Spatial transcriptomic analysis of bone marrow megakaryocytes during SARS-CoV-2 infection. Femurs were analysed from K18-hACE2 mice infected with SARS-CoV2 (500 TCID₅₀) for 3 days (COVID_3d) or 7 days (COVID_7d) or with conditioned media (Mock) for 7 days. Ten-μm sections of paraffin-embedded femurs were used to perform spatial transcriptomic analysis. (A) Schematic illustration of NanoString’s GeoMx Digital Spatial Profiler (DSP) workflow. Different steps are indicated: (1) stain: femur sections were hybridized with UV photocleavable probes from the whole mouse genome and fluorescent morphology markers; (2) ROI were selected based on the fluorescence of interest (CD41) and whether MK were adjacent to sinusoid vessels (ASV-MK) or nonadjacent to sinusoid vessels (NASV-MK); (3) ROI were illuminated with UV light that released the barcodes; (4) Each ROI was collected independently using microcapillaries; (5) Sequencing libraries were generated followed by sequencing and counting. A total of 19 072 genes normalized by third quartile (Q3) were expressed above Limit of Quantitation (LOQ) in at least 10% of ROI. (B) Immunofluorescence staining of bone marrow (femur) using morphology markers. Anti-CD41 antibody (turquoise) was used to stain MK, anti-endomucin antibody (purple) to stain sinusoids, and SYTO dye (blue) to stain nuclei; scale bar, 125 μm. Representative ROI (6-8 megakaryocytes per ROI) are illustrated in the right panel. ROI with ASV-MK is yellow, and ROI with NASV-MK is white. (C, D) Volcano Plots comparing COVID_3d (C) or COVID_7d (D) vs Mock. The log2 fold change of each gene is plotted against its statistical significance (−log10 P value). Red dots represent genes significantly upregulated and blue dots, genes significantly downregulated in MK during COVID-19. Thresholds are indicated with dotted lines. (E) UMAP plot of the short (30) gene signature of MK-ROI colored by class (blue circles: Mock [n = 12]; red circles: COVID_7d [n = 12]). (F) Heat map showing differences of the 30-gene signature between Mock and COVID_7d. Each row represents a MK-ROI, and each column represents a gene obtained from the short signature. (G) Relevant enriched biological processes using the 30-gene signature and their P values are presented. The 30-gene signature was obtained using a machine learning approach. BioDiscML was used to classified “Mock” and “COVID_7d” groups, and the best model was variable feature importance (VFI) optimized with false discovery rate (FDR). MCC for the model was 0.997. (H) PF4 mRNA expression was evaluated in bone barrow by RT-ddPCR. PF4 mRNA copies were normalized with Gapdh mRNA copies (n = 4-5 mice per condition). Results are expressed as mean (± SD). Statistics: unpaired t-test with Welch correction; ^∗P < .05 (n = 4-5 per condition). MK, megakaryocytes; mPF4, mouse platelet factor 4; mRNA, messenger RNA; RT-ddPCR, reverse transcription digital droplet polymerase chain reaction.