Bioorthogonal probe development for highly parallel in vivo imaging

摘要 多重分析技术，如流式细胞术、scRNAseq和空间转录组学， 彻底改变了现代生物学不幸的是，仍然没有可靠的方法来执行高度多路复用， 体内实时成像，以了解细胞和分子在其完整的功能行为 微环境随着时间的推移基于“点击化学”的进展，我们开发了一种新的， 基于体内多路复用技术（SAFE; Ko等人，2022，Nat Biotech，出版中; Ko等人，高级科学， 2022年出版），这是第一次允许活体成像远远超过目前的可能性（~12-20 在可行性研究中提供4 -6通道体内成像，而不是活体显微镜检查提供的4-6通道）。的 新技术利用了特别标记的荧光亲和配体i）其中外源施用的 四嗪在几秒钟内切割抗体和荧光染料之间含有C2 TCO的接头， ii）允许抗体标记的超亮聚合物和吲哚菁染料被洗掉 在成像和再染色的循环之间。本提案的目标是进一步开发和验证 技术，然后用它来获得更深入的时间和空间洞察肿瘤微环境。我们 提出了两个具体目标：i）优化SAFE-IVM技术，以实现更宽（约30通道）的体内复用 成像（新化学，包括新型iTCO、Tz-BHQ、扩展颜色、改进成像室）;以及ii） 对肿瘤微环境及其免疫细胞进行多重成像。后期研究的结果将 是重要的，因为它们将i）建立以黄金为基准的新技术的效用 标准和ii）为新技术的更广泛应用打开了大门。

ABSTRACT Multiplexed analytical techniques such as flow cytometry, scRNAseq and spatial transcriptomics have revolutionized modern biology. Unfortunately, there are still no reliable ways of performing highly multiplexed in vivo real-time imaging to understand the functional behavior of cells and molecules in their intact microenvironment over time. Based on advances in “click chemistries” we have developed a novel, scission based in vivo multiplexing technology (SAFE; Ko et al., 2022, Nat Biotech, in press; Ko et al., Adv Science, 2022 in press), that for the first time allows intravital imaging far beyond what is currently possible (~12-20 channel in vivo imaging in feasibility studies instead of 4-6 channels afforded by intravital microscopy). The new technology utilizes specially labeled fluorescent affinity ligands i) where an exogenously administered tetrazine cleaves a C2TCO containing linker between antibody and fluorochromes in seconds and at non-toxic concentrations and ii) allows antibody-tagged superbright polymeric and indocyanine dyes to be washed away between cycles of imaging and thus re-staining. The goal of this proposal is to further develop and validate the technology and then use it to gain deeper temporal and spatial insight into the tumor microenvironment. We propose two specific aims: i) Optimize the SAFE-IVM technology for broader (~30 channel) multiplexed in vivo imaging (new chemistry including novel iTCO, Tz-BHQ, expand colors, improve imaging chambers); and ii) perform multiplexed imaging of tumor microenvironment and its immune cells. Findings from latter studies will be important because they will i) establish the utility of the new technology benchmarked against gold standards and ii) open the door to broader applicability of the new technology.