评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

摘要

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

摘要

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called “cancer stem cells” (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

引言

There is increasing evidence that ovarian carcinomas are comprised of heterogeneous mixtures of cells and harbor so-called “cancer stem cells” (CSCs) responsible for disease initiation, maintenance and relapse after conventional cytotoxic therapies^1-3. Therefore, the development of molecular strategies targeting ovarian CSCs is an important goal and promises to improve the therapy of ovarian cancer patients.

A pre-requisite for the understanding of the molecular features of CSCs is their reliable isolation from the non-CSCs. However, identification of ovarian CSCs appears challenging. While CD133 expression and aldehyde dehydrogenase (ALDH) activity^4,5 have been reported to mark ovarian CSCs, some data indicate that these markers are unstable⁶. Consistently, in ovarian cancer, other than for example in breast carcinoma⁷, expression of ALDH1 associates with favorable outcome⁸ and expression of the proposed stem cell marker CD44 variant has no prognostic value⁹. More recently, we have shown that expression of the embryonic stem cell protein SOX2 confers stemness to ovarian carcinoma cells¹⁰ and high SOX2 expression associates with clinically aggressive ovarian and breast carcinomas^11,12. Therefore, in this report we use a lentiviral reporter construct containing a red fluorescence protein (RFP) whose expression is controlled by a SOX2 regulatory region, as a method to isolate putative ovarian CSCs.

By definition, CSCs can both self-renew and differentiate, giving rise to all tumor cell types. Putative CSC populations need to be analyzed in functional assays performed in vivo. For obvious reasons, in human cells such functional tests are confined to xenograft assays, comprising mostly transplantation of human tumor cells into immuno-compromised mice^10,13.

An alternative in vitro method was offered by Brent Reynolds and Sam Weiss who firstly reported the so-called neurosphere assay as a surrogate assay evaluating stem potential in neural cells¹⁴. Dontu and colleagues later confirmed the use of this assay for evaluation of stem cell potential in breast cells^15,16. Here, human mammary cells were plated in different numbers in serum-free medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B-27 and heparin and cultured under non-adherent conditions for seven to ten days before sphere formation was scored by microscopy. Following this protocol with some adjustments in cell numbers, growth medium and supplements, several groups have explored in vitro stem cell potential from several cancer types such as breast¹⁷, brain¹⁸, pancreas¹⁹ and colon²⁰ tumors. In ovarian carcinoma, we have recently reported feasibility of the spheres assay and compared its results to those collected in in vivo murine xenograft models¹⁰. We found that overexpression of the stem cell protein SOX2 enhanced both in vitro sphere formation as well as in vivo tumorigenicity of human ovarian carcinoma cells¹⁰. However, the frequency of sphere-initiating cells was higher than the frequency of tumor-initiating cells measured in vivo¹⁰ suggesting that either the sphere assay may lead to false positive results due to technical reasons or, alternatively, the in vivo assay may be inefficient and result in false negative results.

In this report, we analyze multi cell-based ovarian spheres assays in more detail, review the different protocols available in the literature and compare them to a single cell-based assay. We show that the single cell-based assay provides more accurate and reproducible results than multi cell-based assays, which can be highly influenced by the density of plated cells unless methylcellulose is added to the cultures to immobilize cells. However, also in single cell-based assays, in vitro sphere-initiating potential is observed at higher frequency than in vivo tumor-initiating potential.

研究方案

1.代OVCAR-3人卵巢癌细胞的稳定转导与慢病毒包含SOX2调控区记者构造

1. 产生的慢病毒颗粒转染与报道构建识别SOX2调节区如所述^10,21的HEK 293T包装细胞系。
   注:记者进一步构建包含ProteoTuner盾系统的不稳定领域领先tdTomato荧光蛋白质。 Shield1结合不稳定域，从而防止蛋白酶体降解的荧光蛋白^22。
2. 转导OVCAR-3细胞在24小时的时间段的慢病毒颗粒。之后，去除病毒上清并洗涤细胞用磷酸盐缓冲盐水（PBS），培养在完全培养基（RPMI补加了10％FBS，100U / ml青霉素，100微克/ ml链霉素）。
3. 48小时后，10微克/毫升PURO霉素加入到该培养物，并保持5天，以允许选择正确的转导的细胞。

2.准备细胞分选和镀

1. 添加Shield1 1:之前的细胞分选1000稀释24小时。使用稳定转导OVCAR-3细胞，而不Shield1治疗作为阴性对照（ 图1）。从烧瓶中吸出培养基，洗涤细胞用1×PBS和trypsinize细胞用0.05％胰蛋白酶-EDTA进行3分钟。
2. 停止胰蛋白酶通过使用完全培养基（见上），计数细胞数，离心细胞，在300×g离心在RT（15 - 25℃）5分钟。
3. 倒出上清和悬浮细胞仔细0.5 - 1毫升无菌PBS。
4. 使用40微米的细胞滤网过滤帽，以获得单细胞悬浮液。
5. 调整细胞计数到每毫升500万细胞。
6. 制备超低附件96孔板用100μl球体介质（MEGM补充有生长因子，细胞因子，和补充剂，B-27，肝素钠;或DMEM / F12补充有生长因子，细胞因子，和补充剂，B-27，肝素-钠加入或不加入1％甲基纤维素，也见表1）。任选添加抗生素的培养基中以100U / ml青霉素和100μg/ ml链霉素的浓度，以减少可能的污染的风险。
7. 排序RFP +和RFP-细胞进入准备96孔板从上面，每孔1细胞（单细胞系球法）和每孔，分别为100细胞（多基于细胞球试验）。进行排序市售细胞分选仪（见材料 ），使用单节电池模式，排序设置:100μ喷嘴，护套的压力20磅，和产量面膜0，纯度面膜32，相位掩模16。
8. 评估通过显微镜得分含有细胞（对于单基于细胞的测定）的孔并通过在各孔计数的细胞数（对于多基于细胞的测定电镀效率; 图2 的）。
9. 孵育在球体标准条件下培养基的细胞（对于组合物见步骤2.6）在37℃和5％的CO _2。补充每日的bFGF（20毫微克/毫升）和表皮生长因子（20毫微克/毫升）。
10. 一周后，计数使用标准显微镜4X或10X放大率和荧光显微镜检测从综合报告系统的荧光信号出现肿瘤球体的数目。算球体与直径超过100微米的为"大"的球，并且球体，直径50 - 100微米为"小"球（ 图3）。要确保你算真正的球体，而不是细胞群。
    注:在单细胞分析领域形成更容易后10（比7）天文化对微观得分。
11. 计算球形成细胞中的RFP +和分别RFP-细胞单基于细胞的试验（1 96孔板的每个个体实验）或多细胞-BA的比例SED球体测定（1以及为每个单独的实验），为呈现由Shaw 等人^。16
    注:球形成细胞（％）=（球数）/（接种的细胞数）的比例×100

球3.连续传代

1. 放置的各孔在适当的无菌管中并离心含量在300×g离心在室温10分钟。对于多基于细胞球试验，收集从一个很好的球在一起。用PBS离心2分钟长5倍 - 洗井3。对于单细胞为基础的球试验，收集各个领域。由于低数量的细胞，可使用1.5毫升管离心和洗涤步骤。
2. 在200微升0.05％胰蛋白酶EDTA去除上清，重悬沉淀。
3. 为了达到最佳的细胞分离，培养的细胞悬浮液，在37℃下进行5分钟，在软摇床。优化胰蛋白酶消化时间为您的细胞系，以更低的细胞死亡率:如果没有大球体是轻轻用100微升枪头可见磨碎。在的情况下的大球仍然存在，孵育胰酶另外3分钟，然后进行研磨步骤。情况下的单细胞分析确认，以优化的时间的活细胞中的胰蛋白酶消化步骤最佳细胞产量。
4. 以灭活胰蛋白酶，加入500微升完全培养基中，并离心，在300×g离心10分钟，在单细胞测定法，离心额外2分钟的情况。
5. 在球体中小心取出上清和悬浮细胞。
6. 使用一个40微米的细胞滤网过滤帽，以获得单细胞悬浮液。
7. 在使用RFP +和RFP-细胞在多细胞bassed测定的情况下，通过流式细胞仪分析评估的荧光细胞的百分比在每个后良好的再悬浮。
8. 对单个细胞的串行replating测定法，种子，每孔1细胞到一个新的超低附件96孔板制备如上详述。从一个单独的领域，种子大约20个人口井。对于多单元基于初级球体replating测定法，从初级球体的一个孔获得的种子细胞到新的96孔板中。第二天用显微镜计数细胞数。
9. 评估球形成细胞中利用在步骤2.11中所述的式次级球测定的比例。

4.结果分析

1. 分析来自于独立一式三份进行的实验的结果，并使用双面学生t检验来分析正态分布的值和以其他方式采用Mann-Whitney-测试进行统计分析。

结果

在常规球测定中，近40％的RFP + OVCAR-3细胞与RFP-细胞的20％就产生了一个单独的肿瘤球在初级球体测定（ 图4A）。此外，通过RFP +细胞形成球在大小上比由RFP-细胞形成的要大。

当在单一的基于细胞的测定法镀，RFP +细胞也形成为比RFP-细胞更球体，证实上述结果。不过，由于是朝向产生较少球体每镀在单相对于多基于细胞的测定（ 图4A，B）中的倾向，这...

讨论

球体培养物是一种广泛使用的方法，以测定癌症干细胞潜能，并在广泛的人类肿瘤细胞^15,25,26的富集干细胞样细胞。在这些培养条件下，缺乏自我更新能力的癌细胞预期分化并最终发生细胞死亡。尽管它们可能最初形成细胞团，甚至肿瘤球特别是在主试验中，它们不能够维持在串行replating球体形成能力因缺乏自我更新的特性。球形检测作为替代试验，以确定肿瘤干细胞，并评估其在整个肿...

材料

Name	Company	Catalog Number	Comments
Low-attachment plate	Corning	3474
MEGM	Lonza	CC-3151
Insulin	Lonza	CC-4136	SingleQuots™ Kit
Hydrocortisone	Lonza	CC-4136	SingleQuots™ Kit
EGF	Lonza	CC-4136	SingleQuots™ Kit
EGF	Sigma	E9644	end concentration: 20 ng/ml
FGF	PeproTech	100-18B	end concentration: 20 ng/ml
B-27	Invitrogen/ Gibco	17504-044	end concentration: 1X
Heparin-Natrium-25000 IE	Ratiopharm	N68542.02	dilution 1:1,000
Pen/Strep	Gibco	15140-122
FCS	Gibco	10500-064
RPMI 1640	Gibco	21875-034
Trypsin-EDTA	Gibco	25300-054
Dulbecco’s PBS (1X)	Gibco	14190-094
Shield1	Clontech	632189	dilution 1:1,000
DMEM/F12	Gibco	21041-025
DMEM/F12 (powder)	Gibco	42400-010
Methyl cellulose	Sigma	M0387
Puromycin dihydrochloride	Applichem	A2856
Cell sorter	BD		Aria III cell sorter
FACS analyser	BD		Accuri c6 flow cytometer
Microscope	Olympus		IX50 Osiris