Integrated Imaging Tools for Intercellular Chemokine Signalling

趋化因子及其G蛋白偶联受体（GPCR）是细胞间粘附的关键介质。 癌细胞和多种基质细胞类型之间的信号传导。局部癌细胞增殖 侵袭、全身性转移、免疫细胞的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核心作用 使其成为抑制或增强趋化因子的治疗的有吸引力的靶点。 发信号。然而，趋化因子的许多复杂的生物化学仍有待发现， 这是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进行信号传导，并且它们的空间分布复杂 这是因为它们能够结合细胞表面和细胞外基质。此外， 趋化因子通过GPCR和清道夫受体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CXCL 12在一个距离上的短距离，陡峭的局部梯度， 单个细胞通过受体CXCR 4最有效地驱动信号传导和趋化性。使 趋化因子的变革性研究，并最终推进药物靶向的应用 我们建议开发一套新的定量分子成像工具， 为了测量趋化因子分布、关联和表达的时空动态， 在数百个单细胞中传递信号。这些工具将使我们能够测量细胞信号模式 在趋化性和确定潜在的机制，细胞间异质性， 对趋化因子的反应我们将1）生成集成的、多路复用的生物发光， 荧光显微镜工具箱，用于细胞外和细胞内步骤的单细胞成像， 趋化因子信号传导;和2）测试我们的趋化因子成像技术作为可推广的， 这是适用于患者来源的细胞的定量方法。我们将独特地将联合收割机， 单细胞生物发光和荧光显微镜技术来测量许多 细胞用来将趋化因子输入转化为信号输出和趋化性的分子事件。 我们将把我们的多路复用成像读数与先进的图像处理方法结合起来， 生成趋化因子基础研究所需的高维定量数据集， 与其他大规模数据的融合。我们创新的成像工具将允许前所未有的 趋化因子梯度和复杂环境中细胞反应的异质性的观点。 这项技术将改变癌症中趋化因子的研究， 致力于靶向趋化因子的治疗。

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.