3012 – PROBING HEMATOPOIETIC-STROMAL CROSSTALK BY SPATIALLY RESOLVED SINGLE-CELL RNA SEQ

Spatial transcriptomics encompasses a rapidly advancing set of techniques aimed at resolving the spatial organization of mRNA expression down to the micrometer scale. Virtually all of these techniques, however, rely on generation of tissue sections, impeding faithful reconstruction of 3D mRNA expression maps and providing only a static snapshot of the spatial organization. Intravital microscopy (IVM), on the other hand, provides both 3D and dynamic imaging data, enabling real-time visualization of disease progression and response to treatment that can be spatially and temporally heterogeneous. By IVM of the mouse calvarium we have observed the earliest stage of AML proliferation after seeding the bone marrow (BM), from single cells to small clusters, with striking spatial heterogeneity. To examine whether cross-talk with the BM microenvironment impacts AML cell fate, we developed a new experimental platform to differentially profile the BM compartments that support AML quiescence vs. proliferation. The platform utilizes precise femtosecond laser etching of bone to forge a microchannel, through which a micropipette was inserted to aspirate the rare leukemic and surrounding CXCl12+ stromal cells followed by analysis using the SMARTseqV4 protocol. For a more comprehensive assessment of the local microenvironment, we isolated several thousand BM cells for parallel scRNA-seq utilizing the high-throughput 10x genomics platform. The procedure was carried out under image guidance, using video-rate in vivo multi-photon microscopy to identify and aid the isolation of cells with specific dynamic properties from their respective BM micro-environments. This platform integrates spatial and temporal information from IVM with high content molecular information from scRNAseq, defining a new methodology for studying the mechanisms underlying disease progression and response to therapy.