Simultaneous isolation and detection of circulating tumor cells with a microfluidic silicon-nanowire-array integrated with magnetic upconversion nanoprobes

The development of sensitive and convenient methods for detection, enrichment, and analysis of circulating tumor cells (CTCs), which serve as an importance diagnostic indicator for metastatic progression of cancer, has received tremendous attention in recent years. In this work, a new approach characteristic of simultaneous CTC capture and detection is developed by integrating a microfluidic silicon nanowire (SiNW) array with multifunctional magnetic upconversion nanoparticles (MUNPs). The MUNPs were conjugated with anti-EpCAM antibody, thus capable to specifically recognize tumor cells in the blood samples and pull them down under an external magnetic field. The capture efficiency of CTCs was further improved by the integration with a microfluidic SiNW array. Due to the autofluorescence free nature in upconversion luminescence (UCL) imaging, our approach allows for highly sensitive detection of small numbers of tumor cells, which afterward could be collected for further analysis and re-culturing. We have further demonstrated that this approach can be applied to detect CTCs in clinical blood samples from lung cancer patients, and obtained consistent results by analyzing the UCL signals and the clinical outcomes of lung cancer metastasis. Therefore our approach represents a promising platform in CTC capture and detection with potential clinical utilization in cancer diagnosis and prognosis.     

Microdevice for the isolation and enumeration of cancer cells from blood

Cancer metastasis is the main attribute to cancer-related deaths. Furthermore, clinical reports have shown a strong correlation between the disease development and number of circulating tumor cells (CTCs) in the peripheral blood of cancer patients. Here, we present a label-free microdevice capable of isolating cancer cells from whole blood via their distinctively different physical properties such as deformability and size. The isolation efficiency is at least 80% for tests performed on breast and colon cancer cells. Viable isolated cells are also obtained which may give further insights to the understanding of the metastatic process. Contrasting with conventional biochemical techniques, the uniqueness of this microdevice lies in the mechanistic and efficient means of isolating viable cancer cells in blood. The microdevice has the potential to be used for routine monitoring of cancer development and cancer therapy in a clinical setting. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A fast and sensitive method for isolation of detergent-resistant membranes from T cells

We describe a fast and sensitive method for isolation of detergent-resistant membranes (DRMs) from T cells by sucrose density gradient centrifugation using a smaller accumulated centrifugal force in a tabletop ultracentrifuge. Compared to previous reports, this method, which requires less biological material, is faster and permits quantitative separation of DRMs from other cellular membranes with good resolution. The method, which can be completed in 6 h, yields more than 80% of the total content of DRM-associated adaptor molecules LAT (linker for T cell activation), PAG/Cbp (protein associated with glycosphingolipid-enriched microdomains or Csk-binding protein) and LIME (Lck-interacting membrane protein) in low-density fractions using only 2x10(7) T cells.     

Detection of Survivin-expressing circulating cancer cells in the peripheral blood of breast cancer patients by a RT-PCR ELISA

Survivin mRNA expression was detected in 69.2%–93.8% of primary breast carcinomas, but is rarely expressed in normal breast tissues and hematopoietic cells. The objective of this study was to investigate the significance that the detection of Survinin-expressing circulating breast cancer cells in the peripheral blood has on clinical outcomes. The detection method was based on a RT-PCR ELISA technique developed in our laboratory. Sixty-seven breast cancer patients in various stages and 135 normal healthy women were investigated. Survivin-expressing circulating cancer cells were detected in the peripheral blood samples from 34 (50.7%) out of 67 breast cancer patients, but not in the healthy women that were used as controls. The presence of Survivin-expressing circulating breast cancer cells was found to be significantly associated with various clinicopathological parameters such as vessel infiltration, histological grade, tumor size, nodal status, ER/PgR status, Her-2 status and clinical stages of the disease (P < 0.01). During a follow-up period of 36 months, 9 out of 11 (81.8%) breast cancer patients that had a positive Survivin-expressing at the time of the initial assay test suffered a relapse of the disease, whereas recurrence was only found in 2 out of 6 (33.3%) breast cancer patients that had a negative Survivin-expression. Thus, the detection of circulating cancer cells expressing Survivin mRNA could provide valuable information for the prediction of metastasis and recurrence of breast cancer.     

An improved method for the isolation and culture of rat epidermal stem cells

The management of burns and injuries using novel treatment strategies involving epidermal stem cells (ESC) requires a better understanding of the biology of these cells, in particular, their isolation and the maintenance of their unique characteristics in culture. The purpose of this study was to describe an improved method for isolating putative ESC from fetal rat skin and to maintain them long term in culture. Single ESC suspensions were obtained from fetal rat skin by enzyme digestion containing 0.5% neutral protease. The target cells were harvested by rapid adherence on type IV collagen plates and were cultured in complex DMEM. After primary isolation, cells were continuously cultured in K-serum free medium. After reaching 70-80% confluence, the cells were digested with 0.25% trypsin at 37°C for 5-10 minutes, and passaged at a ratio of 1:2. The cultured ESC showed good growth, resulting in cell viability of over 98%. Four days later, clones containing 100-200 cells were detected, showing cobblestone-like characteristics. The rapidly adherent cells were positive for keratin 15, 19 and P63. Eighty three percent of cells expressed β1 integrin. The growth-curve showed that the rapidly adherent cells were in the exponential growth phase. The protocol described in this paper provides a simplified and effective method to isolate and maintain long-term culture of epidermal stem cells from fetal rat skin. This method should be valuable for isolating and studying ESC from various transgenic rat lines that are currently available.     

胎儿骨髓间充质干细胞的分离培养及表面标志的检测

目的 建立胎儿骨髓间充质干细胞（mesenchymal stem cells,MSC)的分离培养方法，并对其表面标志进行检测。方法 采用体外细胞培养技术，分离培养胎儿骨髓MSC，用流式细胞术对其进行鉴定，并研究其增殖及生长特征。结果 流式细胞术证明，MSC具有间质细胞的特征。原代及传代培养显示，胎儿骨髓MSC具有活跃增殖的能力。结论 胎儿骨髓MSC作为组织工程重建的种子细胞具有较强的可行性。     

Immunomagnetic separation for enrichment and sensitive detection of disseminated tumour cells in patients with head and neck SCC.

Screening for malignant cells in the blood and bone marrow was introduced as a strategy for the improved detection of tumour spread and may predict the development of distant metastases. The sensitivity of these approaches depends on several factors, including the choice of antibody for immunocytochemistry (ICC) and the number of cells examined. In this study criteria have been defined for scoring cells reactive with a pan-cytokeratin antibody as tumour, by comparing immunostained cells in clinical samples obtained from head and neck cancer patients and a control group without epithelial malignancy. When leucocyte subfractions are prepared by density gradient separation (DGS) from central venous blood obtained from patients with advanced head and neck squamous cell carcinoma (SCC) and screened by ICC, epithelial tumour cells sediment preferentially with the mononuclear cells but may also be detected in the granulocyte (GC) fraction. Some cases were found to have more tumour cells in the GC fraction. Similar results were seen in model experiments. To increase the sensitivity of the ICC approach, the efficiency of positive immunomagnetic selection (IMS) using Dynabeads coated with an antibody recognizing the Ber-EP4 epitope has been compared with negative IMS using anti-CD45 Dynabeads. Tumour cells were recovered from bone marrow aspirates for 2/17 cases using the positive enrichment technique and for 11/17 patients following negative IMS. These findings justify prospective studies incorporating negative IMS to establish the prognostic significance of these disseminated tumour cells for this group of patients.     

小鼠肝脏窦状内皮细胞分离和培养的一种新方法

目的：建立小鼠肝窦状内皮细胞(liver sinusoidal endothelial cells, LSEC)的分离培养方法, 研究其生物学特性。方法：中性蛋白酶消化小鼠肝脏组织,Percoll密度梯度离心分离消化后的细胞悬液,用内皮细胞筛选培养基及酶差异消化法纯化LSEC;免疫细胞化学染色检测LSEC的表面标志,分析LSEC的超微结构,观察DiI荧光标记的乙酰低密度脂蛋白（acetylated low density lipoprotein,DiI Ac LDL）摄取能力,以体外成血管能力分析LSEC的功能。结果：分离纯化后的细胞呈典型鹅卵石样内皮细胞形态,这些细胞表达VIII因子,不表达CD31,CD90和CK19,具有窦状内皮细胞特征性的窗孔结构;能摄取DiI Ac LDL,并有一定的成血管能力。结论：建立了一种分离、培养LSEC的方法,为研究LSEC的功能奠定了基础。     

MDSCs drive the process of endometriosis by enhancing angiogenesis and are a new potential therapeutic target

Endometriosis affects women of reproductive age via unclear immunological mechanism(s). Myeloid‐derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive and angiogenic properties. Here, we found MDSCs significantly increased in the peripheral blood of patients with endometriosis and in the peritoneal cavity of a mouse model of surgically induced endometriosis. Majority of MDSCs were granulocytic, produced ROS, and arginase, and suppressed T‐cell proliferation. Depletion of MDSCs by antiGr‐1 antibody dramatically suppressed development of endometrial lesions in mice. The chemokines CXCL1, 2, and 5 were expressed at sites of lesion while MDSCs expressed CXCR‐2. These CXC‐chemokines promoted MDSC migration toward endometriotic implants both in vitro and in vivo. Also, CXCR2‐deficient mice show significantly decreased MDSC induction, endometrial lesions, and angiogenesis. Importantly, adoptive transfer of MDSCs into CXCR2‐KO mice restored endometriotic growth and angiogenesis. Together, this study demonstrates that MDSCs play a role in the pathogenesis of endometriosis and identifies a novel CXC‐chemokine and receptor for the recruitment of MDSCs, thereby providing a potential target for endometriosis treatment.     

不同类型中药制剂逆转结直肠癌T细胞免疫抑制及其靶分子的比较研究

目的比较研究不同类型抗瘤中药制剂对结直肠癌所致T细胞免疫抑制的影响,寻找能有效逆转结直肠癌T细胞免疫抑制的抗瘤中药制剂组方。方法分别制备三氧化二砷(As2O3)、川芎嗪(LHC)、黄芪(AMB)、氧化苦参碱(MOX)、猪苓多糖(PUPS)、青蒿琥酯(ART)等6种中药制剂作用后的小鼠结直肠癌Colon26细胞再培养上清;MTT法检测中药制剂作用后对Colon26所致T细胞转化抑制的影响,直接免疫荧光FCM法检测对IL-2Rα及CD3ε+ζ+表达抑制的影响;定量ELISA法测定上清中TGF-β1、VEGF、IL-4、IL-6、IL-10和PGE2含量;多元相关分析中药制剂逆转结直肠癌T细胞免疫抑制的相应靶分子。结果 AMB、PUPS及ART对T细胞转化抑制的下调程度较强(下调约50%),维持时间较长;ART对IL-2Rα表达抑制的下调作用最强(下调约50%),维持时间较长;AMB对CD3ε+ζ+表达抑制的下调作用最强,不但可完全消除表达抑制,还可进一步促进表达,维持时间亦较持久。对Colon26肿瘤细胞所致转化抑制及CD3ε+ζ+表达抑制的逆转,LHC的作用靶分子为TGF-β1和IL-10,其他5种中药制剂的靶分子均为TGF-β1。对IL-2Rα表达抑制的逆转,除AMB以外其他5种中药制剂的靶分子均为TGF-β1。结论通过阻碍肿瘤细胞分泌TGF-β1、IL-10等免疫抑制分子,以逆转肿瘤细胞产生的T细胞免疫抑制,应是中药制剂的新型抗瘤机制之一。     

Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy

The fundamental role of dendritic cells (DC) in initiating and directing the primary immune response is well established. Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and infectious diseases. As blood DC (BDC) physiology differs from that of the DC homologues generated in vitro from monocyte precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package (Version 6.0 AutoPBPC) provides an operator-independent reliable source of mononuclear cells (MNC) for BDC preparation. Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating BDC predicted by blood volume. An average of 66 million (range, 17-179) BDC per 10-l procedure were obtained, largely satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 15 l. BDC were not activated by the isolation procedure and, more importantly, both BDC subsets (CD11c(+)CD123(low) and CD11c(-)CD123(high)) were equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection and demonstrated that fully functional BDC can be obtained by this procedure.     

Development and validation of a duplex quantitative real-time RT-PCR assay for simultaneous detection and quantitation of foot-and-mouth disease viral positive-stranded RNAs and negative-stranded RNAs

A simplified, cost-effective, two-step duplex quantitative real-time RT-PCR assay was shown to detect and quantify foot-and-mouth disease virus positive-stranded RNAs and negative-stranded RNAs simultaneously for improved investigation of the state of virus infection and replication. The primers and Taqman probes were selected from the coding regions of 2B gene and 3D gene respectively, which have the least variations among serotypes. Cells infected acutely, tissue samples and single cell samples were used for evaluation of the assay. At the early stages of virus infection in vitro, the replication level reached a peak at 9 h.p.i. and the negative strands were detectable until 3 h.p.i. The kinetics of ratios of positive strands to negative strands (+RNA/−RNA) in vivo in the liver, kidney and spleen were similar, which demonstrated that the replication dynamics were similar in the three organs. 55 single cell samples out of 187 were positive by both positive strands qPCR and negative strands qPCR, the ratios (+RNA/−RNA) ranged from 15.6 to 1463.4 which showed considerable difference among single cell samples, indicating that active viral replication differs greatly in single cells. A duplex quantitative real-time RT-PCR was validated as effective and reliable.     

一种分离、培养扩增小鼠NK细胞方法的建立

目的:建立小鼠NK细胞的分离及培养扩增的方法。方法:用两步黏附分离法和磁珠活化的细胞分选(MACS)法,从小鼠脾脏的单个核细胞(MNC)中分离NK细胞,计数所得细胞的总数,并用FITC抗小鼠CD3和PE抗小鼠NK1.1单克隆荧光抗体染色后,用流式细胞仪(FACS)检测NK细胞的纯度。以YAC -1为靶细胞,用MTT比色法检测NK细胞的杀伤活性。结果:将两步黏附分离法分离的2×107个脾MNC培养5、10、15、20d,计数细胞的总数并检测CD3-NK1.1 细胞百分率,分别为0.5×107、1.4×107、2.6×107、3.0×107和18.36%、43.44%、55.68%、60.03%。效靶细胞25∶1时,NK细胞的杀伤率分别为54.38%、66.54%、79.38%和83.86%,明显高于脾MNC(41.93%)(P<0.01)。MACS法纯化前后细胞的总数和CD3-NK1.1 细胞的百分率,分别为1.0×108、1.5×106和2.54%、93.60%;但纯化后的细胞在体外培养20d时,只扩增到1.9×106个。结论:用两步黏附分离法分离的小鼠NK细胞,培养2～3wk可获得大量的NK细胞,纯度可达55%～60%;在效靶细胞为25∶1时,NK细胞对YAC1细胞的杀伤率约为80%。     

识别胸腺髓质上皮细胞和胸腺树突状细胞的单链抗体的筛选

目的　利用噬菌体展示技术寻找胸腺基质细胞表面的新抗原。方法　用培养的胸腺基质细胞系MTDC作为靶抗原富集初级噬菌体抗体库 ,从经过富集后的次级抗体库中挑选克隆 ,制备单链抗体 ,并在冰冻切片及培养的细胞系上检测抗体的特异性。结果　从经 4轮富集的次级抗体库中挑选到一个新的克隆。用此克隆制备的单链抗体可同时辨认胸腺髓质上皮细胞亚群和胸腺树突状细胞亚群。结论　胸腺髓质上皮细胞和胸腺树突状细胞均具有异质性 ,同时噬菌体展示技术可以作为寻找细胞表面新分子的强有力工具。     

人上皮性卵巢癌细胞系侧群细胞的肿瘤干细胞样细胞生物学特性及膜蛋白差异表达的鉴定

目的 鉴定上皮性卵巢癌细胞侧群细胞(SP细胞)肿瘤干细胞样细胞生物学特性,并检测侧群细胞内差异蛋白质的表达.方法 流式分选上皮性卵巢癌细胞系SKOV3和A2780具有外泌Hochest33342染料生物学特性的侧群细胞,鉴定其肿瘤干细胞样细胞相关生物学特性.运用细胞培养稳定同位素标记(SILAC)的定量蛋白质组学技术,...