Figure 2.
PMP infiltration in solid tumor allografts and RNA transfer in mice. (A) Immunohistochemistry (IHC) in a 5-μm section from LLC tumor allograft, 21 days. αIIb integrin, red. (B) IHC in a 5-μm section from LLC tumor allograft, 21 days. αIIb integrin, red (top); CD63, green (middle); DAPI, blue (shown in merged image, bottom). αIIb integrin/CD63 overlap appears in some areas in the merged image as yellow. (C) Tumor cells from a resected LLC tumor at 21 days, isolated as described in supplemental Methods. αIIb integrin, red; DAPI, blue. (D-E) Tumor cells from resected LLC allograft 24 hours after transfusion of AO-labeled platelets. (D) αIIb integrin, red; (E) AO, green. Bars (A-E), 10 μm. *PMP− cells. (F) Percentage of cells with cytosolic AO staining, shown ± SEM (n = 3, >100 cells each). (G) Human platelets were transfected with unlabeled siRNA and transfused into mice bearing 20-day LLC tumors. After 24 hours, tumors were resected, digested, cleared of vascular cells with α-CD31 beads, and tumor cells were captured on fibronectin-coated coverslips. (H) Tumor cells ex vivo as in panel G, from mice transfused with human platelets transfected with FAM-siRNA. (I) LLC cells, treated in vitro with PMPs derived from human platelets 48 hours after platelet transfection with unlabeled siRNA. (J) Cells treated as in panel I with PMPs derived from human platelets transfected with FAM-siRNA. (H-J) Human αIIb integrin, red; FAM, green; DAPI, blue. Dashed white lines indicate cell borders as determined from accompanying brightfield images (not shown). Yellow lines indicate x–y plane of z-section. Corresponding z-sections are shown below, minus DAPI stain. Bars (G-J), 7.5 μm; z-stack bars, 1 μm. Asterisks in panels G-J denote apical side of z-section.