在乳腺癌干细胞中Hedgehog信号通路相关基因表达增强

目的 了解Hedgehog信号通路在乳腺癌发生发展中的作用.方法 用免疫磁珠法从无血清培养的乳腺癌悬浮细胞中分选CD44+CD24-细胞和非CD44+CD24-细胞,用real-time RT-PCR法检测Hedgehog信号通路主要分子$HH、PTCH1、SMO和GLI1 mRNA在细胞中的表达,用免疫组织化学法检测上述因子在乳腺癌组织中的表达.结果 分选出的CD44+CIDA-细胞约占乳腺癌悬浮细胞总数的8.25%,分选出的CD44+CD24-细胞表达干细胞标志蛋白ALDHA1和Oct-4;SHH、PTCH1、SMO和GLI1 mRNA在CD44+CD24-细胞中的表达均高于其在非CD44+CD24-细胞中的表达(P＜0.05);SMO和GLI1蛋白在三阴性乳腺癌的表达均高于非三阴性乳腺癌组织(P＜0.05).结论 在乳腺癌干细胞CD44+CD24-细胞中Hedgehog信号通路被激活,抑制癌症干细胞中Hedgehog通路的活化可能会降低或阻止乳腺癌的复发及化疗耐受.     

MCF7细胞中肿瘤干细胞的增殖特征

背景：肿瘤干细胞学说认为,肿瘤干细胞是肿瘤不断增殖的根源,并与肿瘤的浸润转移、耐药现象密切相关。 目的：在单细胞水平研究体外传代培养的MCF7细胞中肿瘤干细胞含量变化规律,认识肿瘤干细胞在体外培养环境下的增殖特点。 方法：利用单细胞分离种植-成瘤性克隆方法对传代培养后不同时间点MCF7细胞中肿瘤干细胞的比例连续检测,流式细胞仪同步检测相应细胞样本中CD44⁺CD24^-/low亚群的含量变化。 结果与结论：传代后培养的MCF7细胞在不同时间点其肿瘤干细胞比例及CD44⁺CD24^-/low亚群比例均呈现有规律的变化。但CD44⁺CD24^-/low亚群变化更为显著(36.84%到81.95%),而肿瘤干细胞含量仅在小范围波动(38.54%~47.39%)。结果提示传代培养的MCF7细胞中,肿瘤干细胞具有通过调节自身增殖来保持其比例相对稳定的特点;CD44⁺CD24^-/low亚群并不代表或者富集MCF7细胞株中的肿瘤干细胞亚群。     

盐霉素通过TGFβ1/Smad信号通路抑制MMP-2、9降低CD44~+/CD24~(-/low)表型乳腺癌干细胞迁移和侵袭能力

目的分析盐霉素对CD44~+ /CD24~(-/low)表型乳腺癌干细胞迁移和侵袭能力的影响,并初步探讨其影响机制。方法通过有血清和无血清培养技术培养人乳腺癌MCF-7细胞株,收集两种细胞,对其CD24、CD44标志物进行荧光染色,采用流式细胞仪检测CD44~+ /CD24~(-/low)亚群细胞的比例;盐霉素培养MCF-7细胞株和CD44~+ /CD24~(-/low)表型乳腺癌干细胞,MTT法筛选出引起CD44~+ /CD24~(-/low)表型乳腺癌干细胞细胞凋亡低于IC50的浓度;Transwell技术检测MCF-7和CD44~+ /CD24~(-/low)表型乳腺癌干细胞的迁移和侵袭能力,以筛选出的浓度诱导CD44~+ /CD24~(-/low)表型乳腺癌干细胞,以排除盐霉素对该细胞增殖抑制作用的影响,Transwell技术检测该细胞迁移和侵袭能力的变化;Western blot技术检测TGFβ1、Smad2、Smad3、p-Smad2、pSmad3、MMP-2、MMP-9蛋白水平的变化。结果 CD44~+ /CD24~(-/low)表型乳腺癌干细胞在无血清培养和有血清培养中的比例分别为(86.93±0.53)%和(19.98±0.62)%(P0.01),CD44~+ /CD24~+ 表型细胞的比例分别是(12.68±0.59)%和(79.90±0.57)%(P0.01);MTT法筛选出低于IC50的浓度是1、3、5、7μmol/L;Transwell技术检测CD44~+ /CD24~(-/low)表型乳腺癌干细胞迁移和侵袭能力明显高于MCF-7细胞株,并随盐霉素浓度的上升呈下降趋势(P0.05)。Western blot技术检测TGFβ1、Smad2、Smad3、p-Smad2、p-Smad3、MMP-2、MMP-9蛋白水平下调(P0.01)。结论盐霉素可能通过TGFβ1/Smad信号通路下调MMP-2、MMP-9蛋白水平,从而降低CD44~+ /CD24~(-/low)表型乳腺癌干细胞迁移和侵袭能力。     

CD44和CD24在鼻咽癌细胞系HK-1中调控STAT3发生磷酸化的分子机制研究

目的 研究CD44和CD24在鼻咽癌细胞系HK-1中调控STAT3发生磷酸化的分子机制.方法 采用流式细胞仪对培养的鼻咽癌HK-1高分化NPC细胞进行分选以获得CD44 +/CD24+ HK1细胞及CD44-/CD24-HK1细胞,通过Western blot、MTT和肿瘤微球形成等实验,分析鼻咽癌阳性肿瘤细胞中P-STAT3的表达,以及STAT3被抑制剂Stattic沉默后,对CD44+/CD24+ HK1和CD44-/CD24-HK1细胞增值能力和肿瘤微球形成能力的影响.结果 鼻咽癌HK-1细胞中可以提取到34.7％的CD44 +/CD24+ HK1细胞和41.5％的CD44-/CD24-HK1细胞,CD44+/CD24+ HK1细胞比CD44 /CD24-HK1细胞表达磷酸化STAT3水平高.STAT3的抑制剂Stattic可以抑制CD44+/CD24+ HK1和CD44-/CD24-HK1细胞磷酸化STAT3的表达,MTT实验显示16μmol/L Stattic明显抑制CD44+/CD24+ HK1和CD44-/CD24-HK1细胞增殖,肿瘤微球形成实验表明Stattic可明显抑制CD44+/CD24+ HK1和CD44-/CD24-HK1细胞微球形成能力,即STAT3在CD44+/CD24+ HK1细胞增殖和鼻咽癌进程中发挥重要的作用.结论 CD44和CD24在鼻咽癌侧群细胞HK-1细胞中,CD44 +/CD24+阳性细胞通过诱导STAT3发生磷酸化来促进鼻咽癌发展,为鼻咽癌肿瘤干细胞的靶向治疗提供了新的靶点,临床治疗可靶向抑制STAT3的表达,从而抑制CD44+/CD24+ HK1细胞增殖和肿瘤微球的形成,最终达到降低鼻咽癌的发生率.     

人乳腺肿瘤干细胞分离培养及鉴定

目的:体外分离、培养乳腺肿瘤干细胞,并鉴定其生物学特性.方法:收集临床术后乳腺癌患者的肿瘤组织,经机械剪切联合酶消化法获得肿瘤细胞并培养,流式细胞仪鉴定细胞表面抗原,MTT比色法检测细胞生长曲线,实时定量PCR(qPCR)鉴定细胞Sox2、Nanog等基因表达,免疫荧光检测细胞特异性蛋白Bcl-2、孕激素受体(PR)的表达.结果:所获得的细胞呈克隆球样形态生长,鉴定结果发现其具有CD44+/CD24-表型,该细胞高表达Sox2、Nanog基因,且Bcl-2、PR蛋白均阳性表达.结论:人乳腺肿瘤组织中存在具有自我更新和增殖能力、表达CD44 +/CD24-的乳腺肿瘤干细胞,并能在体外长期培养.     

脂肪间充质干细胞源exosomes对乳腺癌细胞系MCF7中microRNA表达谱的影响

目的观察脂肪间充质干细胞源exosomes的生物学特性,初步探讨其对乳腺癌细胞系MCF7中microRNAs表达谱的影响。方法收集脂肪间充质干细胞培养上清液,以超滤和超离的方法提取exosomes;电镜下观察形态;用Western blot检测exosomes表面蛋白标志物的表达情况;用Dil染料标记exosomes后加入到乳腺癌细胞MCF7上清中,荧光显微镜下观察MCF7对exosomes的内吞作用;用microRNA芯片检测MCF7在exosomes处理24 h后microRNAs表达谱的变化以及exosomes中含有的microRNAs种类。结果脂肪间充质干细胞分泌30~100 nm的exosomes,exosomes表达CD63和HSP70,可被乳腺癌细胞MCF7内吞;exosomes作用24 h,可引起MCF7中244个microRNAs表达发生变化(其中174个表达上调,70个表达下调,倍数2倍);对脂肪间充质干细胞exosomes中microRNAs的分析提示MCF7中microRNAs的上调大部分是内源性的,不是exosomes输入的。结论脂肪间充质干细胞源exosomes可以诱导乳腺癌细胞MCF7microRNA表达谱发生变化,揭示了间充质干细胞影响乳腺癌细胞发生和发展的一个新机制。     

受体型酪氨酸磷酸酶D在乳腺癌干细胞特性中的影响

目的探讨受体型酪氨酸磷酸酶D(protein tyrosine phosphatase receptor type D,PTPRD)对乳腺癌干细胞特性的影响。方法采用小RNA干扰技术下调PTPRD在乳腺癌细胞系MDA-MB231中的表达;用乳腺球成球实验检测干细胞的自我更新能力;用全克隆形成实验检测细胞全克隆形成能力;用流式细胞技术检测CD44~+/CD24~-细胞比例;用小鼠成瘤实验分析乳腺癌细胞在小鼠中的成瘤能力;用免疫磁珠法分选CD44~+/CD24~-干细胞群及非干细胞群,用免疫荧光技术检测PTPRD在干细胞及非干细胞中的表达;用Western blot法检测蛋白表达。结果 PTPRD下调促进干细胞标记分子ALDH1及OCT-4的表达。乳腺癌干细胞中PTPRD表达显著低于非干细胞(P 0. 05)。PTPRD下调后,乳腺癌细胞的乳腺球形成数(147±3. 51)显著高于对照组(106±12. 5)(P 0. 05);全克隆形成比例[(35. 9±3. 4)%]显著高于对照组[(11. 2±5. 3)%](P 0. 05); CD44~+/CD24~-细胞的比例[(2. 88±1. 2)%]显著高于对照组[(0. 6±0. 4)%];乳腺癌细胞在裸鼠中成瘤能力显著上调(P 0. 05)。结论乳腺癌干细胞中PTPRD低表达,PTPRD可抑制乳腺癌干细胞的自我更新能力。     

CD44+/CD24-细胞在乳腺癌组织及细胞系中的数量与分布

目的 检测CD44+/CIY24-细胞在乳腺癌组织及细胞系中的分布及数量,探讨其与乳腺癌常用标志物表达和乳腺癌分子亚型的关系.方法 采用免疫组织化学SP双染及单染法,分别检测了60例乳腺浸润性导管癌中的CD44及C1724的共表达情况和ER、PR、HER2、人雌激素诱导蛋白PS2、bcl-2、nm23的单独表达情况,同时检测了三种乳腺痛细胞系(MCF-7、MDA-MB-468及MDA-MB-231)中CD44及CD24的表达情况.结果 不同病例标本中CD44+/C1724-细胞的数量差异较大,分布无明显规律,总阳性率为65.0%;CD44+/CD24-细胞数量与患者年龄、肿瘤大小、淋巴结转移情况及ER、PR、HER-2、人雌激素诱导蛋白PS2、bcl-2、nm23表达情况无关(P均>0.05);CD44+/CD24-细胞数量与乳腺癌分子亚型无关.CD44+/CIY24-细胞在MCF-7、MDA-MB-468及MDA-MB-231细胞系中的比例分别为<1%、5%及>80%.结论 CD44+/CD24-细胞存在于部分乳腺癌组织及细胞系中,其数量及分布与乳腺癌的分子亚型和临床病理参数无直接关系.     

神经干细胞体外培养鉴定及诱导分化的表型特征

背景：神经干细胞促进受损中枢神经系统结构和功能再修复具有广阔的应用前景,而进行神经干细胞体外培养鉴定及诱导分化表型的研究是实现这一应用的基础。 目的：观察神经干细胞在体外培养条件下的生物学特性和分化表型特点。 方法：从新生小鼠海马、嗅球提取神经干细胞。选取3代后稳定的神经干细胞采用BrdU进行标记,并进行BrdU+巢蛋白+Hochest33258免疫荧光复合染色对神经干细胞进行鉴定。体外诱导促使神经干细胞贴壁分化,对分化产生的子代细胞进行BrdU、β-微管蛋白Ⅲ、胶质纤维酸性蛋白、Hochest33258复合免疫荧光染色确定分化表型。 结果与结论：来源于新生鼠海马及嗅球的细胞连续传代培养后可形成稳定悬浮的类球状细胞团,且BrdU+巢蛋白免疫荧光双染阳性。神经干细胞体外诱导贴壁分化后可产生β-微管蛋白Ⅲ、胶质纤维酸性蛋白阳性的子代细胞。以上结果表明体外培养的神经干细胞具有很强的自我增殖更新的能力,在培养过程中趋向于形成稳定的神经球,经体外诱导通过不对称细胞增殖、分化产生神经元和星形胶质细胞等细胞表型。     

无血清悬浮培养法富集结肠癌干细胞

目的初步富集结肠癌干细胞球,并对其进行鉴定。方法无血清悬浮培养初步富集结肠癌干细胞,流式细胞术,细胞分化实验,NOD/SCID小鼠致瘤实验鉴定其生物学特性。结果结肠癌细胞株CW-2细胞能在SFM中存活、增殖并形成肿瘤干细胞球;分离后细胞中CD44+EPCAM+癌干细胞含量为60.39%;肿瘤干细胞球的增殖能力、致瘤能力均强于含血清培养细胞(P0.05)。结论无血清悬浮培养可以初步富集结肠癌干细胞。     

Effect of fibroblasts on breast cancer cell mammosphere formation and regulation of stem cell-related gene expression

The purpose of this study was to investigate the regulatory effects of breast cancer fibroblasts (BCFs) vs. normal mammary fibroblasts (NMFs) on mammosphere formation and stem cell-related gene expression in breast cancer cells. Breast cancer cells (MCF-7) were cultured in suspension to generate primary and secondary mammospheres. The proportion of CD44+/CD24low/- cells was assessed by flow cytometry (FCM), and Wnt1, Notch1, β-catenin, CXCR4, SOX2 and ALDH3A1 gene expression was detected by quantitative real-time PCR. The fibroblasts from either breast cancer tissue or normal mammary tissue were purified from tissue specimens and co-cultured with breast cancer cells. The mammosphere formation efficacy was approximately 180/10,000 MCF-7 cells. FCM analysis showed that, compared to the 2.1% positive expression in the MCF-7 monolayer culture cells, the expression of CD44+/CD24low/- in MCF-7 mammosphere cells was significantly elevated to 10.4% (P<0.01). The proportion of the CD44+/CD24low/- subpopulation of the cells in mammospheres was nearly 5-fold higher than that of general MCF-7 cells. Compared with MCF-7 monolayer culture cells, mammosphere cells showed significantly (P<0.01) enhanced expression of Wnt1 [fold-change (FC), 2.25], Notch1 (FC, 2.45), β-catenin (FC, 1.72), CXCR4 (FC, 4.68), SOX2 (FC, 4.25) and ALDH3A1 (FC, 5.38). When BCFs were co-cultured with MCF-7 cells under mammosphere culture conditions, the length of time of mammosphere formation decreased, the volume of the mammo-spheres increased and the mammosphere-forming efficiency (MFE) was higher than that of NMFs and the control group. Both the BCF and NMF groups showed enhanced gene expression for the following genes: Wnt1 (FC, 3.18 and 1.27, respectively), β-catenin (FC, 1.75 and 1.22, respectively), Notch1 (FC, 2.09 and 1.31, respectively), CXCR4 (FC, 2.77 and 1.33, respectively), SOX2 (FC, 2.77 and 1.80, respectively) and ALDH3A1 (FC, 5.23 and 1.85, respectively). Cancer fibroblast cells can promote the MFE and up-regulate stem cell-related gene expression in breast cancer cells.     

Distinct patterns of promoter CpG island methylation of breast cancer subtypes are associated with stem cell phenotypes

Although DNA methylation profiles in breast cancer have been connected to breast cancer molecular subtype, there have been no studies of the association of DNA methylation with stem cell phenotype. This study was designed to evaluate the promoter CpG island methylation of 15 genes in relation to breast cancer subtype, and to investigate whether the patterns of CpG island methylation in each subtype are associated with their cancer stem cell phenotype represented by CD44+/CD24- and ALDH1 expression. We performed MethyLight analysis of the methylation status of 15 promoter CpG island loci involved in breast cancer progression (APC, DLEC1, GRIN2B, GSTP1, HOXA1, HOXA10, IGF2, MT1G, RARB, RASSF1A, RUNX3, SCGB3A1, SFRP1, SFRP4, and TMEFF2) and determined cancer stem cell phenotype by CD44/CD24 and ALDH1 immunohistochemistry in 36 luminal A, 33 luminal B, 30 luminal-HER2, 40 HER2 enriched, and 40 basal-like subtypes of breast cancer. The number of CpG island loci methylated differed significantly between subtypes, and was highest in the luminal-HER2 subtype and lowest in the basal-like subtype. Methylation frequencies and levels in 12 of the 15 genes differed significantly between subtypes, and the basal-like subtype had significantly lower methylation frequencies and levels in nine of the genes than the other subtypes. CD44+/CD24- and ALDH1+ putative stem cell populations were most enriched in the basal-like subtype. Methylation of promoter CpG islands was significantly lower in CD44+/CD24-cell (+) tumors than in CD44+/CD24-cell (-) tumors, even within the basal-like subtype. ALDH1 (+) tumors were also less methylated than ALDH1 (-) tumors. Our findings showed that promoter CpG island methylation was different in relation to breast cancer subtype and stem cell phenotype of tumor, suggesting that breast cancers have distinct patterns of CpG island methylation according to molecular subtypes and these are associated with different stem cell phenotypes of the tumor.     

Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells

ABSTRACT: BACKGROUND: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. METHODS: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44+CD24+ESA+ cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. RESULTS: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44+CD24+ESA+ cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44+CD24-ESA+ cells. CONCLUSIONS: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.     

STAT3 mediates resistance of CD44+CD24-/low breast cancer stem cells to tamoxifen in vitro

We sought to determine whether STAT3 mediated tamoxifen resistance of breast cancer stem cells in vitro.The capacities for mammosphere formation and STAT3 expression of CD44+CD24-/low MCF-7 and MCF-7 were observed.The CD44+CD24-/low subpopulation ratio and its sensitivity to adriamycin were analyzed in MCF-7 and TAM resistant(TAM-R) cells.Cell cycle,apoptosis,STAT3 and phospho-STAT3 changes were observed af-ter treatment with tamoxifen.Small interference RNA-mediated knockdown of STAT3 in TAM-R cells was also performed.CD44+CD24-/low MCF-7 showed higher capacities for mammosphere formation and STAT3 expression than total MCF-7.The CD44+CD24-/low subpopulation was also upregulated in TAM-R cells with less sensitivity to adriamycin than MCF-7.Cell cycle changes,anti-apoptotic effects and STAT3 changes were also found.Mean-while,the knock-down of STAT3 in TAM-R resulted in an increase in sensitivity to tamoxifen.It is concluded that STAT3 plays an essential role in breast cancer stem cells,which correlated with tamoxifen resistance.     

Isolation and identification of cancer stem-like cells from murine melanoma cell lines

In current study, cancer stem-like cells in the murine melanoma B16F10 cells were investigated. CD phenotypes of the B16F10 cells were analyzed by flow cytometry, and the specific CD phenotype cells from the B16F10 cells were isolated by MACS. Then we used colony formation assay in soft agar media, the cell growth assay in serum-free culture media as well as the tumorigenicity investigation of the specific CD phenotype cells in C57BL/6 mice, respectively, to identify cancer stem-like cells in the B16F10 cells. The results showed that the B16F10 cells could form spherical clones in serum-free culture media, and the rate of clonegenesis of CD133^＋, CD44^＋ and CD44^＋CD133^＋ cells was higher than that of CD133^-, CD44^- and CD44^＋CD133^＋ cells in soft agar media, respectively. The tumorigenic potential of CD133^＋, CD44^＋, CD44^＋CD133^＋ cells and CD44^＋CD133^＋CD24^＋ cells was stronger than that of CD133^-, CD44^-, CD44^＋CD133^- cells and CD44^＋CD133^＋CD24^- cells in mice, respectively. In conclusion, the CD44^＋CD133^＋CD24^＋ cells have some biological properties of cancer stem-like cells or are highly similar to the characteristics of cancer stem cells （CSC）. These results provide an important method for identifying cancer stem-like cells in B16F10 cells and for further cancer target therapy. Cellular ＆ Molecular Immunology.     

Stem cell-related markers in primary breast cancers and associated metastatic lesions

It has been reported previously that: (1) normal-breast epithelial cells that are CD24-/44+ express higher levels of stem/progenitor cell-associated genes; (2) cancer cells that have undergone epithelial to mesenchymal transition display CD24-/44+ cell-surface expression, a marker for breast cancer stem cells; (3) loss of E-cadherin is a preliminary step in epithelial to mesenchymal transition; and (4) vimentin is a marker of mesenchymal phenotype. We hypothesized that stem cell subpopulations would be more frequent in metastatic than in primary tumors. Therefore we assessed by immunohistochemical analysis, tissue microarrays containing tissue from primary and associated metastatic breast cancers for expression of CD24, CD44, E-cadherin and vimentin to evaluate candidate cancer-initiating cell populations in breast cancer subtypes and metastatic lesions. The occurrence of CD24-/44+ and CD24+/44- cells did not differ in primary vs matched lymph node or distant and locoregional metastatic lesions; E-cadherin expression was decreased in primary vs lymph node metastases (P=0.018) but not decreased in distant and locoregional metastases relative to primary tumor, whereas vimentin, was more frequently expressed in lymph node and distant and locoregional metastases (P=0.013, P=0.004) than in matched primary cancers. Thus, the frequency of CD24-/44+ cells does not differ in metastases relative to the primary breast cancer but differs by tumor stage and subtype.     

CD44+CD24-/low乳腺癌干细胞活性与多药耐药的相关性

BACKGROUND:Tumor stem cells are found to be involved in the recurrence, metastasis and drug resistance of the tumor. OBJECTIVE:To explore the relationship between cell activity and multidrug resistance of CD44⁺CD24^-/low breast cancer stem cells. METHODS: CD44⁺CD24^-/low breast cancer stem cells sorted from multidrug resistant breast cancer cell line MCF-7/ADR were detected as percentage using flow cytometry. P-gp fluorescence intensity of the cell membrane and MDR mRNA expression in sorted cells and MCF-7/ADR were detected using flow cytometry and RT-PCR, respectively. RESULTS AND CONCLUSION:After sorting by flow cytometry, the proportion of CD44⁺CD24^-/low breast cancer stem cells was more than 90%, indicating that the sorted cells could meet the needs of the subsequent experiment. CD44⁺CD24^-/low cell subsets exhibited stronger ability to form microspheres than non- CD44⁺CD24^-/low cell subsets. The P-gp fluorescence intensity and MDR mRNA expression of CD44⁺CD24^-/low cells were significantly higher than those of MFC-7/ADR cell line (P < 0.05). These experimental findings suggest that CD44⁺CD24^-/low breast cancer stem cells sorted from MCF-7/ADR cell lines have a strong ability to form cell microspheres in vitro, and significantly raise the level of P-gp protein and MDR mRNA expression, which may be one of the causes of multidrug resistance.     

The role of ALDH1A1 in contributing to breast tumour aggressiveness: A study conducted in an African population

Aldehyde dehydrogenase 1 member A1 (ALDH1A1) is one of the most well studied breast cancer stem cells. Its expression has been associated with poor clinicopathological features and clinical outcomes in several studies. This paper studies the expression of ALDH1A1 and its combination with CD44⁺/CD24^−/low breast cancer stem cell and their association with clinicopathological parameters and molecular subtypes.MethodTissue Microarray was constructed from 222 Formalin Fixed Paraffin Embedded (FFPE) breast cancer tissues. The expression of ALDH1A1, CD44 and CD24 were assessed by Immunohistochemistry (IHC). The association of ALDH1A1 and its association with clinicopathological parameters, molecular subtypes, CD44 and CD24 were studied in an African population. The association between CD44⁺/CD24^−/low/ALDH1⁺ and the clinicopathological phenotypes were also studied.ResultsA high ALDH1A1 expression of 90% was recorded in this study. No association was found between ALDH1A1 and clinicopathological parameters. ALDH1A1 was positively associated with CD24 (r = 0.228, OR-4.599 95% CI- 1.751–12.076, p = 0.001) and CD44 (r = 0.228, OR-5.538 95%CI- 1.841–16.662, p = 0.001) but not associated with CD44⁺/CD24^−/low (r = 0.134, OR- 2.720 95%CI- 0.959–7.710, p = 0.052). CD44⁺/CD24⁻/ALDH1⁺ however had significant associations with Age (p- 0.020, r = 0.161, OR- 2.771, 95%CI 1.147–6.697), Gender (p = 0.004, OR- 15.333 95%CI 1.339–175.54), Tumour grade (p = 0.005, r = 0.197, OR-3.913 95%CI 1.421–10.776) and clinical prognostic staging (p = 0.014, r = 0.182, OR-3.028 95%CI- 1.217–7.536). There was no association between CD44⁺/CD24⁻/ALDH1⁺ and the molecular subtypes.ConclusionThe high expression of ALDH1A1 in breast cancer makes it an important target for targeted therapy. This study further confirms the increased tumourigenicity of CD44⁺/CD24⁻/ALDH1⁺ combination phenotype and its association with increased tumour grade and clinical prognostic stage. Survival studies of ALDH1A1 and other breast cancer stem cells in African populations are strongly recommended to help further understand their effect on tumour aggressiveness.