Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells

We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein–protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0–5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein–protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks.Although signal transduction inhibitors occasionally offer clinical benefit for cancer patients (1), signal flux emanating from oncogenes is often distributed through multiple pathways (2), potentially underlying the failure of most such inhibitors (3). Measuring signal flux through multiple pathways, in response to signal transduction inhibitors, may help uncover network interactions that contribute to therapeutic resistance and that are not predicted by analyzing pathways in isolation (4). The cellular and molecular complexity of a solid tumor microenvironment (5) suggests the need to study signaling in individual cancer cells.Protein–protein interactions within signaling pathways are often elucidated by assessing the levels of relevant pathway proteins in model and tumor-derived cell lines and with various genetic and molecular perturbations. Such interactions, and the implied signaling networks, may also be elucidated via quantitative measurements of multiple pathway-related proteins within single cells (6). At the single-cell level, inhibitory and activating protein–protein relationships, as well as stochastic (single-cell) fluctuations, are revealed. However, most techniques for profiling signaling pathways (7, 8) require large numbers of cells. Single-cell immunostaining (9) is promising, and some flow cytometry (6) techniques are relevant, as discussed below.We describe quantitative, multiplex assays of intracellular signaling proteins from single cancer cells using a platform called the single-cell barcode chip (SCBC). The SCBC is simple in concept: A single or defined number of cells is isolated within an approximately 2 nL volume microchamber that contains an antibody array (10) for the capture and detection of a panel of proteins. The SCBC design (11) permits lysis of each individual trapped cell.Intracellular staining flow cytometry can assay up to 11 phosphoproteins from single cells (6). Our SCBC can profile a similar size panel, but only for approximately 100 single cells per chip. Each protein is assayed twice, yielding some statistical assessment for each experiment. The SCBC is a relatively simple platform and only requires a few hundred cells per assay.We used the SCBC to study signal transduction in glioblastoma multiforme (GBM), a primary malignant brain tumor (12). GBM has been genetically characterized, yet the nature of signaling pathways downstream of key oncogenic mutations, such as epidermal growth factor receptor activating mutation (EGFRvIII) and phosphatase and tensin homolog (PTEN) tumor suppressor gene loss associated with receptor tyrosine kinase (RTK)/PI3K signaling, are incompletely understood (13–15). Single-cell experiments may also help resolve the characteristic heterogeneity of GBM.We interrogated 11 proteins directly or potentially associated with PI3K signaling (see SI Appendix, Methods I) through three isogenic GBM cell lines: U87 (expressing wild-type p53, mutant PTEN, and low levels of wild-type EGFR, no EGFRvIII) (16, 17), U87 EGFRvIII (U87 cells stably expressing EGFRvIII deletion mutant), and U87 EGFRvIII PTEN (U87 cells coexpressing EGFRvIII and PTEN) (18). Fig. 1 diagrams this biology. Each cell line was investigated under conditions of standard cell culture, in response to EGF stimulation, and after erlotinib treatment followed by EGF stimulation. The proteins assayed represented RTKs and proteins signifying activation of PI3K and MAPK signaling. They were (p- denotes phosphorylation) p-Src, p-mammalian target of rapamycin (p-mTOR), p-p70 ribosomal protein S6 kinase (p-p70S6K), p-glycogen synthase kinase-3 (p-GSK-3α/β), p-p38 mitogen activated protein kinase (p-p38α), p-extracellular regulated kinase (p-ERK), p-c-Jun N-terminal kinase (p-JNK2), p-platelet derived growth factor receptor β (p-PDGFRβ), p-vascular endothelial growth factor receptor 2 (p-VEGFR2), tumor protein 53 (P53), and total EGFR.Open in a separate windowFig. 1.The PI3K pathway activated by EGF-stimulated EGFR or by the constitutively activated EGFRvIII. All proteins in light blue with central yellow background were assayed. Orange background proteins were expressed in the cell lines U87 EGFRvIII or U87 EGFRvIII PTEN. The oval, yellow background components are the investigated molecular perturbations.     

Analysis of 13 cases of venous compromise in 178 radial forearm free flaps for intraoral reconstruction

The purpose of this study was to analyse the causes of venous compromise and flap failure in radial forearm free flap (RFFF) surgery for intraoral reconstruction. One hundred seventy-eight RFFF reconstructions were reviewed retrospectively for intraoral defects. Of the 13 flaps with venous obstruction, 9 flaps were salvaged, and 4 were lost, with a salvage rate of 69.2%. Eleven venous occlusions occurred within the first 72h. The main reasons for venous failure were mechanical obstruction or technical errors due to inadequate pedicle length and geometry, inadequate venous drainage, compression and kinking of the vein. The main cause of failure for oropharynx reconstruction was unrecognized vascular events due to the lack of reliable monitoring for buried flap. Oozing of dusky blood from the flap margin may be directly related to venous congestion in the early postoperative period and a late indication of a change in skin colour. In conclusion, a thorough operative plan, including carefully selected drainage vein for the flap and recipient vessels, adequate pedicle length and geometry, precise surgical technique, avoidance of haematoma, and expert monitoring of buried flaps may improve the success rate of RFFF transfer in intraoral reconstruction.     

嗜酸乳杆菌对人舌癌细胞增殖的影响

目的研究嗜酸乳杆菌对人舌癌细胞Tca8113增殖及细胞周期的影响。方法体外培养Tca8113细胞,分别将不同稀释度（原液和4、16倍稀释）的嗜酸乳杆菌上清液、灭活菌液和无细胞提取物与Tca8113细胞共培养,采用倒置显微镜观察细胞形态并行细胞计数,磺酰罗丹明B（SRB）法测定细胞增殖率,流式细胞术分析嗜酸乳杆菌各组分对Tca8113细胞增殖及细胞周期的影响,激光扫描共聚焦显微镜（CLSM）检测细胞内自由基和Ca2+含量。结果嗜酸乳杆菌各组分作用于Tca8113细胞48 h后,在倒置显微镜下观察,细胞由菱形、多角形、铺路石状变为细长形。细胞计数与SRB实验分析：在不同稀释度同一培养时间与不同培养时间同一稀释度培养条件下,嗜酸乳杆菌各组分均可明显抑制Tca8113细胞增殖,抑制力随稀释度增加而降低,随培养时间延长而增强。流式细胞术分析：嗜酸乳杆菌各组分作用Tca8113细胞48 h后,细胞增殖指数降低（P<0.01）。CLSM检测：嗜酸乳杆菌各组分作用Tca8113细胞48 h后细胞内自由基和Ca2+含量均升高（P<0.01）。结论嗜酸乳杆菌代谢产物、灭活菌液、无细胞提取物均可抑制Tca8113细胞增殖,可能与菌体及其代谢产物引起细胞内自由基含量增多、Ca2+超载有关。     

模拟口腔环境下温控型镍钛弓丝力学性能的实验研究

目的研究温控型镍钛弓丝在不同温度人工唾液环境下的力学性能变化,为临床合理、有效地使用温控型镍钛弓丝提供参考。方法利用Instron万能材料力学实验机在25、33、37、60 ℃人工唾液中对4种温控型镍钛弓丝进行改良部分牙弓托槽弯曲实验。将4种温控型镍钛弓丝加载挠曲至3 mm后卸载,绘制弓丝卸载的载荷-挠曲曲线,计算卸载至2.5 mm与0.5 mm之间的卸载刚度,结果采用单因素方差分析法进行统计分析。结果随着温度的升高,4种弓丝的卸载力值均有不同程度的升高;同种弓丝的卸载刚度在25、33、37 ℃时十分接近,而在60 ℃时卸载刚度均有明显增大。结论在本实验条件下,4种温控型镍钛弓丝表现出不同的力学性能,但均表现出临床所需的超弹性与形状记忆效应,也均表现出明显的温度敏感性。