Figure 2

Lpd is required for lung metastasis from orthotopic mammary tumors. (a–e) NOD/SCID/IL2Rγ-null mice were injected orthotopically with LM2 cells stably expressing Ctrl-shRNA or Lpd-shRNA1 or Lpd-shRNA2. Tumors were allowed to grow for 6±0.5 weeks. (a) Primary tumor weights at the time of killing of individual mice are shown. Data are represented as mean±s.e.m. Number of animals per group is indicated in (b). (b) The number of mice that presented with visible metastases in the lung is indicated (mice with lung metastases/total number of mice analyzed). (c) Representative images of whole left pulmonary lobe from LM2 (control or knockdown) tumor-bearing mice with ZsGreen-positive metastatic foci (top panel). Scale bar, 5 mm. Representative lung sections stained with H&E; arrowhead indicates the presence of metastatic foci (bottom panel). Scale bar, 20 μm. (d) Numbers of ZsGreen-positive metastatic foci in the left pulmonary lobe were counted. Quantification of data shown in (c, top panel). Data are represented as mean±s.e.m. One-way ANOVA; Dunnett’s; *P⩽0.05. Number of animals per group is indicated in (b). (e) Representative images of paraffin tissue sections stained with Masson’s trichrome of primary tumors to show local invasion. Scale bar, 20 μm. (f–i) Tumor cell motility in vivo monitored by multi-photon confocal imaging. (f) Image shows a Ctrl-shRNA ZsGreen tumor. Cyan=ZsGreen-positive cells, red=collagen fibers. One motile Ctrl-shRNA-expressing tumor cell is outlined. Scale bar, 40 μm. (g) The average numbers of motile cells per field were determined. Data are represented as mean±s.e.m.. Unpaired t-test; *P⩽0.05, n=5 mice per group. (h) The average numbers of cells extending protrusions per field were determined. Data are represented as mean±s.e.m.; unpaired t-test; *P⩽0.05; n=5 mice per group. (i) Directionality of the motile cells (net path/total path) was determined. Data are represented as mean±s.e.m.; unpaired t-test; *P⩽0.05; n=5 mice per group. (j, k) Red fluorescently labeled MDA-MB-231 cells overexpressing GFP-Lpd or GFP as a control were implanted into the perivitelline cavity of zebrafish embryos and dissemination to the trunk region was quantified 2 days after injection. (j) Image of representative zebrafish trunks 2 days after injection. Arrows point to seeded tumor cells. Scale bar, 500 μm. (k) Quantified data from (j) represented as mean number of seeded tumor cells per mm of trunk length. Mean±s.e.m; data from 53 fish embryos for GFP and from 49 fish embryos for GFP-Lpd from 3 independent experiments; t-test; **P⩽0.01. See also Supplementary Figure S2.