Top hits from (A) transcriptomic, (B, D) proteomic and (C) secretomic analyses.

<p>Top hits from (A) transcriptomic, (B, D) proteomic and (C) secretomic analyses.</p

Bone marrow increases the migration of metastatic OS through uPA/uPAR.

<p><b>(A)</b> Migration of metastatic and non-metastatic OS cell lines in the presence of uPA-rich medium (20–80 U/mL) collected from bone marrow cell cultures (BMC). Percentage migration is normalized against KHOS control. Bars: SEM. ***<i>P</i> <0.0001, **<i>P</i> = 0.0084. Experiments were performed in triplicate at least twice. <b>(B)</b> Immunohistochemistry of the same FFPE section of mouse bone showing expression of uPA in bone marrow cells (right panels). Left panels: negative rabbit IgG (Dako), 2 μg/mL. Right panels: rabbit anti-human uPA (H-140) (Santa Cruz Biotechnology), 1:100 (2 μg/mL). The top panels show strong uPA staining in an area in the proximal epiphysis near the epiphyseal line, an area of rapid bone turnover, rich in osteoblasts and osteoclasts which secrete uPA. The bottom panels show and area in the diaphysis in the border of the medullary cavity and compact bone, where osteoblasts and osteoclasts are not so abundant. Magnification: 10X. <b>(C)</b> Activity of uPA in various batches (A-D) of bone marrow conditioned medium measured by a uPA Activity Assay Kit (Merck Millipore). <b>(D)</b> Migration of KHOS cells in the presence of normal growth medium (control) and each of the batches (A-D) of bone marrow conditioned medium in (C). Percentage migration is normalized against KHOS control. Bars: SEM. *<i>P</i> < 0.02, **<i>P</i> < 0.009. Experiments were performed in triplicate at least twice. <b>(E)</b> Linear regression of uPA activity <i>vs</i>. migration. Slope = 0.7589 ± 0.3693; Y-intercept when X = 0.0 is -65.61 ± -53.78; X-intercept when Y = 0.0 is 86.45.</p

Model of uPA/uPAR signaling in OS and the bone microenvironment.

<p>Osteosarcoma cells express high levels of uPA mRNA and secrete uPA into the surrounding microenvironment. They also express abundant uPAR on their cell membranes. The OS-secreted uPA acts in an autocrine fashion by binding to OS uPAR and promoting OS cell migration, invasion and metastasis by activating intracellular signaling. In addition, bone marrow cells in the surrounding microenvironment contribute in a paracrine fashion to both increase uPA/uPAR expression by OS cells, and to the pool of secreted uPA in the microenvironment, to further increase metastasis. </p

The uPA/uPAR system regulates OS migration.

<p><b>(A)</b> Increase in migration over basal levels of metastatic and non-metastatic OS cells in the presence of uPA-rich (200 U/mL) conditioned medium collected from metastatic KHOS cells after 24 h culture. Bars: SEM. <b>(B)</b> Increase in migration of metastatic KHOS cells in the presence of 0.5 and 1.0 μg/mL of rh-uPA (R&D Systems). Bars: SEM. *<i>P</i> < 0.02, **<i>P</i> < 0.002. <b>(C)</b> Decrease in migration of metastatic KHOS cells in the presence of a neutralizing mAb against uPAR (American Diagnostica). Control: LEAF Purified Mouse IgG (BioLegend). Bars: SEM. **<i>P</i> < 0.005. <b>(D)</b> Decrease in migration of metastatic KHOS cells in the presence of a non-toxic concentration (80 μM) of the uPA inhibitor, amiloride. Bars: SEM. **<i>P</i> = 0.0014. Experiments were performed in triplicate at least twice.</p

uPAR silencing inhibits migration and metastasis of OS.

<p><b>(A)</b> Migration of KHOS WT and KHOS uPAR-KD in the absence (control) or presence of 100 nM uPA (5.4 μg/mL). Percentage migration is normalized against KHOS. Bars: SEM. ***<i>P</i> < 0.0002. <b>(B)</b> Migration of KHOS wild-type and uPAR-KD cells in the presence of growth medium (control), 100 nM uPA amino terminal fragment (ATF) or high molecular weight (HMW) uPA. Percentage migration is normalized against KHOS. Migration experiments were performed in triplicate at least twice. Bars: SEM. ***<i>P</i> ≤ 0.0002. <b>(C)</b> Reduction of Erk1/2 phosphorylation in uPAR-KD cells but not in WT after 24 h treatment with 100 nM rh-uPA. <b>(D)</b> Metastatic burden (% area of lung covered by metastatic lesions/% total lung area) in mice (<i>n</i> = 5) injected with KHOS WT, uPAR-KD or uPAR-SCR cells. *<i>P =</i> 0.013. Bars: SEM. <b>(E)</b> Quantitative image analysis of DAB-stained FFPE sections of KHOS WT primary tumour and the corresponding lungs after immunohistochemistry with goat anti-human uPAR (Santa Cruz), 1:100. <b>(F)</b> Western blot showing expression of CD63 in KHOS-secreted extracellular vesicles (EV) but not in KHOS whole cell extract (WCE), and expression of uPA in both EV and WCE.</p

Inhibition of OS metastasis by the uPA inhibitor, WX-340.

<p><b>(A)</b> Metastatic burden (% area covered by metastatic lesions/% total lung area) in animals treated with vehicle (control) (<i>n</i> = 14) or treated with 10 mg/kg WX-340 (<i>n</i> = 15) i.p. 3 times/week. Left: scatter plot. Right: Bars: SEM. ***<i>P</i> = 0.0004. <b>(B)</b> Average tumour growth of mice in (A).</p