Multiplexing Angiogenic Receptor Quantification via Quantum Dots
Multiplexing Angiogenic Receptor Quantification via Quantum Dots
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DOI：
10.1021/acs.analchem.9b00238
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发表时间：
2019-06-18
影响因子：
7.4
通讯作者：
Imoukhuede, P. I.
中科院分区：
文献类型：
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作者：
Chen, Si;Imoukhuede, P. I.
临床和生物医学研究寻求单细胞定量，以更好地了解它们在复杂的多细胞环境中的作用。最近，血管内皮生长因子受体（VEGF）的定量提供了重要的见解内皮细胞的特性和肿瘤微环境中的反应。然而，用于定量质膜受体酪氨酸激酶（RTK）的现有技术缺乏多路复用能力，限制了详细的表征。在这里，我们利用量子点（Qdot）的独特光谱特性来优化和双重量化人脐静脉内皮细胞（HUVEC）上的VEGFR 1和VEGFR 2。为了实现这种定量，我们通过缓冲液优化减少Qdot缀合的抗体和细胞之间的非特异性结合。其次，我们通过检查Qdot结合抗体与五种受体的结合来确定最佳标记条件：VEGF受体（VEGFR 1和VEGFR 2），其辅助受体神经纤毛蛋白1（NRP 1）和血小板衍生生长因子受体（PDGFR α和PDGFR β）。我们确定800-20 000是可以实现精确Qdot启用量化的动态范围。通过这些优化，我们证明了每个HUVEC测量1 100个VEGFR 1和6 900个VEGFR 2。我们通过24小时VEGF-A处理诱导了类似于90%的VEGFR 1上调和类似于30%的VEGFR 2下调（165）。我们观察到VEGF-B-167处理24小时后VEGFR 1或VEGFR 2浓度没有变化。我们进一步应用Qdots分析HUVEC异质性，并观察到24 h VEGF-A（165）处理诱导VEGFR 2异质性降低15%，但VEGFR 1异质性几乎没有变化。我们观察到VEGF-B-167对VEGFR 1或VEGFR 2的异质性几乎没有诱导变化。总的来说，我们展示了使用Qdots在单水平上量化两个或多个RTK的实验和分析策略，这将有助于为生物系统提供新的见解。
Clinical and biomedical research seeks single-cell quantification to better understand their roles in a complex, multicell environment. Recently, quantification of vascular endothelial growth factor receptors (VEGFRs) provided important insights into endothelial cell characteristics and response in tumor microenvironments. However, existing technologies for quantifying plasma membrane receptor tyrosine kinases (RTKs) lack multiplexing capabilities, limiting detailed characterization. Here, we use the unique spectral properties of quantum dots (Qdots) to optimize and dually quantify VEGFR1 and VEGFR2 on human umbilical vein endothelial cells (HUVECs). To enable this quantification, we reduce nonspecific binding between Qdot-conjugated antibodies and cells via buffer optimization. Second, we identify optimal labeling conditions by examining Qdot-conjugated antibody binding to five receptors: VEGFRs (VEGFR1 and VEGFR2), their coreceptor neuropilin1 (NRP1), and platelet-derived growth factor receptor (PDGFR alpha and PDGFR beta). We establish that 800-20 000 is the dynamic range where accurate Qdot-enabled quantification can be achieved. Through these optimizations, we demonstrate measurement of 1 100 VEGFR1 and 6 900 VEGFR2 per HUVEC. We induce similar to 90% upregulation of VEGFR1 and similar to 30% downregulation of VEGFR2 concentration via 24 h VEGF-A(165) treatment. We observe no change in VEGFR1 or VEGFR2 concentration with 24 h VEGF-B-167 treatment. We further apply Qdots to analyze HUVEC heterogeneity and observe that 24 h VEGF-A(165) treatment induces a similar to 15% decrease in VEGFR2 heterogeneity, but little to no change in VEGFR1 heterogeneity. We observe that VEGF-B-167 induces little to no change in either VEGFR1 or VEGFR2 heterogeneity. Overall, we demonstrate experimental and analytical strategies for quantifying two or more RTKs at single-level using Qdots, which will help provide new insights into biological systems.