们分离非干细胞并使其发生细胞凋亡逆转时, csc 就会出现在最初的非干细胞中癌细胞群, 这表明非干细胞可以促成在细胞凋亡逆转过程中 csc 的百分比增加。
本文旨在展示细胞凋亡逆转后从乳腺癌细胞向乳腺 csc 样细胞的过渡, 并通过流式细胞术检测这种转变。乳腺非干细胞最初是通过流式细胞仪分离 cd44–/cd24+乳腺癌细胞来制备的。然后, 细胞凋亡是由形态学变化在显微镜下诱导和确认的。随后, 利用流式细胞仪分离了 ccasase 的凋亡细胞阳性标记, 并在没有凋亡诱导剂的情况下进行进一步培养, 以实现细胞凋亡逆转。经过7天的流式细胞仪分析, 将倒立的细胞用 csc 标记染色。具有 cd44+/cd24标记的细胞在逆转的人群中重新出现, 这表明在细胞凋亡逆转过程中发生了从非干细胞向 csc 样细胞的过渡。
在多种癌细胞系以及在体外使用不同凋亡刺激治疗的正常原代细胞中观察到细胞凋亡逆转 12,13。这一过程也在体内16、17、19 中被追踪到. 通过使用本手稿中所述的技术, 可以获得许多关于不同癌症疾病模型中癌症复发的潜在机制和 csc 起源的信息。
Protocol
1. 乳腺癌非干式癌细胞的制备 培养 mcf-7 和 mda-mb-231 细胞在10毫升的无酚罗斯威尔公园纪念研究所 (rpmi) 1640 培养基补充10% 热灭活胎牛血清 (fbs) 在一个100毫米的菜。在10毫升的苯酚红素 rpmi 1640 培养基中培养 t47d, 辅以 2 mm l-谷氨酰胺、10% 的热灭活 fbs 和 1% v/v 青霉素-链霉素 (ps), 在100毫米的盘子中。在37°c 空气细胞培养孵化器中培养细胞,在37°c 下培养细胞2/95%。?…
Representative Results
为了观察从乳腺癌非干细胞向乳腺癌样细胞的过渡, 需要对 cd44-/cd24+乳腺癌细胞进行首次分类。对于在原始人群中含有 csc 标记的 mcf-7 细胞系约占0.15 的细胞 (图 1), 这一步骤有助于排除在细胞凋亡逆转过程中 csc 富集的可能性。相反, 如果原始填充中没有带有 csc 标记的单元格, 例如 t47d 单元格 (图 1), 则可以省略?…
Discussion
该协议描述了一种直接和明确的方法来检测乳房非干细胞过渡到乳腺癌样细胞的细胞凋亡逆转的结果。应用体外哺乳动物球形成法和免疫缺陷小鼠体内异种移植法 (18,24, 26)可帮助确认这些逆转细胞的 csc 特性 27,42,43,<sup class="xref…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到创新科技署创新科技基金的支持: 由农业生物技术国家重点实验室 (中大)、罗桂成生物医学研究基金及李希山基金会提供资助。y. x. 得到了中大研究生学生的支持。
Materials
| MCF-7 | American Type Culture Collection (ATCC) | HTB-22 | |
| MDA-MB-231 | American Type Culture Collection (ATCC) | HTB-26 | |
| T47D | American Type Culture Collection (ATCC) | HTB-133 | |
| Reagent | |||
| 0.05% trypsin-EDTA | Invitrogen | 25300054 | |
| 0.25% trypsin-EDTA | Invitrogen | 25200072 | |
| Alexa Fluor 680 annexin V conjugate | Invitrogen | A35109 | |
| bovine serum albumin | USB | 9048-46-8 | |
| CaCl2 · 2H2O | Sigma-Aldrich | C-5080 | |
| CellEvent caspase-3/7 green fluorescent dye | Invitrogen | C10423 | |
| dimethyl sulfoxide | Sigma-Aldrich | D2650 | |
| Fc block | Miltenyi Biotec | 130-059-901 | |
| fetal bovine serum | Invitrogen | 16000044 | heat-inactivated |
| HEPES | USB | 16926 | |
| Hoechst 33342 | Invitrogen | H3570 | |
| L-glutamine | Invitrogen | 25030081 | |
| Mitotracker Red CMXRos | Invitrogen | M7512 | |
| monoclonal antibodies against human CD24 | BD Biosciences | 555428 | PE Clone:ML5 Lot:5049759 RRID:AB_395822 |
| monoclonal antibodies against human CD44 | BD Biosciences | 560531 | PERCP-CY5.5 Clone:G44-26 Lot:7230770 RRID:AB_1727485 |
| NaCl | Sigma-Aldrich | 31434 | |
| paclitaxel | Sigma-Aldrich | T7402 | |
| PE Mouse IgG2a, κ Isotype Control | BD Biosciences | 554648 | Clone:G155-178 (RUO) RRID:AB_395491 |
| Penicillin-Streptomycin | Invitrogen | 15070-063 | |
| PerCP-Cy5.5 Mouse IgG2b, κ Isotype Control | BD Biosciences | 558304 | Clone:27-35 RRID:AB_647257 |
| phosphate buffered saline | Thermo Fisher Scientific | 21600010 | |
| propidium iodide | Invitrogen | P1304MP | |
| Roswell Park Memorial Institute 1640 medium | Invitrogen | 11835055 | phenol red-free |
| sodium azide | Sigma-Aldrich | S2002 | |
| staurosporine | Sigma-Aldrich | S4400 | |
| Equipment | |||
| 100 mm culture dish | Greiner Bio-One | 664160 | |
| 12-well tissue culture plates | Thermo Fisher Scientific | 150628 | |
| Cell Strainer 40-μm nylon mesh | BD Biosciences | 08-771-1 | |
| FACSuite software bundle v1.0 | BD Biosciences | 651360 | |
| FACSVerse | BD Biosciences | 651155 | |
| FluoView FV1000 confocal microscope | Olympus | IX81 | 60X objective |
| FV10-ASW Viewer software Ver.4.2b | Olympus | – | |
| round-bottom polystyrene 12 × 75 mm tubes | BD Biosciences | 352003 | |
| S3e sorter | Bio-Rad | 1451006 |
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Cite This Article
Xu, Y., So, C., Lam, H., Fung, M., Tsang, S. Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal. J. Vis. Exp. (143), e58642, doi:10.3791/58642 (2019).