Spatial Transcriptomics reveals that cells that experience intratumoral hypoxia retain hypoxia-associated transcriptional programs after invading into oxygenated tumor regions.

To understand the role of hypoxia in metastatic progression, we sought to spatially map gene expression in hypoxic cells compared with cells that migrated to oxygenated tumor regions. We adapted the Visium spatial transcriptomics protocol to perform concurrent transcriptional profiling and fluorescent imaging of our hypoxia fate-mapping system. Hypoxia fate-mapping MDA-MB-231 tumors (Godet et. al, 2019) were harvested 18 days post-implantation and immediately frozen in clear OCT. 10 μm tumor slices were placed within the fiducial frame of the Visium slide. The tissue was pre-fixed in cold methanol and imaged at 10X magnification using a Cytation5 in the RFP and GFP channels to acquire native fluorescence. The slide was immediately transferred to cold methanol with DAPI. DAPI staining was imaged at 10X using Cytation5.