乳腺癌细胞迁移的调节与转移

细胞迁移是乳腺癌侵袭和转移中的关键步骤之一.癌细胞在迁移过程中主要受到Rho GTPases的调节,发生肌动蛋白骨架重组,获得定向迁移的能力;高迁移能力的癌细胞通过与胞外基质成分相互作用,为迁移创造合适的微环境;最后迁移的癌细胞在靶器官的趋化作用下在特定部位驻足生长,这些环节共同作用导致乳腺癌转移.研究细胞迁移复杂的分子机制将为控制乳腺癌转移提供新的策略.     

Circulating Tumor Cell Clustering Shapes DNA Methylation to Enable Metastasis Seeding

1. Download high-res image (249KB)
2. Download full-size image

     

EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far–including the gold standard CellSearch—rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAM^low/neg cell line and EpCAM^neg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAM^pos (e.g. MCF7, SKBR3) and EpCAM^low/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAM^neg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAM^pos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAM^low/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAM^low/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAM^neg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAM^neg CTCs could be identified [range of 1–24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAM^neg dual-positive (CK^pos/CD45^pos) cells could be traced in 28 out of 29 samples [range 1–480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAM^neg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAM^neg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.     

Microenvironment Promotes Tumor Cell Reprogramming in Human Breast Cancer Cell Lines

The microenvironment drives mammary gland development and function, and may influence significantly both malignant behavior and cell growth of mammary cancer cells. By restoring context, and forcing cells to properly interpret native signals from the microenvironment, the cancer cell aberrant behavior can be quelled, and organization re-established. In order to restore functional and morphological differentiation, human mammary MCF-7 and MDA-MB-231 cancer cells were allowed to grow in a culture medium filled with a 10% of the albumen (EW, Egg White) from unfertilized chicken egg. That unique microenvironment behaves akin a 3D culture and induces MCF-7 cells to produce acini and branching duct-like structures, distinctive of mammary gland differentiation. EW-treated MDA-MB-231 cells developed buds of acini and duct-like structures. Both MCF-7 and MDA-MB-231 cells produced β-casein, a key milk component. Furthermore, E-cadherin expression was reactivated in MDA-MB-231 cells, as a consequence of the increased cdh1 expression; meanwhile β-catenin – a key cytoskeleton component – was displaced behind the inner cell membrane. Such modification hinders the epithelial-mesenchymal transition in MDA-MB-231 cells. This differentiating pathway is supported by the contemporary down-regulation of canonical pluripotency markers (Klf4, Nanog). Given that egg-conditioned medium behaves as a 3D-medium, it is likely that cancer phenotype reversion could be ascribed to the changed interactions between cells and their microenvironment.     

乳腺癌细胞株中MKK4 的表达及其对细胞转移的影响和机理研究

目的:观察乳腺癌细胞株中MKK4蛋白的表达水平,研究MKK4蛋白表达对乳腺癌细胞运动能力及EMT标志物的影响,确定MKK4在肿瘤细胞EMT转化及肿瘤转移中的作用,为肿瘤转移机制研究提供一定的基础资料,为肿瘤防治奠定一定的理论基础。方法:通过体外细胞培养技术收集系列乳腺癌细胞株的培养裂解液,利用Western blot技术检测细胞培养裂解液中MKK4及EMT标志物的表达水平,构建MKK4表达水平与细胞转移能力的对应图;采用siRNA技术,干扰MKK4高表达乳腺癌细胞株MKK4的表达,Western blot技术观察MKK4低表达后,EMT标志物的变化,同时,构建MKK4质粒,转染MKK4低表达乳腺癌细胞株,Western blot技术观察MKK4高表达后,EMT标志物的变化。并采用MTT法、Transwell、划痕法观察MKK4高表达后细胞增殖、运动、迁移等能力的变化。结果:乳腺癌细胞株中MKK4蛋白的表达水平与乳腺癌细胞运动能力有一定的相关性,并与EMT标志物的表达具有相关性,MKK4蛋白表达越高,细胞运动能力越差。干扰或转染技术影响乳腺癌细胞株中MKK4的表达后,细胞EMT标志物的表达也相应变化,乳腺癌细胞株中MKK4高表达后,其细胞增殖明显抑制,运动迁移能力也相应下降。结论:乳腺癌细胞中MKK4蛋白的表达水平与乳腺癌细胞EMT转化及运动能力有一定的相关性,MKK4蛋白表达越高,细胞运动能力越差。     

乳腺癌细胞株中MKK4的表达及其对细胞转移的影响和机理研究

目的：观察乳腺癌细胞株中MKK4蛋白的表达水平,研究MKK4蛋白表达对乳腺癌细胞运动能力及EMT标志物的影响,确定MKK4在肿瘤细胞EMT转化及肿瘤转移中的作用,为肿瘤转移机制研究提供一定的基础资料,为肿瘤防治奠定一定的理论基础。方法：通过体外细胞培养技术收集系列乳腺癌细胞株的培养裂解液,利用Westernblot技术检测细胞培养裂解液中MKK4及EMT标志物的表达水平,构建M鼬（4表达水平与细胞转移能力的对应图;采用siRNA技术,干扰MKK4高表达乳腺癌细胞株MKK4的表达,Westernblot技术观察MKK4低表达后,EMT标志物的变化,同时,构建MKK4质粒,转染MKK4低表达乳腺癌细胞株,Westernblot技术观察MKK4高表达后,EMT标志物的变化。并采用MTT法、Transwell、划痕法观察MKK4高表达后细胞增殖、运动、迁移等能力的变化。结果：乳腺癌细胞株中MKK4蛋白的表达水平与乳腺癌细胞运动能力有一定的相关性,并与EMT标志物的表达具有相关性,MKK4蛋白表达越高,细胞运动能力越差。干扰或转染技术影响乳腺癌细胞株中MKK4的表达后,细胞EMT标志物的表达也相应变化,乳腺癌细胞株中MKK4高表达后,其细胞增殖明显抑制,运动迁移能力也相应下降。结论：乳腺癌细胞中MKK4蛋白的表达水平与乳腺癌细胞EMT转化及运动能力有一定的相关性,MKK4蛋白表达越高,细胞运动能力越差。     

A Novel 3-D Mineralized Tumor Model to Study Breast Cancer Bone Metastasis

Background

Metastatic bone disease is a frequent cause of morbidity in patients with advanced breast cancer, but the role of the bone mineral hydroxyapatite (HA) in this process remains unclear. We have developed a novel mineralized 3-D tumor model and have employed this culture system to systematically investigate the pro-metastatic role of HA under physiologically relevant conditions in vitro.

Methodology/Principal Findings

MDA-MB231 breast cancer cells were cultured within non-mineralized or mineralized polymeric scaffolds fabricated by a gas foaming-particulate leaching technique. Tumor cell adhesion, proliferation, and secretion of pro-osteoclastic interleukin-8 (IL-8) was increased in mineralized tumor models as compared to non-mineralized tumor models, and IL-8 secretion was more pronounced for bone-specific MDA-MB231 subpopulations relative to lung-specific breast cancer cells. These differences were pathologically significant as conditioned media collected from mineralized tumor models promoted osteoclastogenesis in an IL-8 dependent manner. Finally, drug testing and signaling studies with transforming growth factor beta (TGFβ) confirmed the clinical relevance of our culture system and revealed that breast cancer cell behavior is broadly affected by HA.

Conclusions/Significance

Our results indicate that HA promotes features associated with the neoplastic and metastatic growth of breast carcinoma cells in bone and that IL-8 may play an important role in this process. The developed mineralized tumor models may help to reveal the underlying cellular and molecular mechanisms that may ultimately enable more efficacious therapy of patients with advanced breast cancer.     

Detection of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer

PURPOSE: Gastric cancer studies indicated a potential correlation between circulating tumor cells (CTCs) in peripheral blood and tumor relapse/metastasis. The prevalence and significance of circulating tumor microemboli (CTM) in gastric cancer remain unknown. We investigated the prevalence and prognostic value of CTCs and CTM for progression-free survival (PFS) and overall survival (OS) in gastric cancer patients. METHODS:Eighty-one gastric cancer patients consented to provide 5 ml of peripheral blood before systematic therapy. CTCs and CTM were isolated using isolation by size of epithelial tumor cells and characterized by cytopathologists. For 41 stage IV gastric cancer patients, CTM was investigated as a potential biomarker to predict prognosis. RESULTS:CTCs were detected in 51 patients; the average count was 1.81. In clinical stage I, II, III, and IV patients, the average CTC counts were 1.40, 0.67, 1.24, and 2.71, respectively. CTM were detected in 3 of 33 clinical stage I to IIIb patients, at an average of 0.12 (0-2). CTM were detected in 13 of 53 clinical stage IIIc to IV patients, at an average of 1.26 (0-22). In stage IV patients, CTM positivity correlated with the CA125 level. PFS and OS in CTM-positive patients were significantly lower than in CTM-negative patients (P < .001). CTM positivity was an independent factor for determining the PFS (P = .016) and OS (P = .003) of stage IV patients in multivariate analysis. Using markers of the epithelial-mesenchymal transition, single CTCs were divided into three phenotypes including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. For CTM, CK?/Vimentin+/CD45? and CK+/Vimentin+/CD45? phenotypes were observed, but the CK+/Vimentin?/CD45? CTM phenotype was not. CA125 was detected in gastric cancer cell lines BGC823 and MGC803. CONCLUSIONS: In stage IV patients, CTM positivity was correlated with serum CA125 level. CTM were an independent predictor of shorter PFS and OS in stage IV patients. Thus, CTM detection may be a useful tool to predict prognosis in stage IV patients.     

乳腺癌干细胞和乳腺癌

肿瘤干细胞既包含干细胞的特性也包含肿瘤细胞的特性。乳腺癌起源于乳腺癌干细胞的说法能够合理地解释乳腺癌的不均一性及其治疗后的复发,这些变异的干细胞可能作为肿瘤预防策略的靶标。而且,由于乳腺癌干细胞能够抵抗辐射治疗和化学治疗,所以要想更好地治疗乳腺癌就需要寻找针对这些干细胞的靶标。我们综述了乳腺癌干细胞的发现、富集和分离、相关的信号途径,以及在乳腺癌治疗中的应用。     

Circulating Tumor Cell Composition in Renal Cell Carcinoma

PurposeDue to their minimal-invasive yet potentially current character circulating tumor cells (CTC) might be useful as a “liquid biopsy” in solid tumors. However, successful application in metastatic renal cell carcinoma (mRCC) has been very limited so far. High plasticity and heterogeneity of CTC morphology challenges currently available enrichment and detection techniques with EpCAM as the usual surface marker being underrepresented in mRCC. We recently described a method that enables us to identify and characterize non-hematopoietic cells in the peripheral blood stream with varying characteristics and define CTC subgroups that distinctly associate to clinical parameters. With this pilot study we wanted to scrutinize feasibility of this approach and its potential usage in clinical studies.ResultsWe detected CTC with epithelial, mesenchymal, stem cell-like or mixed-cell characteristics at different time-points during anti-angiogenic therapy. The presence and quantity of N-cadherin-positive or CD133-positive CTC was associated with inferior PFS. There was an inverse correlation between high expression of HIF1A, VEGFA, VEGFR and FGFR and the presence of N-cadherin-positive and CD133-positive CTC.ConclusionsPatients with mRCC exhibit distinct CTC profiles that may implicate differences in therapeutic outcome. Prospective evaluation of phenotypic and genetic CTC profiling as prognostic and predictive biomarker in mRCC is warranted.     

Downregulation of MTAP promotes Tumor Growth and Metastasis by regulating ODC Activity in Breast Cancer

5''-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway and has been reported to suppress tumorigenesis. The MTAP gene is located at 9p21, a chromosome region often deleted in breast cancer (BC). However, the clinical and biological significance of MTAP in BC is still unclear. Here, we reported that MTAP was frequently downregulated in 41% (35/85) of primary BCs and 89% (8/9) of BC cell lines. Low expression of MTAP was significantly correlated with a poor survival of BC patients (P=0.0334). Functional studies showed that MTAP was able to suppress both in vitro and in vivo tumorigenic ability of BC cells, including migration, invasion, angiogenesis, tumor growth and metastasis in nude mice with orthotopic xenograft tumor of BC. Mechanistically, we found that downregulation of MTAP could increase the polyamine levels by activating ornithine decarboxylase (ODC). By treating the MTAP-repressing BC cells with specific ODC inhibitor Difluoromethylornithine (DFMO) or treating the MTAP-overexpressing BC cells with additional putrescine, metastasis-promoting or -suppressing phenotype of these MTAP-manipulated cells was significantly reversed, respectively. Taken together, our data suggested that MTAP has a critical metastasis-suppressive role by tightly regulating ODC activity in BC cells, which may serve as a prominent novel therapeutic target for advanced breast cancer treatment.     

乳腺癌患者循环肿瘤细胞与癌干细胞相关性的初步研究

目的：检测乳腺癌患者外周血单核细胞（PBMC）中循环肿瘤细胞（CTC）和具有癌干细胞（CSC）标志的CTC（CSC-CTC）,探讨患者外周血微转移与CSC的相关性。方法：患者和健康者PBMC与磁珠偶联上皮细胞黏附分子单抗孵育后,用磁性分离法富集PBMC中的上皮细胞。以CK＋为患者PBMC中CTC标志,用流式细胞术（FCM）检测健康者和患者的PBMC中CK＋细胞及CK＋/CD44＋/CD24-细胞含量,并比较各组间CTC、CSC-CTC含量的差异。结果：用FCM在73.07%的患者中检测到CTC,在19例检测到CTC的患者中18例有CSC-CTC（94.74%）,CTC中CSC数量比例平均为19.01%,且患者PBMC中CTC和CSC-CTC比例与临床TNM分期相关。结论：初步建立了患者外周血CSC-CTC的检测方法,结果显示乳腺癌患者外周血微转移中有CSC-CTC的参与,临床分期越晚的患者CTC和CSC-CTC的数量越多。     

Circulating Tumor Cells Count and Morphological Features in Breast,Colorectal and Prostate Cancer

Background

Presence of circulating tumor cells (CTC) in patients with metastatic breast, colorectal and prostate cancer is indicative for poor prognosis. An automated CTC (aCTC) algorithm developed previously to eliminate the variability in manual counting of CTC (mCTC) was used to extract morphological features. Here we validated the aCTC algorithm on CTC images from prostate, breast and colorectal cancer patients and investigated the role of quantitative morphological parameters.

Methodology

Stored images of samples from patients with prostate, breast and colorectal cancer, healthy controls, benign breast and colorectal tumors were obtained using the CellSearch system. Images were analyzed for the presence of aCTC and their morphological parameters measured and correlated with survival.

Results

Overall survival hazard ratio was not significantly different for aCTC and mCTC. The number of CTC correlated strongest with survival, whereas CTC size, roundness and apoptosis features reached significance in univariate analysis, but not in multivariate analysis. One aCTC/7.5 ml of blood was found in 7 of 204 healthy controls and 9 of 694 benign tumors. In one patient with benign tumor 2 and another 9 aCTC were detected.

Significance of the study

CTC can be identified and morphological features extracted by an algorithm on images stored by the CellSearch system and strongly correlate with clinical outcome in metastatic breast, colorectal and prostate cancer.     

Efficacy of Lapatinib in Therapy-Resistant HER2-Positive Circulating Tumor Cells in Metastatic Breast Cancer

Background

To evaluate the efficacy of lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, in therapy-resistant HER2-positive CTCs in metastatic breast cancer (MBC).

Patients and Methods

Patients with MBC and HER2-positive CTCs despite disease stabilization or response to prior therapy, received lapatinib 1500 mg daily in monthly cycles, till disease progression or CTC increase. CTC monitoring was performed by immunofluorescent microscopy using cytospins of peripheral blood mononuclear cells (PBMCs) double stained for HER2 or EGFR and cytokeratin.

Results

A total of 120 cycles were administered in 22 patients; median age was 62.5 years, 15 (68.2%) patients were post-menopausal and 20 (90.1%) had HER2-negative primary tumors. At the end of the second course, HER2-positive CTC counts decreased in 76.2% of patients; the median number of HER2-positive CTCs/patient also declined significantly (p = 0.013), however the decrease was significant only among patients presenting disease stabilization (p = 0.018) but not among those with disease progression during lapatinib treatment. No objective responses were observed. All CTC-positive patients harbored EGFR-positive CTCs on progression compared to 62.5% at baseline (p = 0.054). The ratio of EGFR-positive CTCs/total CTCs detected in all patients increased from 17.1% at baseline to 37.6% on progression, whereas the mean percentage of HER2-negative CTCs/patient increased from 2.4% to 30.6% (p = 0.03).

Conclusions

The above results indicate that lapatinib is effective in decreasing HER2-positive CTCs in patients with MBC irrespectively of the HER2 status of the primary tumor and imply the feasibility of monitoring the molecular changes on CTCs during treatment with targeted agents.

Trial Registration

Clinical trial.gov NCT00694252     

乳腺癌转移相关基因的筛选与生物信息学分析

乳腺癌是女性最常见的恶性肿瘤,转移与复发是乳腺癌患者死亡的主要原因. 研究与乳腺癌细胞转移相关的分子靶点对预防乳腺癌术后复发、提高疗效有重要意义. 本研究以3组乳腺癌转移相关的基因表达谱数据(GSE2034, GSE2603, GSE12276)为分析材料,采用GeneSpring软件筛选乳腺癌原发瘤与转移瘤芯片数据的差异表达基因,结合生物信息学工具PATHER、STRING、pSTIING和文献挖掘工具iHOP对差异基因及其相互作用关系进行分析. 结果显示,共筛选出乳腺癌转移共同差异基因147个,其中表达上调93个,表达下调54个. 这些差异基因主要涉及细胞周期与增殖、细胞粘附、细胞迁移、血管形成及信号转导等生物通路和生物过程. 差异基因编码蛋白间的相互作用主要集中在14个蛋白,且在更为复杂的网络图谱中仍可见其中9个基因（CXCR4、MMP1、MMP2、MMP3、CTGF、COL1A1、MEF2C、PTGS2及SPARC）在重要的节点位置. 文献挖掘发现,COL1A1基因可能为新发现的乳腺癌转移候选基因,为乳腺癌转移的发病机制提供新的思路,也为转移性乳腺癌的分子诊断和个体化治疗奠定基础.