Fig 1: hfRPE cultures are resistant to UAM accumulation. UAM imaged with 488-nm excitation and 500 to 535 nm emission. (A) Fourteen daily RegOS feedings with PS and MFG-E8 (MF) produced UAM in ARPE-19 cultures but not hfRPE cultures; n = 6. (B) Consumption of an OS bolus, as measured by rhodopsin remaining, is greater in hfRPE than ARPE-19 cultures; n = 3. (C) Fourteen daily oxOS feedings with PS and MF, but not with media only or RegOS feedings, produced UAM in hfRPE cultures. UAM accumulation was dose-dependent (5 versus 20 daily oxOS feedings). Micrograph: UAM autofluorescence (green) and CF647-conjugated phalloidin stain of actin (purple). Scale bar: 10 μm. n = 26 for media versus RegOS versus oxOS graph. n = 6 for dose-response graph. (D) UAM accumulation is phagocytosis-dependent. Accumulation markedly increases in the presence of phagocytosis bridging ligands PS and MF; n = 17. ns, nonsignificant. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: vSSCs drive the preferential metastasis of breast cancer to the vertebrae.a, Representative bioluminescence images showing metastasis of PY8119 cells to long bones and vertebrae 4 weeks after injection via the caudal artery. Scale bar, 5 mm. b-d, Quantification of metastasis rates to long bones and vertebrae 3–4 weeks after caudal artery injection with PY8119 (b, 4 weeks, n=32), 4T1.2 (c, 4 weeks, n=30), and E0771 cells (d, 3 weeks, n=25). Chi-square test. e, Schematic diagram of the early seeding experiment. f. Representative flow cytometry plots showing the initially seeding cancer cells in long bones and vertebrae after caudal artery injection. g, Quantification of the early seeding cancer cells in vertebrae and long bone 20h after cancer cells injection. n=13, data are mean±s.d., unpaired, two-tailed Student’s t test. h, Representative histology images of three independent experiments showing the early seeding cancer cells in vertebrae and long bones. i, Schematic diagram of the bone organoid metastasis experiment. j, Representative fluorescent histology images showing PY8119 (Green) metastasis to lbSSC-derived and vSSC-derived bone organoids (Red) 3 weeks after cancer cells injection, scale bar 200 µm. k, Quantification of the metastasis rate of PY8119 cells to lbSSC-derived and vSSC-derived bone organoids 3 weeks after cancer cells injection. n=40. Chi-square test. l, Representative bioluminescence images showing PY8119 metastasis in lbSSC-derived and vSSC-derived bone organoids 3 weeks after cancer cells injection. Scale bar, 5 mm. m, Quantification of the metastasis rate of PY8119 cells to WT or Stat3zic1 vertebrae 4 weeks after cancer cells injection. n=31 for WT mice and n=21 for Stat3zic1 mice. Chi-square test. n&o, Quantification (n) and representative images (o) of PY8119 transwell migration in the presence of the indicated concentrations of recombinant MFGE8 in the lower chamber. n=4, data are mean±s.d., one-way ANOVA followed by Tukey’s multiple comparison test. p&q, Quantification (p) and representative images (q) of PY8119 transwell migration in the presence of bone marrow stromal cells in the lower chamber isolated from long bones or vertebrae of 3-week-old WT or Mfge8-/- mice. n=4 or 5, data are mean±s.d., two-way ANOVA followed by Tukey’s multiple comparison test. r, Representative bioluminescence images showing cancer metastasis in long bones and vertebrae of WT and Mfge8-/- mice 4 weeks after PY8119 cells injection through caudal artery. Scale bar, 5 mm. s, Quantification of the metastasis rate to long bones and vertebrae 4 weeks after caudal artery injection with PY8119 cells, n=51 for WT mice and n=61for Mfge8-/- mice. Chi-square test. t, Quantification of the early seeding of cancer cells in the vertebrae and long bones of WT and Mfge8-/- mice 20h after cancer cells injection. n=11 for WT and n=10 for Mfge8-/- mice, data are mean±s.d. Two-way ANOVA followed by Tukey’s multiple comparison test. u, Representative flow cytometry plots showing the early seeding of cancer cells in long bone and vertebrae of WT and Mfge8-/- mice 20h after cancer cells injection. v, Quantification of the metastasis rate of PY8119 cells to WT or Mfge8-/- vSSC-derived bone organoids 3 weeks after cancer cells injection. n=54. Chi-square test. w, Representative bioluminescence images showing metastasis of PY8119 cells to WT or Mfge8-/- vSSC-derived bone organoids 3 weeks after caudal artery injection. Scale bar, 5 mm.
Fig 3: Identification of vSSC secreted proteins mediating breast cancer metastatic tropism.a, Heatmap showing the top differentially expressed secreted proteins in Zic1-lineage vSSCs versus lbSSCs. b, qPCR analysis of candidate vertebral-derived metastasis tropism factors in FACS isolated Zic1-lineage vSSCs (Lin-THY1-6C3-CD200+CD105-EMB-GFP+) and lbSSCs (Lin-THY1-6C3-CD200+CD105-EMB-). n=4, data are presented as mean±s.d., unpaired, two-tailed Student’s t test. c, Quantification of bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of L5 vertebrae in 8-week-old Mfge8-/- and WT mice. n=4, data are presented as mean±s.d., unpaired, two-tailed Student’s t test.
Supplier Page from Sino Biological, Inc. for Human MFG-E8 / lactadherin / MFGE8 Protein (His Tag)