CD24+CD44+胰腺癌细胞的获取及其干细胞特性的初步鉴定

背景与目的:近年研究发现,肿瘤内存在极小一部分细胞,决定肿瘤的恶性表型及生物学特性,被称为"肿瘤干细胞".本研究拟从人原发胰腺癌组织中找到具有干细胞特性的胰腺癌细胞,为今后靶向干预胰腺癌干细胞,解决胰腺癌治疗的临床难题提供研究基础.方法:将人原发胰腺癌组织种植于NOD/SCID小鼠成瘤,对移植瘤细胞进行流式细胞分选;将流式细胞仪分选获得的细胞用无血清、含有生长因子的DMEM/F12培养基培养,观察细胞的生长、传代及体外干细胞球形成能力;将细胞接种于非肥胖型糖尿病/重症联合免疫缺陷型(Nonobese diabetic/severe combined immunodeficiency,NOD/SCID)小鼠,观察、比较成瘤率,免疫组化法检测干细胞移植瘤肿瘤分化相关抗原Ki67、CK7及甲基化调控因子MBD1的表达;免疫荧光检测细胞Hedgehog、BMI-1的表达.结果:人原发胰腺癌组织种植于NOD/SCID小鼠可部分成瘤,对移植瘤细胞进行流式细胞分选,获得CD24+CD44+细胞(获得率0.6%～1.8%);将CD24+CD44+细胞用无血清、含有生长因子的DMEM/F12培养基培养,细胞呈悬浮球状生长,可连续传代并能再次形成干细胞球;将102个CD24+CD44+细胞接种于NOD/SCID小鼠,成瘤率12.5%(1/8小鼠成瘤),将103个CD24+CD44+细胞接种于NOD/SCID小鼠,4周后100%成瘤,而同样数量的CD24 CD44胰腺癌细胞则无法成瘤(P＜0.05),免疫组化法检测干细胞移植瘤中部分细胞表达Ki67、CK7及MBD1阳性;免疫荧光检测证实CD24+CD44+细胞阳性表达Hedgehog、BMI-1,表达量分别高于CD24-CD44-细胞9.95倍和2.74倍(P＜0.05).结论:所获得的CD24+CD44+细胞具有胰腺癌干细胞特性,可作为今后胰腺癌干细胞研究的实验基础.     

人口腔上皮样癌KB细胞及其SP细胞在NOD/SCID小鼠皮下成瘤性的研究

目的:研究KB细胞及其SP细胞在NOD/SCID小鼠中的生长.方法:用流式细胞仪分离SP细胞,分别用KB细胞和分离出的SP细胞种植NOD/SCID小鼠皮下,观察移植瘤的生长.结果:注射1×106、1×107个KB细胞的鼠产生移植瘤,注射1×104 、1×105个KB细胞的鼠均未产生移植瘤.注射1×105个SP细胞的鼠产生移植瘤,注射1×102、1×103、1×104个SP细胞的鼠均未产生移植瘤.结论:SP细胞产生移植瘤的能力比KB细胞强.移植瘤组织病理特点与人口腔粘膜癌基本一致.     

人卵巢癌细胞系干/祖细胞分离及其鉴定

目的:自人卵巢癌细胞系SK-OV-3中分离干/祖细胞并进行鉴定。方法:采用无血清球形体形成法从SKOV-3中分离培养卵巢癌干/祖细胞;采用实时定量PCR和蛋折质印迹法测定球形体细胞干/祖细胞相关标志ABCG2、Oct-4、Nanog基因和蛋白的表达;流式细胞仪检测其耐药性;双层软琼脂检测其克隆形成能力;NOD/SCID小鼠检测其体内致瘤性。结果:球形体细胞表达干/祖细胞相关标志Oct-4、ABCG2、Nanog;对顺铂高耐药;在双层软琼脂上克隆形成率达(13.67±1.48)%;1 000个球形体形成细胞就能在NOD/SCID鼠中成瘤。结论:采用无血清培养基中球形体形成法从SKOV-3细胞系中可以分离出具有干/祖特性的卵巢癌细胞,可为今后研究卵巢癌的发生、发展、复发及其化疗药物筛选提供简便实用的体外模型。     

人滑膜肉瘤肿瘤干样细胞分离与鉴定

目的:分离鉴定滑膜肉瘤（SW982）肿瘤干样细胞。方法:体外培养人滑膜肉瘤细胞系SW982,用CD133标记方法检测SW982中肿瘤干细胞的数量,用免疫磁珠分选法进行分选,分选后所得CD133+和CD133-细胞群分别进行以下实验。通过无血清悬浮培养基培养获得CD133+悬浮细胞球,并将其进行重新贴壁、重新成球以检测其自我更新能力。顺铂（cisplatin,CDDP）和阿霉素（doxorubicin,DXR）分别处理CD133+与CD133-贴壁细胞,CD133+贴壁细胞与CD133+悬浮细胞球,MTS检测每两种细胞药物敏感性。Real-time PCR及Western blotting 检测CD133+和CD133-贴壁细胞干细胞相关基因ABCG2、Bmi1、c-Myc、Nanog、Oct3/4、Sox2表达情况。将CD133+和CD133-贴壁细胞分别接种于6~8周雌性BALB/c裸鼠,观察成瘤情况,并将接种后所得肿瘤进行免疫组化,观察接种瘤含CD133情况。结果:SW982细胞系中CD133含量为8.59%,CDDP、DXR对CD133+和CD133-贴壁细胞抑制具有浓度依赖性。CD133+和CD133-细胞均表达干细胞相关基因ABCG2、Bmi1、c-Myc、Nanog、Oct3/4、Sox2。相对于CD133-细胞,CD133+细胞ABCG2、Nanog、Oct3/4、Sox2表达明显升高。CD133+细胞裸鼠体内成瘤率高于CD133-细胞,CD133+接种瘤含CD133较CD133-接种瘤多。结论:SW982细胞系CD133+细胞具有高自我更新能力、高耐药性、高表达干细胞相关基因、高成瘤性,是肿瘤干细胞。     

苦参碱对高表达ABCG2人耐药鼻咽癌细胞NKG2D配体表达的诱导作用

目的:探讨苦参碱对高表达三磷酸腺苷(ATP)结合转运蛋白G超家族成员2(ABCG2)人耐药鼻咽癌细胞CNE2/DDP(简写作ABCG2aHighCNE2/DDP)NKG2D配体表达的诱导作用及其对NK细胞杀伤敏感性的机制.方法:利用免疫磁珠技术分离ABCG2HighCNE2/DDP细胞及NK细胞,流式细胞技术检测分离后细胞纯度及经苦参碱处理前后靶细胞NKG2D配体表达率,LDH释放测定法检测经苦参碱处理前后ABCG2HighCNE2/DDPP细胞对NK细胞的杀伤敏感性.结果:ABCG2HighCNE2/DDP细胞分离后ABCG2表达率为(91.40±2.32)%.分选后NK细胞CD3-CD16+CD56+细胞的纯度达90%以上,经苦参碱处理之后靶细胞MICA、MICB、ULBP1、ULBP2、ULBP3表达率,由药物处理之前的(2.92±0.33)%、(4.27±0.33)%、(5.80±0.62)%、(11.10±3.15)%、(7.75±1.14)%分别上升到(11.30±0.89)%、(14.29±2.61)%、(12.56±1.06)%、(43.24±4.43)%、(12.77±1.06)%.在效靶比为10:1、20:1时,NK细胞对苦参碱处理前后ABCG2HighCNE2/DDP细胞的杀伤率分别为(15.32±1.34)%、(27.26±6.81)%及(28.53±1.37)%、(42.72±2.80)%.处理前后杀伤率有显著性差异(F=29.05,P=0.000).结论:苦参碱通过诱导肿瘤细胞高表达NKG2D配体(MICA/B、ULBP1-3).使肿瘤细胞对NK细胞的杀伤敏感性增强.     

骨肉瘤生物治疗基础研究的现状

随着新辅助化疗的广泛应用和手术技术的进展,骨肉瘤患者的5年生存率得到了很大提高,但受肿瘤细胞耐药机制和免疫逃逸的影响,目前骨肉瘤的疗效处于“瓶颈”状态。近期的研究显示,骨肉瘤细胞中存在着一些表达间充质干细胞和胚胎干细胞表面标志和表型的干细胞样肿瘤细胞,它们具有自我更新、广泛增殖的特点;同时,自骨肉瘤细胞分选出表达高水平ABCG2、能在NOD/SCID小鼠成瘤的SP细胞,显示骨肉瘤细胞耐药可能与SP细胞相关。研究针对上述标志物和降低ABCG2表达的治疗方法或药物,将有望进一步提高骨肉瘤的疗效。此外,研究主动和被动免疫疗法,以及针对Src蛋白酪氨酸激酶等靶点的靶向治疗也将为提高骨肉瘤的疗效提供新的途径。     

肝癌增强子-miRNA调控作用对的识别与特征分析

目的：探讨肝癌和正常肝组织中增强子与miRNA形成的调控关系及调控miRNA的增强子的特征,筛选出由增强子调控的差异表达miRNA并探讨其与肝癌治疗靶点的相关性。方法：基于TCGA与FANTOM5 数据库,对肝癌和正常肝组织中共417 个样本的增强子与miRNA进行共表达与3D基因组分析得到调控关系。通过ENCODE数据库中肝癌与正常肝组织的组蛋白修饰与转录因子的ChIP-seq 数据分析调控miRNA的增强子上信号值的差异。筛选由增强子调控的差异表达的miRNA,并对患者的生存期与治疗靶点进行相关性分析。结果：在肝癌与正常肝组织中分别识别增强子-miRNA 作用对93 对和40 对。ChIP-seq 数据比对分析发现,肝癌中组蛋白修饰H3K27ac、H3K4me1 和H3K4me3 信号在调控miRNA增强子区域显著高于不调控miRNA的增强子区域（|rho|>0.3,P<0.05）;多种转录因子在肝癌相关增强子中的富集显著低于正常肝组织（|rho|>0.3,P<0.05）。对增强子调控的miRNA进行差异表达分析,识别了6个与肝癌患者生存相关miRNA（hsa-miR-4664、hsa-miR-5003、hsa-miR-1915、hsa-miR-3619、hsa-miR-4745、hsa-miR-6728）,发现这些miRNA 与87 个用于靶向治疗的基因以及8 个免疫检查点基因显著相关（|rho|>0.1,FDR<0.05）。结论：成功在肝癌中识别出增强子-miRNA调控作用对与调控miRNA的增强子的特征,并筛选出由增强子调控的与肝癌患者生存和治疗靶点相关的miRNA,为后续深入开展肝癌的基础与临床研究提供了参考依据。     

分子靶向药物诱导耐药鼻咽癌CNE2/DDP细胞NKG2DLs的表达

目的：探讨不同分子靶向药物对高表达与低表达ATP结合转运蛋白G超家族成员2（ATPbinding cassette superfamily G member 2,ABCG2）的人耐药鼻咽癌CNE2/DDP细胞（分别简写为ABCG2highCNE2/DDP和ABCG2lowCNE2/DDP）表面NKG2D配体（natural killer group 2 member D ligands, NKG2DLs）表达的诱导作用及其对NK细胞杀伤敏感性的影响。方法：免疫磁珠法分选ABCG2 highCNE2/DDP、ABCG2lowCNE2/DDP细胞及NK细胞。流式细胞术检测分选细胞的纯度和不同分子靶向药物（硼替佐米、索拉非尼、舒尼替尼）处理前后ABCG2highCNE2/DDP和ABCG2lowCNE2/DDP细胞NKG2DLs的表达率。LDH释放法检测不同药物处理前后ABCG2highCNE2/DDP和ABCG2lowCNE2/DDP细胞对NK细胞杀伤的敏感性。结果：ABCG2 highCNE2/DDP和ABCG2lowCNE2/DDP细胞表面ABCG2的表达率分别为（91.40±2.32）%和（1.70±0.24）%。分选后NK细胞中CD3-CD16+CD56+细胞的比例达90%以上。药物处理前,ABCG2highCNE2/DDP和ABCG2lowCNE2/DDP细胞MICA、MICB、ULBP1、ULBP2和ULBP3呈弱表达;经不同分子靶向药物处理后,5种NKG2DLs的表达率均明显上升(P<001),以舒尼替尼处理后NKG2DLs的表达率升高最明显。随着NKG2DLs表达的上调,ABCG2highCNE2/DDP和ABCG2lowCNE2/DDP细胞对NK细胞杀伤的敏感性也随之升高。结论：不同分子靶向药物可诱导耐药鼻咽癌CNE2/DDP细胞NKG2DLs的表达,以舒尼替尼的诱导作用最强,且肿瘤细胞NKG2DLs的表达与其对NK细胞杀伤敏感性之间存在线性关系。     

硼替佐米诱导ABCG_2~(High)耐药鼻咽癌细胞高表达NKG2D配体的实验研究

目的 探讨硼替佐米诱导高表达ABCG2耐药鼻咽癌细胞对Allo-NK细胞杀伤敏感性的机制.方法 利用免疫磁珠技术分离ABCG2High CNE2/DDP细胞及Allo-NK细胞,流式细胞技术检测分离后细胞纯度及经硼替佐米处理前后靶细胞NKG2D配体表达率,LDH释放测定法检测经硼替佐米处理前后ABCG2HighCNE2/DDP细胞对Allo-NK细胞的杀伤敏感性.结果 ABCG2HighCNE2/DDP细胞分离后ABCG2表达率为(91.40±2.32)%,分选后NK细胞CD3-CD16+CD56+细胞的纯度达90%以上.经硼替佐米处理之后靶细胞MICA、MICB、ULBP1、ULBP2、ULBP3表达率,由药物处理之前的(2.92±0.33)%、(4.27±0.33)%、(5.80±0.62)%、(11.10±3.15)%、(7.75±1.14)%分别上升到(17.52±2.04)%、(12.53±3.68)%、(15.24±2.91)%、(62.02±6.85)%、(35.69±3.23)%.在效靶比为10:1、20:1、Allo-NK细胞对硼替佐米处理前后ABCG2HighCNE2/DDP细胞的杀伤率分别为(15.32±13.86)%、(27.26±6.81)%及(35.06±5.10)%、(52.34±4.78)%.处理前后杀伤率差异有统计学意义(F=26.03,P=0.000).结论 硼替佐米通过诱导肿瘤细胞高表达NKG2D配体(MICA/B、ULBP1-3),使肿瘤细胞对Allo-NK细胞的杀伤敏感性增强.     

人肺腺癌吉非替尼获得性耐药株H1975细胞与敏感株PC9细胞miRNA谱表达差异

[目的]分析人肺腺癌吉非替尼耐药细胞株H1975(epidermal growth factor receptor,EGFR基因双突变)和人肺腺癌吉非替尼敏感细胞株PC9(EGFR单突变)细胞株微小RNA(microRNA,miRNA)表达谱的差异.[方法]用Agilent human miRNA芯片分别检测H1975细胞与PC9细胞株的miRNA表达谱,Agilent Feature Extraction软件分析并筛选表达差异达5倍以上的miRNA,生物学软件分析表达差异miRNA的可能靶基因.实时定量PCR(qRT-PCR)验证miRNA芯片结果.[结果]与PC9细胞相比,H1975细胞株中22个miRNA表达上调＞5倍,24个miRNA表达下调＞5倍.qRT-PCR结果验证了芯片的结果(上调的如hsa-miR-489,hsa-miR-210和hsa-miR-21等;下调的如hsa-miR-205,hsa-miR-200c和hsa-miR-155等),同时发现,其中33个异常表达的miRNA有潜在的靶基因,靶基因超过100个的有24个miRNA.[结论]H1975吉非替尼耐药与miRNA谱异常表达有关,miRNA可能通过调控靶基因而参与EGFR-TKI的获得性耐药.     

肝癌细胞系Huh-7中CD133阳性细胞亚群的分离及其特性的研究

目的：研究CD133在人肝癌细胞系Huh-7中的表达,初步探讨肝癌中CD133阳性细胞亚群的干细胞特性。方法：流式细胞荧光激活分选技术纯化肝癌细胞系Huh-7中CD133肿瘤细胞,体外培养并观察其增殖、体内成瘤及分化能力。结果i流式细胞仪检测肝癌细胞系Huh-7中有62．3％的细胞CD133呈阳性表达,流式细胞荧光激活分选的CD133肿瘤细胞在无血清培养基中第3、5、7d的吸光度（OD值）分别为0．310、0．362、0．564,均高于相同条件下未分选细胞和CD133阴性细胞;CD133阳性细胞亚群成瘤能力明显强于阴性细胞亚群;CD133阳性细胞亚群在培养体系中的比例逐日下降,至培养的第8天,由第1天的92．3％下降至61．4％。结论：肝癌细胞系Huh-7中CD133阳性细胞亚群比其它细胞亚群具有较强增殖、成瘤和分化能力,是具有肝癌干细胞特性的细胞亚群。     

腺病毒介导microRNA-99a过表达抑制肝癌细胞生长

目的:利用腺病毒介导microrRNA-99a(miR-99a)的过表达,观察miR-99a对肝癌细胞生长的抑制作用,探讨一种腺病毒介导miRNA治疗肿瘤的新方法。方法:qRT-PCR检测正常肝细胞系L-02和肝癌细胞系HepG2、SMMC-7721、Hep3B和Huh7细胞中miR-99a的表达,构建含miR-99a的重组5型腺病毒Ad5-miR-99a。用空载腺病毒Ad-blank和Ad5-miR-99a分别感染Huh7细胞,MTT法和集落形成实验检测miR-99a过表达对Huh7细胞生长的抑制作用。结果:与正常肝细胞L02和其他肝癌细胞相比,miR-99a在肝癌Huh7细胞中表达量相对最低(P<0.01)。成功构建重组腺病毒Ad5-miR-99a,Ad5-miR-99a感染Huh7细胞后,miR-99a可在Huh7细胞中稳定高表达。集落形成实验和MTT实验显示,相对于Ad-blank组,Ad5-miR-99a可显著抑制Huh7细胞的生长和集落形成(P<0.01)。结论:成功构建重组腺病毒Ad5-miR-99a,miR-99a能有效抑制肝癌细胞的增殖,Ad5-miR-99a有可能成为治疗肝癌的新药物。     

宫颈癌侧群细胞化疗耐受性的体外研究

目的：观察化疗药物干预对人宫颈癌细胞系Hela侧群细胞和非侧群细胞中ABCG2、ABCC2基因表达变化的影响,探讨两群细胞化疗耐受性的差异。方法：Hoechst 33342免疫荧光染色与流式细胞术（ FACS）相结合分选宫颈癌细胞系Hela中的侧群细胞和非侧群细胞。给予顺铂干预,利用RT-PCR观察两群细胞化疗前后ABCG2、ABCC2基因的表达差异,以及细胞凋亡情况。结果：与非侧群细胞相比,侧群细胞 ABCG2、ABCC2基因表达更高,但差异不明显;顺铂干预后,两组细胞ABCG2、ABCC2基因表达均有升高,组间比较侧群细胞中ABCG2、ABCC2明显高于非侧群细胞,差异有统计学意义;侧群细胞对于IC50附近两个顺铂浓度0.95μg/ml和1.9μg/ml的耐受能力较好,细胞没有因药物的加入而大量凋亡,细胞凋亡率反而略有下降。非侧群细胞对于IC50附近0.95μg/ml和1.9μg/ml顺铂浓度的耐受能力较差,细胞大量凋亡。结论：来源于宫颈癌Hela细胞中的侧群细胞相对于非侧群细胞顺铂化疗耐受性更强,这种化疗耐受可能与侧群细胞 ABCG2、ABCC2的高表达抑制细胞凋亡有关。     

hsa-miR-520h downregulates ABCG2 in pancreatic cancer cells to inhibit migration,invasion, and side populations

Background:

Expression of ABCG2 is normally absent or low in the pancreas, but high in human pancreatic cancer cells. The mechanism by which ABCG2 is altered in human cancers remains unknown.

Methods:

We investigated ABCG2 expression in four pancreatic cancer cell lines, and used three microRNA (miRNA) target prediction programmes, and information from the existing literature to predict and identify hsa-miR-520h as an miRNA that targets ABCG2. The function of this miRNA was investigated by transient transfection of the pancreatic cancer cell line PANC-1 with oligonucleotides that mimic hsa-miR-520h.

Results:

Results showed that both mRNA and protein levels of ABCG2 were reduced, indicating that it was a target of hsa-miR-520h. Introduction of hsa-miR-520h mimics into PANC-1 cells also resulted in inhibition of cell migration and invasion, and reduction of side population cells. Cell proliferation, cell cycle progression and apoptosis were not affected.

Conclusions:

We propose that the effects of hsa-miR-520h may be, at least in part, caused by its regulation of ABCG2. Thus, our findings provide a new insight into the function of miRNA in the regulation of ABCG2 expression in pancreatic cancer. Gene therapy using miRNA mimics may therefore be useful as a pancreatic cancer therapy.     

RNA-electroporated CD40-activated B cells induce functional T-cell responses against HepG2 cells

Hepatocellular carcinoma (HCC) is one of the incurable tumours in the world. Cell-based immunotherapy, in which antigen-loaded antigen-presenting cells (APCs) are able to elicit T cell responses, has become an alternative treatment for liver cancer. Here, we used HepG2 cells' total RNA-electroporated CD40 ligand-activated B (CD40-B) cells as alternative APC for induction of specific CD8⁺ T-cell responses. The antigen-presenting ability of CD40-B cells was determined by phenotypic analysis, showing a polyclonal, strongly activated B-cell population with high expression of co-stimulatory molecules. To demonstrate the ability of total RNA extracted from HepG2 cells electroporated CD40-B cells to induce CD8⁺ T-cell responses, these RNA-loaded cells were co-cultured with autologous peripheral blood mononuclear cells for 7 days followed by analysis of T-cell antigen specificity. These experiments showed that CD40-B cells electroporated with HepG2 cells' total RNA are capable of activating antigen-specific interferon-γ-producing CD8⁺ T cells, and these T cells activated by CD40-B cells show a killing effect on HepG2 cells. These findings demonstrated that the carcinoma cell derived total RNA-electroporated CD40-B cells could be used as alternative APC for the induction of antigen-specific CD8⁺ T-cell responses, which might be used in HCC immunotherapy.     

沉默ABCG2基因对人肺腺癌细胞GLC-82体内增殖影响的定量研究

目的探讨ABCG2基因沉默对人肺腺癌细胞GLC-82体内增殖的影响。方法将人肺腺癌细胞GLC-82、绿色增强荧光蛋白（EGFP）标记的细胞GLC-82／EGFP^＋与ABCG2基因沉默的细胞GLC-82／EGFP^＋／ABCG2^-分别接种于裸鼠皮下,用免疫组化SP法及银染法,检测成瘤后瘤组织中细胞增殖指数Ki-67的变化与核仁组成区嗜银蛋白（AgNOR）在瘤细胞中的变化,测试肿瘤细胞AgNOR颗粒截面数、单个AgNOR颗粒面积、单个核AgNOR总面积,并用图像分析系统对ABCG2蛋白在瘤细胞中的表达进行定量分析。结果裸鼠体内GLC-82／EGFP^＋／ABCG2^-细胞成瘤组织与GLC-82、GLC-82／EGFP^＋细胞成瘤组织相比,前者ABCG2蛋白的表达明显减弱（P〈0．05）,Ki-67增殖指数显著降低（P〈0．05）且瘤细胞AgNORs颗粒截面数、单个核内AgNOR总面积明显减少,而单个AgNORs颗粒面积增大,差异有显著性（P〈0．05）。结论沉默ABCG2基因能抑制人肺腺癌细胞GLC-82在体内的增殖。     

Isolation of Side Population Cells and Detection of ABCG2 from SW480

Objective:Side population cells(SP cells)are a new type of stem cells.They mainly express ABCG2/BCRP1 and have the ability to eliminate DNA dye Hoechst33342.Many studies showed that side population cells were able of self-renewal,differentiation and carcinogenesis in cancers.Our investigation aimed at isolation of side population cells and ABCG2 positive subpopulation from colon cancer cell line SW480 and identification of their characteristics of cancer stem cells.Methods:side population cells and non-side population cells of colon cancer cell line SW480 were isolated with DNA dye Hoechst33342 and their cell cycles were measured by flow cytometry.Expression of ABCG2 of SW480 was measured by immunohistochemistry and immunofluorescence,and its proportion was measured by flow cytometry.Results:SW480 contained 2.29% side population cells.The fraction of side population cells decreased greatly to 0.40% by treatment with verapamil.The fraction of side population cells in S-G2M cell cycle was 16.14%,which was much lower than the fraction(34.05%)of non-side population cells in S-G2M.In SW480,ABCG2 positive cells,which proportion was 9.66%,were small,circular or oval,lack of psuedopods,similar to poor differentiation.On the contrary,the ABCG2 negative cells were large,polygonal,with many psuedopods,similar to high differentiation.Conclusion:our assay identified that side population cells did exist in SW480 and had a quiescence characteristic of stem cells.ABCG2 positive subpopulation occupied about 9.66% of SW480 and may have the ability to promote cell self-renewal and inhibit cell differentiation.Therefore,to isolate ABCG2 positive subpopulation from side population cells may be an alternative to study colorectal cancer stem cells.     

Stemness and anti‐cancer drug resistance in ATP‐binding cassette subfamily G member 2 highly expressed pancreatic cancer is induced in 3D culture conditions

The expression of ATP‐binding cassette subfamily G member 2 (ABCG2) is related to tumorigenic cancer stem cells (CSC) in several cancers. However, the effects of ABCG2 on CSC‐related malignant characteristics in pancreatic ductal adenocarcinoma (PDAC) are not well elucidated. In this study, we compared the characteristics of low (ABCG2?) and high (ABCG2+)‐ABCG2‐expressing PDAC cells after cell sorting. In adherent culture condition, human PDAC cells, PANC‐1, contained approximately 10% ABCG2+ cell populations, and ABCG2+ cells displayed more and longer microvilli compared with ABCG2? cells. Unexpectedly, ABCG2+ cells did not show significant drug resistance against fluorouracil, gemcitabine and vincristine, and ABCG2? cells exhibited higher sphere formation ability and stemness marker expression than those of ABCG2+ cells. Cell growth and motility was greater in ABCG2? cells compared with ABCG2+ cells. In contrast, epithelial‐mesenchymal transition ability between ABCG2? and ABCG2+ cells was comparable. In 3D culture conditions, spheres derived from ABCG2? cells generated a large number of ABCG2+ cells, and the expression levels of stemness markers in these spheres were higher than spheres from ABCG2+ cells. Furthermore, spheres containing large populations of ABCG2+ cells exhibited high resistance against anti‐cancer drugs presumably depending on ABCG2. ABCG2+ cells in PDAC in adherent culture are not correlated with stemness and malignant behaviors, but ABCG2+ cells derived from ABCG2? cells after sphere formation have stemness characteristics and anti‐cancer drug resistance. These findings suggest that ABCG2? cells generate ABCG2+ cells and the malignant potential of ABCG2+ cells in PDAC varies depending on their environments.