Tumor-derived uPA recruits MDSCs and augments tumor growth. DA-3/TM tumor cells were infected with a retrovirus encoding murine uPA shRNA, then selected, and cloned. Three clones were analyzed (DA-3/TM-shuPA8, -shuPA52, and -shuPA56). (A) Semiquantitative PCR for uPA and β-actin message was performed on RNA from DA-3/TM cells and the uPA knockdown clones. Relative density of uPA to β-actin is presented. (B) Conditioned media from tumor cells that were plated in serum-free media for 3 days were analyzed on an uPA-specific zymography gel. As a positive control, 50 ng recombinant uPA (Rt.uPA) was loaded in parallel. The high-molecular-weight (HMW) and low-molecular-weight (LMW) degradation product of uPA can be detected on the gel. The density of the HMW uPA bands is presented. (C) To analyze recruitment of MDSCs to the various tumor cells, 2 × 10⁶ tumor cells were injected intraperitoneally, and 18 hours later IP cells were harvested and stained for MDSC analysis by flow cytometry. (D) Tumor development was monitored by implanting BALB/c mice with 10⁶ cells subcutaneously. and measuring tumors every 3 to 4 days. (E) To determine whether systemic uPA can augment tumor development, BALB/c mice were injected with 10⁶ DA-3/TM cells subcutaneously, and either 1 μg recombinant uPA or saline intraperitoneally every other day for 3 weeks while tracking tumor development every 3 to 4 days. Error bars representing SEM and P values are provided. *P < .001 between DA-3/TM-shuPA8 and DA-3/TM-shuPA56. **P < .05 between DA-3/TM-shuPA52 and DA-3/TM-shuPA56.