Fig 2: Tumor cell migration, invasion, and lung colonization in response to platelets and activated platelet releasate.Time-lapse quantification of (A) migrating or (C) invading Met-1 cells toward the releasate of activated platelets. n = 4 mice per condition for migration and n = 8 mice per condition for invasion. Error = 95% confidence interval of the difference. Control indicates serum-free medium as the attractant. Endpoint quantification of the number of (B) migratory or (D) invaded cells per well. n = 4 mice per condition for invasion and n = 8 mice per condition for invasion. P values were determined by unpaired two-sample t tests. (E and F) Cytokine profiling of pooled (n = 5) activated releasate from the platelets of either 13-week-old PyMT or WT mice. (G) Experimental design for AT-3 tumor cell lung colonization following the transfusion of either PyMT- or WT-derived platelets into Mpl-/- recipients. Schematic created using BioRender.com. (H) Representative gating strategy to quantify platelets (CD41+, FSClow) from blood sampled before and after platelet transfusion. FSC, forward scatter. (I) Fold change in platelet count 2 hours after transfusion. P values were determined by unpaired two-sample t tests. (J) Selection criteria and (K) quantification of CMFDA+ AT-3 cells in the lungs of Mpl-/- mice 48 hours after intravenous injection. P values were determined by unpaired two-sample t tests; n = 5 recipient mice per condition.

Fig 3: Single-dose application of the cobra venom factor (CVF (1x)) enhances the formation of platelet-neutrophil aggregates (PNAs) and the mobilization of neutrophils to the peripheral blood. Scatter plots show flow cytometry analyses of (a) the number of neutrophils per microliter of blood, (b) the number of platelets per microliter of blood measured by the differential blood count, and (c) the percentage of PNA formation relative to the total number of neutrophils in phosphate-buffered saline (PBS)- and CVF (1x)-treated mice 27 h after injection, respectively, 3 h after double femoral artery ligation (FAL) or a double sham operation (sham) of both femoral arteries. For the PNA analysis, platelets were detected by an anti-CD41 antibody, and neutrophils were identified by anti-CD11b and anti-Gr-1 antibodies. All values are the means ± S.E.M., n = 4 mice per group, * p < 0.05 by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test.

Fig 4: Transcriptomic profiling of MKs from a model of murine breast cancer.CD41+ DAPIlow cells were isolated from the BM of a 13-week-old PyMT mouse and a WT littermate for scRNA-seq. (A) Dimensionality reduction using Uniform Manifold Approximation and Projection (UMAP) of pooled PyMT and WT samples. Cells of MK lineage (orange) were separated into five clusters (G1 = 1699, G2/M = 1395, MK1 = 871, MK2 = 537, and MK3 = 344 cells) based on differential gene expression. Gray cells from non-MK clusters (n = 4184 cells) were excluded. (B) Overlay of PyMT (red, n = 3817) and WT (blue, n = 5213) cells. (C) Heatmaps clustered according to average log fold change depicting the top 50 differentially expressed genes between PyMT and WT cells in MK1, MK2, and MK3. Scatterplots depict gene expression of all recorded transcripts. Red lines model no change in gene expression between WT and PyMT samples. (D) Diseases and functions associated with differentially expressed genes in MKs from PyMT mice. (E) Expression levels of thrombospondin 1 (Thbs1), cathepsin G (Ctsg), S100a8, and S100a9 mRNA transcripts among MK subclusters.

Fig 5: Morphological characterization of MKs from mice with breast cancer.Spontaneous tumor growth within the mammary fat pads of (A) MMTV-PyMT (PyMT) mice and tumor growth in mice orthotopically inoculated with either Met-1 (B) or AT-3 (C) cells. Arrows depict age of PyMT animals or days post-inoculation upon sacrifice. n = 5 to 10 mice per condition. (D to F) Femurs from PyMT and wild-type (WT) mice, or orthotopically injected animals were immunofluorescently stained for MKs (CD41, cyan), nuclei (DAPI, gray), and blood vessels (laminin, magenta). Phosphate-buffered saline (PBS)–injected mice were used as orthotopic tumor-naïve controls. Scale bars, 50 µm; n = 4 to 5 mice per condition. P values were determined by two-sample t tests. (G to I) Polyploidization of native MKs from either PyMT and WT mice or orthotopic Met-1 and AT-3 models. n = 4 to 5 mice per condition. *P < 0.05 and **P < 0.01, as determined by one-way ANOVA.