Spitalny Leslie, Falco Natalie, England Whitney, Allred Tyler, Spitale Robert C
Department of Pharmaceutical Sciences, University of California Irvine California 92697 USA
Department of Chemistry, University of California Irvine California 92697 USA.
RSC Chem Biol. 2025 Jan 20;6(3):404-411. doi: 10.1039/d4cb00262h. eCollection 2025 Mar 5.
The architecture of cells and the tissue they form within multicellular organisms are highly complex and dynamic. Cells optimize their function within tissue microenvironments by expressing specific subsets of RNAs. Advances in cell tagging methods enable spatial understanding of RNA expression when merged with transcriptomics. However, these techniques are currently limited by the spatial resolution of the tagging, the number of RNAs that can be sequenced, and multiplexing to isolate spatially-distinct cells within the same tissue landscape. To address these limitations, we developed CrossSeq, which employs photocrosslinking fluorescent probes and confocal microscopy activation to demarcate user-defined regions of interest on fixed cells for multiplexed spatial transcriptomic analysis. We investigate phenyl azide and diazirine crosslinking scaffolds and define their photoactivity profiles. We then deploy the aryl azide scaffold with three fluorophores for multiplexing on glyoxal fixed cells and analyze the defined populations using flow cytometry. Finally, we apply CrossSeq to investigate an MDA-MB-231-LM2 metastatic cancer migration model to evaluate changes in gene expression at the migratory cell front the exterior population. We anticipate this new technology will be a valuable tool addition as it will enable easier access to spatial transcriptomic analysis for the scientific community using conventional microscopy and analysis techniques.
多细胞生物体内细胞及其形成的组织的结构高度复杂且动态变化。细胞通过表达特定的RNA子集在组织微环境中优化其功能。细胞标记方法的进展使得与转录组学相结合时能够在空间上理解RNA表达。然而，这些技术目前受到标记的空间分辨率、可测序的RNA数量以及在同一组织环境中分离空间上不同细胞的多路复用的限制。为了解决这些限制，我们开发了CrossSeq，它采用光交联荧光探针和共聚焦显微镜激活来在固定细胞上划定用户定义的感兴趣区域，用于多路复用空间转录组分析。我们研究了苯基叠氮化物和重氮烷基交联支架，并确定了它们的光活性谱。然后，我们将带有三种荧光团的芳基叠氮化物支架用于乙二醛固定细胞的多路复用，并使用流式细胞术分析定义的群体。最后，我们应用CrossSeq研究MDA-MB-231-LM2转移性癌症迁移模型，以评估迁移细胞前沿（外部群体）基因表达的变化。我们预计这项新技术将成为一个有价值的工具，因为它将使科学界能够使用传统显微镜和分析技术更轻松地进行空间转录组分析。
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