Integrated Imaging Tools for Intercellular Chemokine Signalling

趋化因子及其G蛋白偶联受体(GPCRs)是细胞间相互作用的关键介质 癌细胞和多种基质细胞类型之间的信号转导。癌细胞增殖，局部 侵袭、全身转移、免疫细胞募集和血管生成都受到 趋化因子的时空动态。趋化因子在肿瘤生物学中的中心作用 使它们成为抑制或增强趋化因子的有吸引力的治疗目标 发信号。然而，趋化因子的许多复杂的生物化学仍有待发现， 由于追踪它们在生命系统中的存在和功能的技术挑战。 趋化因子在非常低的水平上有效地发出信号，而且它们的空间分布是复杂的。 由于它们能够结合到细胞表面和细胞外基质上。此外，企业的内部化 GPCRs和清道夫受体产生的趋化因子通过以下途径形成局部趋化因子梯度 隔离和降解趋化因子。实验证据和计算模型 我们的实验室表明，趋化因子CXCL12的短距离、陡峭的局部梯度在 单细胞通过受体CXCR4最有效地驱动信号和趋化作用。要启用 趋化因子的变革性研究和最终推进药物靶向的应用 关于癌症中的趋化因子，我们建议开发一套新的定量分子成像工具 以测量趋化因子的空间和时间动态分布、关联和 在数百个单细胞中发出信号。这些工具将使我们能够测量细胞信号模式 在趋化过程中，并确定潜在的机制细胞间的异质性 对趋化因子的反应。我们将1)产生集成的、多路传输的生物发光 用于细胞外和细胞内步骤单细胞成像的荧光显微镜工具箱 趋化因子信号；以及2)测试我们的趋化因子成像技术作为一种通用的、 定量方法适用于患者来源的细胞。我们将独一无二地将动态、 单细胞生物发光和荧光显微镜技术 细胞用来将趋化因子输入转化为信号输出和趋化的分子事件。 我们将把我们的多路传输图像读数与先进的图像处理方法相结合，以 生成趋化因子基础研究所需的高维量化数据集 与其他大规模数据的集成。我们的创新成像工具将允许前所未有的 复杂环境中细胞反应的趋化因子梯度和异质性的观点。 这项技术将改变癌症中趋化因子的研究，并推动正在进行的 针对趋化因子进行治疗的努力。

Chemokines and their G-protein coupled receptors (GPCRs) are key mediators of intercellular signaling between cancer cells and multiple stromal cell types. Cancer cell proliferation, local invasion, systemic metastasis, recruitment of immune cells and angiogenesis all are regulated by spatial and temporal dynamics of chemokines. The central role of chemokines in tumor biology has made them an attractive target for therapies designed to inhibit or enhance chemokine signaling. However, much of the complex biochemistry of chemokines remains to be discovered, due to the technical challenges of tracing their presence and function in living systems. Chemokines are effective at signaling at very low levels, and their spatial distribution is complex due to their ability to bind to cell surfaces and extracellular matrix. Furthermore, internalization of chemokines by GPCRs and scavenger receptors shape local chemokine gradients by sequestering and degrading chemokines. Experimental evidence and computational modeling by our lab show that short-range, steep local gradients of chemokine CXCL12 over distances of a single cell most effectively drive signaling and chemotaxis through receptor CXCR4. To enable transformative studies of chemokines and ultimately advance applications of drugs targeting chemokines in cancer, we propose to develop a new suite of quantitative molecular imaging tools to measure the spatial and temporal dynamics of chemokine distribution, association, and signaling in hundreds of single cells. These tools will allow us to measure cell signaling patterns during chemotaxis and identify potential mechanisms underlying intercellular heterogeneity in responses to chemokines. We will 1) Generate an integrated, multiplexed bioluminescence and fluorescence microscopy toolbox for single-cell imaging of extracellular and intracellular steps in chemokine signaling; and 2) Test our chemokine imaging technology as a generalizable, quantitative approach applicable to patient-derived cells. We will uniquely combine dynamic, single-cell bioluminescence and fluorescence microscopies technologies to measure numerous molecular events cells use to convert chemokine inputs into signaling outputs and chemotaxis. We will couple our multiplexed imaging readouts with advanced image processing methods to generate high-dimensional quantitative data sets needed for basic studies of chemokines and integration with other large-scale data. Our innovative imaging tools will allow an unprecedented view of chemokine gradients and heterogeneity of cellular responses in complex environments. This technology will transform investigations of chemokines in cancer and advance ongoing efforts to target chemokines for therapy.