High resolution mapping of the tumor microenvironment using integrated single-cell, spatial and in situ analysis [scRNA-seq and Visium]

Single-cell and spatial technologies that profile gene expression across a whole tissue are revolutionizing the resolution of molecular states in clinical tissue samples. Current commercially available technologies provide whole transcriptome single-cell, whole transcriptome spatial context, or high resolution in situ targeted gene expression analysis. Here, we combine these technologies to explore tissue heterogeneity in large, FFPE human breast cancer sections. In one sample, we identify three molecularly distinct tumor subtypes, enabling us to define cell neighborhoods and biomarkers in the progression towards invasive carcinoma. In another sample, we identify rare boundary cells that sit at the critical myoepithelial border confining the spread of malignant cells. We demonstrate that each technology alone provides information about molecular signatures relevant to understanding cancer heterogeneity. However, it is the integration of these technologies that leads to even deeper insights, ushering in discoveries that will progress oncology research and the development of diagnostics and therapeutics. Two formalin-fixed & paraffin-embedded (FFPE) breast cancer tissue blocks were obtained from Discovery Life Sciences. Sample #1 was annotated by a pathologist to be T2N1M0, Stage II-B, ER+/HER2+/PR−. Sample #2 was characterized as stage pT2 pN1a pMX, ER−/HER2+/PR−. Corresponding dissociated tumor cells for Sample #1, fresh frozen (FF) in liquid nitrogen, were also sampled from the same 2.5 cm biopsy. For the Chromium Flex workflow, two 25 μm curls were pooled as a single replicate. 5 μm sections from Sample #1 were taken from the FFPE tissue using a microtome. Two replicate 5 μm sections were taken each for Visium CytAssist and Xenium. A 5 μm section was also taken from Sample #2 for Xenium.