Fig. 6: Tumor-derived PDGF-C is required for metastatic outgrowth.
All experiments involved ZR-75-1-mChLuc2 cells inoculated into young NSG mice. a, Orthotopic inoculation (TB), with NTB controls (n = 5 mice per group). Left, ex vivo IVIS imaging of lungs (days 34–43; n = 4 TB mice as one mouse was used for tumor cell isolation; scale bar, radiance, p s−1 cm−2 sr−1). Right, TB lungs stained for human lamin A/C (scale bar, 250 µm). Tumor cells sorted from lungs (n = 1 TB mouse) were treated with control medium or 4T1 or fibroblast (MRC5 or 3T3) CM. Number of cells per colony (days 7 and 14). b, Intravenous inoculation (TB; n = 6 mice). Left, ex vivo IVIS imaging of lungs (day 29; scale bar, radiance, p s−1 cm−2 sr−1). NTB control mice were from a. Right, human lamin A/C staining of lungs, liver and hind leg bones (scale bars; lungs, 250 µm; liver and bones, 500 µm). c, Cells transduced with shNTC1, shNTC2, shPDGFC2 or shPDGFC5 were injected intravenously (shNTCs and shPDGFCs; n = 7 or 8 mice per group, respectively). Top left, whole-body IVIS signal (day 28; scale bar, counts). Top right, ex vivo IVIS signal in livers (day 29). Bottom left, quantification of metastatic deposits and DTCs with representative human lamin A/C-stained liver sections (scale bar, 5 mm). Only three shPDGFC5 hind leg bone samples were available. d, Mice were treated daily for 5 d with vehicle, bleomycin or rPDGF-C. On day 12, ZR-75-1-mChLuc2 cells were inoculated intravenously (i.v.) (n = 5 vehicle- or rPDGF-C-treated mice; n = 4 bleomycin-treated mice). Mice were injected with EdU (day 37) and culled 24 h later. Percentage of EdU+ human (lamin A/C+) tumor cells, representative images (scale bar, 50 µm). e, Cells carrying EV or overexpressing (o/e) Pdgfc cells were injected orthotopically (n = 5 mice per group). Mice were culled on days 45–60. Left, tumor growth for individual mice. Right, quantification of lung metastatic deposits, with representative human lamin A/C staining (scale bar, 125 µm). Data are presented as mean values; ±s.e.m. (a, left; b–e, right); two-tailed t-test (a–c, middle right), one-way ANOVA with multiple comparisons (d) or two-tailed Mann–Whitney U-test (c, top, middle left, bottom; e).
