Fig 2: Immunofluorescence images of uPA and uPAR expression in osteosarcoma cell lines.Both diffuse cytoplasmic and nuclear uPA and uPAR positivity are seen in one human osteosarcoma cell line (A) and two canine cell lines D17 (B) and Dharma (C). Nuclear expression was strong in uPA and weak to moderate in uPAR in all three cell lines. Nuclei were stained with DAPI (blue). Primary antibody was omitted in negative control (NC). Images taken at 400x.

Fig 3: Kaplan-Meier curves for dogs with low or high serum uPA activity.Kaplan-Meier curves for progression free survival (PFS) of dogs with osteosarcoma treated with a limb amputation and carboplatin chemotherapy. Median PFS was 266 days for dogs with low serum uPA activity (n = 20, solid line) and 227 days for dogs with high serum uPA activity (n = 5, dashed line; p = 0.043).

Fig 4: Representative tissue microarray cores with uPA and uPAR immunohistochemical labeling.(A) A core with negative uPA/uPAR expression. Osteosarcoma cells had clear cytoplasm and nuclei with no immunoreactivity. (B) Diffuse and weak cytoplasmic uPA expression (1+) is seen in majority of the osteosarcoma cells with nuclear positivity. (C) Osteosarcoma cells diffusely showed strong cytoplasmic uPA expression (2+) throughout the core with nuclear positivity. (D) Strong distinct nuclear uPA expression is present with weak cytoplasmic labeling (1+). (E) Weak to moderate uPAR expression (1+) is present in cytoplasm of the osteosarcoma cells. (F) Cytoplasmic uPAR expression is strong (2+) and seen diffusely in this core sample. (G) Nuclei of most osteosarcoma cells are positive for uPAR expression with weak cytoplasmic positivity (1+). (H) Negative control with primary antibody omission. Images taken at 400x.

Fig 5: Kaplan-Meier curves for dogs with low or high serum uPA.Kaplan-Meier curves for progression free survival (PFS) of dogs with osteosarcoma treated with a limb amputation and carboplatin chemotherapy. (A) Kaplan-Meier curves for the 26 dogs in the discovery cohort. Median PFS was 266 days for dogs with low serum uPA (n = 17, solid line) versus 105 days for dogs with high serum uPA (n = 9, dashed line; p = 0.003). (B) Kaplan-Meier curves for the 23 dogs in the validation cohort. Median PFS was 490 days for dogs with low serum uPA (n = 7, solid line) versus 167 days for dogs with high serum uPA (n = 16; dashed line; p = 0.16). (C) Kaplan-Meier curves for all 49 dogs combining the discovery and validation cohorts. Median PFS was 266 days for dogs with low serum uPA (n = 24, solid line) versus 128 days for dogs with high serum uPA (n = 25, dashed line; p = 0.003).