靶向肿瘤干细胞治疗肿瘤

具有独特的分子表达、表面标志物、干性相关信号通路和代谢模式等方面特征的肿瘤干细胞(cancer stem cell,CSC)因其具有高致瘤、高转移、高治疗抵抗能力,可能是多种类型恶性肿瘤生长、转移、治疗抵抗的关键因素,也是肿瘤发生和复发的重要根源.正常干细胞在产生了第一个致癌突变之后将逐步发展成为癌前干细胞和CSC,随...     

Aspirin counteracts cancer stem cell features,desmoplasia and gemcitabine resistance in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia, the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment, although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features, stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines, non-malignant cells, 3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays, flow cytometry, colony and spheroid formation assays, Western blot analysis, antibody protein arrays, electrophoretic mobility shift assays (EMSAs), immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression, inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly, aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice. These results highlight aspirin as an effective, inexpensive and well-tolerated co-treatment to target inflammation, desmoplasia and CSC features PDA.     

干细胞标志物在卵巢癌SKOV3细胞侧群细胞中的表达

目的：检测干细胞相关标志物在卵巢癌SKOV3细胞系侧群（side population,SP）细胞和非侧群（Non-SP）细胞中的表达差异,确定卵巢癌干细胞特异性标志物。 方法： Hoechst 33342染色检测SKOV3细胞中SP细胞的比例,流式细胞术检测SKOV3细胞的SP和Non-SP细胞中干细胞标志物CD133、CD117、CD44、ABCG2、ALDH1 的表达情况,荧光定量PCR检测SP和Non-SP细胞中 ABCG2、ALDH2、NANOG、OCT4、SOX2、CD133、CD117 mRNA的表达水平。 结果： SKOV3细胞中SP细胞比例为(1.56±0.35)%。 ALDH1、ABCG2在SP细胞中表达率分别为（87.3±5.76）%、（29.48±4.43）%,在Non-SP的表达率分别为（5.32±0.47）%、（3.01±1.69）%,差异有统计学意义（ P <0.01）;CD44在这两种细胞亚群中的表达率均高于99%（ P >005）;CD133、CD117在这两种细胞亚群中均不表达。 ALDH1、ABCG2、 NANOG、OCT4和SOX2 mRNA在SP细胞中的表达分别是Non-SP细胞的21.03倍（ P =0.001）、3.14倍（ P =0.001）、23.94倍（ P =0.001）、10.73倍（ P =0.009）和21.46倍( P =0001), CD133、CD117 mRNA在两种细胞亚群中均不表达。 结论： 人卵巢癌细胞株SKOV3中存在SP细胞亚群, ALDH1、ABCG2和NANOG、OCT4、SOX2 mRNA可能是卵巢癌干细胞标志物,为诊断及治疗卵巢癌提供了潜在的靶点。     

Inhibition of glucose turnover by 3-bromopyruvate counteracts pancreatic cancer stem cell features and sensitizes cells to gemcitabine

According to the cancer stem cell (CSC) hypothesis, the aggressive growth and early metastasis of pancreatic ductal adenocarcinoma (PDA) is due to the activity of CSCs, which are not targeted by current therapies. Otto Warburg suggested that the growth of cancer cells is driven by a high glucose metabolism. Here, we investigated whether glycolysis inhibition targets CSCs and thus may enhance therapeutic efficacy. Four established and 3 primary PDA cell lines, non-malignant cells, and 3 patient-tumor-derived CSC-enriched spheroidal cultures were analyzed by glucose turnover measurements, MTT and ATP assays, flow cytometry of ALDH1 activity and annexin positivity, colony and spheroid formation, western blotting, electrophoretic mobility shift assay, xenotransplantation, and immunohistochemistry. The effect of siRNA-mediated inhibition of LDH-A and LDH-B was also investigated. The PDA cells exhibited a high glucose metabolism, and glucose withdrawal or LDH inhibition by siRNA prevented growth and colony formation. Treatment with the anti-glycolytic agent 3-bromopyruvate almost completely blocked cell viability, self-renewal potential, NF-κB binding activity, and stem cell-related signaling and reverted gemcitabine resistance. 3-bromopyruvate was less effective in weakly malignant PDA cells and did not affect non-malignant cells, predicting minimal side effects. 3-bromopyruvate inhibited in vivo tumor engraftment and growth on chicken eggs and mice and enhanced the efficacy of gemcitabine by influencing the expression of markers of proliferation, apoptosis, self-renewal, and metastasis. Most importantly, primary CSC-enriched spheroidal cultures were eliminated by 3-bromopyruvate. These findings propose that CSCs may be specifically dependent on a high glucose turnover and suggest 3-bromopyruvate for therapeutic intervention.     

类固醇激素在乳腺癌中的作用及对肿瘤干细胞的影响

类固醇激素尤其雌激素和孕激素在乳腺癌的发病、进展和治疗中起着重要作用。与此同时,大量的研究证实肿瘤干细胞在乳腺癌发生、进展和转移过程中也起着关键作用。在乳腺癌临床治疗中,尽管肿瘤细胞经过常规抗内分泌治疗后受到了明显的抑制,但仅需少量的乳腺肿瘤干细胞(Breast cancer stem cells,BCSCs)存在,仍可维持肿瘤的再生长。以往大量的研究多集中在激素对正常乳腺发育及其与乳腺癌的相关性方面,最近,更多的研究观察激素对乳腺肿瘤干细胞活性影响,这为乳腺癌治疗和肿瘤干细胞研究开辟了新的方向。本文全面讨论类固醇激素(雌激素和孕激素)在乳腺癌中的作用及其对乳腺肿瘤干细胞的活性调节。     

The expression of stem cell protein Piwil2 and piR-932 in breast cancer

BackgroundTo investigate the expression status of PIWIL2 and piR-932 in breast cancer stem cells and the role they could play in tumor cell growth and metastasis through Latexin.MethodsCD44⁺/CD24^? tumor cells (CSC) from clinical specimens were sorted using flow cytometry. PIWIL2 expression status was detected in CSC cells by microarray analysis and 1086 breast cancer specimens by Western blot and immunohistochemistry staining. piR-932 expression was also detected in CSC cells by piRNA microarray assay. The relationship between the PIWIL2 protein and clinico-pathological parameters and prognosis was subsequently determined.ResultsCSC cells are more likely to generate new tumors in mice and cell microspheres that are deficient in NOD/SCID compared to the control group. PIWIL2 protein was expressed higher in CSC cells compared to the control cells. In total, 334 (30.76%) of the 1086 breast cases showed high PIWIL2 expression. PIWIL2 was observed to be related to age, tumor size, histological type, tumor stage, and lymph node metastasis (all P < 0.05). Furthermore, we have found that one of the Piwi-interacting RNAs (piRNAs) called piR-932 expressed significantly higher in the breast cancer cells that were induced to EMT, and it could form immune complexes through immunoprecipitation with PIWIL2; in PIWIL2+ breast cancer stem cells, Latexin expression significantly reduced because of its promoter region CpG island methylation.ConclusionsThese results suggest that the combination of piR-932 and PIWIL2 may be a positive regulator in the process of breast cancer stem cells through promoting the methylation of Latexin, and they both could be the potential targets for blocking the metastasis of breast cancer.     

乳腺癌干细胞的分选鉴定

乳腺癌干细胞是一群未分化,具有自我更新、多向分化潜能的细胞。化疗及放疗抵抗性、缺氧耐受性、高致瘤性、高侵袭转移性是这群细胞的特征,成为乳腺癌难以根除、日后复发的一个重要原因。因此正确分选鉴定乳腺癌干细胞是乳腺癌干细胞研究的关键。本文简要介绍了侧群细胞分选技术、细胞表面分子标记的筛选技术、ALDEFLUOR分析法、原位检测法等乳腺癌干细胞分选技术和体外细胞的微球体培养法、有限稀释法及体内动物模型等鉴定技术的研究进展,为后续相关研究提供重要的参考方法,从而探索治疗乳腺癌的新方法。     

Complexity of cancer stem cells

Heterogeneity of tumor tissue has been accounted for in recent years by a hierarchy‐based model in which cancer stem cells (CSCs) have the ability both to self‐renew and to give rise to differentiated tumor cells and are responsible for the overall organization of a tumor. Research into CSCs has progressed rapidly and concomitantly with recent advances in the biology of normal tissue stem cells, resulting in the identification of CSCs in a wide range of human tumors. Studies of mouse models of human cancer have provided further insight into the characteristics of CSCs as well as a basis for the development of novel therapies targeted to these cells. However, recent studies have revealed complexities, such as plasticity of stem cell properties and clonal diversity of CSCs, in certain tumor types that have led to revision of the original CSC model. In this review, we summarize the history of the discovery and characterization of CSCs, as well as address recent advances that have revealed the complexity of these cells and their therapeutic implications.     

Evaluating the link between stem cells and breast cancer

Mammary stem cells have recently been identified and purified on the basis of surface antigens and transplantation assays. In addition, recent reports have identified a small sub-population of highly tumorigenic cells within primary and metastatic breast tumors and in a number of breast cancer cell lines. This suggests that, similarly to its normal physiological counterpart, a cancer stem cell may be at the origin of breast cancer. These observations have dramatic biological and clinical implications, as they dictate a revision of our understanding of breast cancer and of our therapeutic strategies. The aim of this article is to review recent data regarding normal mammary epithelial stem cells and evidence in support of the cancer stem cell hypothesis in the breast, and to provide further insight into how taking this subpopulation of cells into account may affect the way we treat epithelial cancers in the future.     

乳腺癌细胞系中肿瘤干细胞相关亚群的初步研究

目的 通过乳腺癌细胞系及其干细胞的培养,化疗药干预和流式细胞仪筛选鉴定,探讨不同乳腺癌细胞系中CD44⁺CD24^-/low细胞比例,及富集乳腺癌干细胞相关亚群的方法。方法 通过细胞培养乳腺癌细胞系MCF-7、MDA-MB-231,观察生长曲线,比较化疗药物干预下生长情况;利用流式细胞仪检测两种乳腺癌细胞系中CD44⁺CD24^-/low细胞比例;无血清悬浮培养,化疗药(多西紫杉醇、表阿霉素)干预这两种乳腺癌细胞系,观察其是否形成细胞球。结果 (1)MDA-MB-231细胞系倍增时间短,生长速率高于MCF-7细胞系;(2)MCF-7细胞系中可能存在较大比例肿瘤干细胞,其对化疗抵制,能自我更新;(3)化疗敏感性用两独立样本t检验,MCF-7细胞,差异没有统计学意义;MDA-MB-231细胞,差异有统计学意义,提示MDA-MB-231细胞系对该方案化疗较敏感;(4)无血清悬浮培养,MDA-MB-231细胞系未发现明显细胞球;MCF-7细胞系初次无血清培养约6天出现细胞球。加入化疗药筛选后两种细胞,见大部分肿瘤细胞逐渐死亡,未发现明显细胞球;(5)流式细胞仪检测,MCF-7、MDA-MB-231两种细胞系中主要是CD44⁺CD24⁺亚群和CD44^-CD24⁺亚群,CD44⁺CD24^-/low细胞比例分别2.07%和0.20%。结论 (1)MDA-MB-231细胞系增值较快,恶性度相对较高,其对TA联合化疗药物较敏感;MCF-7细胞系中可能存在少量肿瘤干细胞,对化疗抵制,能自我更新;(2)无血清培养液培养MCF-7细胞系能形成悬浮细胞球;流式细胞仪检测两种细胞系中CD44⁺CD24^-/low细胞比例小;(3)CD44⁺CD24^-/low表型可能不是乳腺癌干细胞唯一特异性的表面标志。     

小白菊内酯体内杀伤小鼠乳腺癌肿瘤干细胞

目的： 探讨小白菊内酯（parthenolide,PTL）对小鼠乳腺癌肿瘤干细胞（cancer stem cell,CSC）的杀伤作用,为临床应用PTL治疗乳腺癌提供实验依据。 方法： 采用5-氟尿嘧啶（5-fluorouracil, 5-FU）化疗法制备富含CSC的小鼠4T1细胞乳腺癌模型,随机分为对照组、5-FU组、PTL组。4周后脱颈处死小鼠,检测各组小鼠肿瘤的体积和重量,流式细胞术检测小鼠肿瘤组织中CD44+CD24-/low细胞比例,Hoechst33342染色法检测侧群（side population,SP）细胞的比例,免疫组化法检测CD55和乙醛脱氢酶1（aldehyde dehydrogenase1,ALDH1）蛋白的表达,倒置显微镜观察乳腺癌细胞微球体的形成。 结果： 成功制备富含CSC的小鼠乳腺癌细胞移植瘤模型,PTL可下调小鼠肿瘤组织中CD44+CD24-/low细胞的比例\[（42.5±3.7）% vs （68.7±32）%,P<0.05\],有效降低荷瘤小鼠肿瘤组织中SP细胞的比例\[（39.2±1.8）% vs （61.3±2.6）%,P<0.05\],下调小鼠移植瘤组织中CD55和ALDH1蛋白的表达\[（18.9±1.5）% vs （30.1±1.3）%,（8.1±2.3）% vs （18.0±1.4）%;均P<0.05\],抑制小鼠肿瘤细胞在无血清培养条件下形成微球体,并可抑制小鼠移植瘤的体积和重量\[（0.625±0.159）cm3 vs （1.715±0184）cm3,（1.467±0.373）g vs （3.367±0.398）g;均P<0.05\]。 结论： PTL在荷瘤小鼠体内可以明显降低肿瘤组织CSC含量,提示PTL可用来靶向杀伤乳腺癌CSC。     

Specific inhibition of Notch1 signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells

Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly, Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs, with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival.     

Targeting of cancer stem cells by differentiation therapy

Chemoresistance is a hallmark of cancer stem cells (CSCs). To develop novel therapeutic strategies that target CSCs, we established osteosarcoma‐initiating (OSi) cells by introducing the c‐Myc gene into bone marrow stromal cells derived from Ink4a/Arf KO mice. These OSi cells include bipotent committed cells (similar to osteochondral progenitor cells) with a high tumorigenic activity as well as tripotent cells (similar to mesenchymal stem cells) of low tumorigenicity. We recently showed that the tripotent OSi cells are highly resistant to chemotherapeutic agents, and that depolymerization of the actin cytoskeleton in these cells induces their terminal adipocyte differentiation and suppresses their tumorigenicity. We here provide an overview of modulation of actin cytoskeleton dynamics associated with terminal adipocyte differentiation in osteosarcoma as well as discuss the prospects for new therapeutic strategies that target chemoresistant CSCs by inducing their differentiation.     

Implications of understanding cancer stem cell (CSC) biology in head and neck squamous cell cancer

Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer in the world. Effective therapeutic modalities such as surgery, radiation, chemotherapy and combinations of each are used in the management of this disease. Efforts are ongoing throughout the world to improve early detection and prevention of HNSCCs. Often, treatment fails to obtain total cancer cure and this is more likely with advanced stage disease. In recent years it appears that one of the key determinants of treatment failure may be the presence of cancer stem cells (CSC) that 'escape' currently available therapies. CSCs form a minute portion of the total tumour burden but may play a disproportionately important role in determining outcomes. Molecular mechanisms which underlie the genesis of CSCs are yet not fully understood and their detection within the total tumour bulk remains a challenge. Specific markers like Aldehyde dehydrogenase 1 (ALDH1), CD44 and Bmi-1 have shown early promising results both in CSC detection and in guiding treatment protocols. CSCs have been shown to be relatively resistant to standard treatment modalities. It is hoped that developing robust in vitro and in vivo experimental models of CSCs might provide a means of devising more effective therapeutic strategies.     

Liposome encapsulated curcumin-difluorinated (CDF) inhibits the growth of cisplatin resistant head and neck cancer stem cells

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with 600,000 new cases every year worldwide. Although chemotherapeutics exist, five-year survival is only 50%. New strategies to overcome drug resistance are required to improve HNSCC treatment. Curcumin-difluorinated (CDF), a synthetic analog of curcumin, was packaged in liposomes and used to evaluate growth inhibition of cisplatin resistant HNSCC cell lines CCL-23R and UM-SCC-1R generated from the parental cell lines CCL-23 and UM-SCC-1 respectively. Growth inhibition in vitro and expression levels of the CD44 (cancer stem cell marker), cytokines, and growth factors were investigated after liposomal CDF treatment. The in vivo growth inhibitory effect of liposomal CDF was evaluated in the nude mice xenograft tumor model of UM-SCC-1R and the inhibition of CD44 was measured. Treatment of the resistant cell lines in vitro with liposomal CDF resulted in a statistically significant growth inhibition (p < 0.05). The nude mice xenograft study showed a statistically significant tumor growth inhibition of UM-SCC-1R cells and a reduction in the expression of CD44 (p < 0.05), indicating an inhibitory effect of liposomal CDF on CSCs. Our results demonstrate that delivery of CDF through liposomes may be an effective method for the treatment of cisplatin resistant HNSCC.     

Cancer stem cells in human gastrointestinal cancer

Cancer stem cells (CSCs) are thought to be responsible for tumor initiation, drug and radiation resistance, invasive growth, metastasis, and tumor relapse, which are the main causes of cancer‐related deaths. Gastrointestinal cancers are the most common malignancies and still the most frequent cause of cancer‐related mortality worldwide. Because gastrointestinal CSCs are also thought to be resistant to conventional therapies, an effective and novel cancer treatment is imperative. The first reported CSCs in a gastrointestinal tumor were found in colorectal cancer in 2007. Subsequently, CSCs were reported in other gastrointestinal cancers, such as esophagus, stomach, liver, and pancreas. Specific phenotypes could be used to distinguish CSCs from non‐CSCs. For example, gastrointestinal CSCs express unique surface markers, exist in a side‐population fraction, show high aldehyde dehydrogenase‐1 activity, form tumorspheres when cultured in non‐adherent conditions, and demonstrate high tumorigenic potential in immunocompromised mice. The signal transduction pathways in gastrointestinal CSCs are similar to those involved in normal embryonic development. Moreover, CSCs are modified by the aberrant expression of several microRNAs. Thus, it is very difficult to target gastrointestinal CSCs. This review focuses on the current research on gastrointestinal CSCs and future strategies to abolish the gastrointestinal CSC phenotype.     

Targeting cancer stem cells by curcumin and clinical applications

Curcumin is a well-known dietary polyphenol derived from the rhizomes of turmeric, an Indian spice. The anticancer effect of curcumin has been demonstrated in many cell and animal studies, and recent research has shown that curcumin can target cancer stem cells (CSCs). CSCs are proposed to be responsible for initiating and maintaining cancer, and contribute to recurrence and drug resistance. A number of studies have suggested that curcumin has the potential to target CSCs through regulation of CSC self-renewal pathways (Wnt/β-catenin, Notch, sonic hedgehog) and specific microRNAs involved in acquisition of epithelial–mesenchymal transition (EMT). The potential impact of curcumin, alone or in combination with other anticancer agents, on CSCs was evaluated as well. Furthermore, the safety and tolerability of curcumin have been well-established by numerous clinical studies. Importantly, the low bioavailability of curcumin has been dramatically improved through the use of structural analogues or special formulations. More clinical trials are underway to investigate the efficacy of this promising agent in cancer chemoprevention and therapy. In this article, we review the effects of curcumin on CSC self-renewal pathways and specific microRNAs, as well as its safety and efficacy in recent human studies. In conclusion, curcumin could be a very promising adjunct to traditional cancer treatments.     

乳腺癌干细胞研究进展

乳腺癌干细胞与乳腺癌的放疗抵抗性和化疗耐受性有关,特定表面分子标志与不同乳腺癌分子分型相关.目前,针对乳腺癌干细胞的抑制剂正逐步进入临床试验研究并在临床上使用.乳腺癌干细胞同时也是转移的源泉,它和循环肿瘤细胞之间存在一些共同点.总之,探究乳腺癌干细胞的各种特性有利于开发其在临床的应用.