Figure 1. Schematic representation of experimental approach.

Histological slides of 24 surgical resection specimens from patients with PDAC were stained with DAPI (‘nuclear’), anti-pan-CK (‘epithelial’), anti-α-SMA (‘fibroblast’) and anti-CD45 (‘immune’) to identify tumor cells and primarily to define three domains: ‘tumor-proximal stroma’ (PS), ‘tumor-distant stroma’ (DS) and ‘immune-enriched’ (I). The NanoString Immuno-oncology RNA probe set, in combination with a custom panel of 10 fibroblast-targeted RNA probes, was used to interrogate four areas (termed ‘Regions of Interest’; ROI) from each of the three domains using the NanoString GeoMx Digital Spatial Profiler (DSP) platform. The transcriptional profile of spatially-defined tumor-proximal or tumor-distant fibroblast cells was subsequently aligned to scRNA-Seq datasets from three additional patients with PDAC.