Fig 2: M1 and M2 macrophage characterization in tumoral-conditioned medium. (A,B) qRT-PCR relative expression levels of TyrRS and SPARC in M1 and M2 macrophages cultured in control medium (white bars) or B16F10-derived tumoral medium (black bars) for 24 h. The analysis was performed twice, with three replicates each time. Data are mean±s.e.m. Statistical significance was calculated using a t-test, with significant differences between compared groups noted by *P<0.05. (C-N) Immunostaining against TyrRS or SPARC (both shown in magenta) of M1 (red) and M2 (green) macrophages cultured in control medium (C,D,I,J) or in two different types of tumor-conditioned medium: B16F10-derived tumoral medium (E,F,K,L) or A549-derived tumoral medium (G,H,M,N) for 24 h. DAPI is shown in blue. Arrows indicate cells positive for the specific staining. (O) Quantification of SPARC and TyrRS mean fluorescent intensity staining under different cultured conditions; 10 to 30 individual cells were analyzed in each case. Statistical significance was calculated using one-way ANOVA Bonferroni's Multiple Comparison Test, with significant differences between compared groups noted by *P<0.05, **P<0.01. (P) Relative expression levels of c-Myc in M1 and M2 macrophages cultured in control medium (white bars) or B16F10-derived tumoral medium (black bars) for 24 h.

Fig 3: SPARC inhibits osteoclastogenesis in tumor cells. Induction of osteoclastogenesis in human mesenchymal stem cell-derived osteoblasts by CM from SUM1315-bo cells. Differentiated multinucleated osteoclasts were revealed by TRAP staining. (A) Representative images of TRAP-staining. (B) Quantification of TRAP-positive cells. Data are presented as the mean ± standard deviation. **P<0.01 with two-sided Student's t-test. CM, conditioned medium; TRAP, tartrate-resistant acid phosphatase; SPARC, secreted protein acidic and rich in cysteine; SPARC-over, SPARC overexpressing SUM1315-bo.

Fig 4: Effect of integrin avß3 blockade and ZEB1 knockdown on the SPARC-induced E-cadherin downregulation and enhanced migration (A). Representative Western blot of ZEB1, E-cadherin and SPARC in LNCaP cells transduced with a lentiviral vector carrying a short hairpin RNA against ZEB1 (shZEB1) or a scrambled sequence (shScr) (B). Quantification of the optic density of the Western blot shown in (A). Protein expression normalized to ß-actin and control cells (shSCR) (C,D). Relative expression of E-cadherin measured by RT-qPCR in LNCaP shScramble (C) and LNCaP shZEB1 cells stimulated with 1 µg/mL SPARC and 50 µM RGD for 6 h. E-cadherin expression normalized to pumilio and control cells (first column) using the ??Ct method (E,F). Transwell migration assay of LNCaP shScramble (E) and LNCaP shZEB1 (F) cells stimulated with 1 µg/mL SPARC and 50 µM RGD for 24 h (G,H). Representative images of wound healing assay of LNCaP shScramble (G) and LNCaP shZEB1 (H) cells stimulated with 1 µg/mL SPARC and 50 µM RGD for 24 h (I,J). Quantification of the percentage of the wound area covered after 24 h (B–F,I,J). Relative migrations were compared with their own control (basal condition) (B–F). Data are expressed as mean ± SD (n = 3). ns = p > 0.05; * = p = 0.05; ** = p = 0.01; *** = p = 0.001; (B)—Mann–Whitney U test; (C–F,I,J)—Kruskal–Wallis test.

Fig 5: Effect of SPARC on migration and invasion of breast cancer cells. (A) siRNA of SPARC was transfected into SUM1315, which was confirmed at the mRNA and protein levels. (B) Migration and Invasiveness of SPARC knockdown SUM1315 and control cells was assessed using Transwell assays. The invasiveness through 8-µm pore Transwell inserts was significantly increased in SPARC knockdown SUM1315 compared with the negative control. Magnification, ×100. (C) Constructed expression vector pEGFP-C2-SPARC was stably transfected into SUM1315-bo. Transfection was confirmed at the mRNA and protein levels. (D) Migration and Invasiveness of SPARC-over and control cells were assessed by Transwell assays. The invasiveness through 8-µm pore Transwell inserts was revealed to be significantly lower in SPARC-over compared with the controls. Magnification, ×100. *P<0.05. SPARC, secreted protein acidic and rich in cysteine; siRNA, small interfering RNA; control, negative control; SPARC-over, SPARC overexpressing SUM1315-bo; OD, optical density.