Spatial Transcriptomics of Pancreatic Tumors Reveal Distinct Compartmentalisation of Neutrophil Subsets [Spatial Transcriptomics]

Neutrophils are increasingly recognized as key players in the tumour immune response, and are associated with poor clinical outcomes. Multiple studies have characterised various neutrophil states within the tumor, with associated pro- and anti- tumoral functions. However, it is unknown if this heterogeneity of neutrophils is contributed by their surrounding tumor microenivironment. To answer this, we performed 10X Visium spatial transcriptomics on a murine model of pancreatic cancer. We discovered that terminally differentiated T3 neutrophils localise primarily in regions near hypoxic and glycolytic areas, while transitional immature T1 and mature T2 neutrophils localise towards the peripheral regions of the tumors. This results points to a spatially dependent role of T3 neutrophils, which may be critical to carry out pro-tumorigenic functions. Frozen samples were quartered before embedding in optimal cutting temperature‒filled molds and sectioned to a thickness of 10 μm at −20 °C with cryotome (CM3050S, Leica). To ensure capture of tumor-infiltrating neutrophils, cryosections were first screened for Ly6G immunofluorescence before mounting on chilled Visium Tissue Optimization Slides (1000193, 10X Genomics) and Visium Spatial Gene Expression Slides (1000184, 10X Genomics). For processing, we fixed tissue sections onto the slide in chilled methanol and prepared them for immunostaining according to manufacturer’s protocol. Sections were stained with Rabbit anti-wide spectrum cytokeratin (Abcam) and Rat Ly6G (1A8, Biolegend) for 30 min. Subsequently, sections were washed with wash buffer for 5 times before secondary staining with Anti-Rabbit Alexa Fluor 488 (Invitrogen) and Anti-Rat Alexa Fluor 555 (Invitrogen) and DAPI for 30 minutes. Upon completion of immunostaining, slides were scanned with the slide scanner (EVOS Auto FL2, Thermo Scientific). After immunofluorescence imaging, Visium Spatial Gene Expression libraries were prepared according to the manufacturer’s protocol. The libraries were sequenced on either HiSeq X or NovaSeq 6000 S4 (Illumina) at PE150 and a read depth of 50,000 read-pairs per tissue-covered spot.