Fig 2: MMP-13 potentiates OC differentiation. A. Western blotting analysis of MMP-13 expression in supernatants of wild type MDA-MB-231, scrambled shRNA and MMP-13 shRNA (clones #1, #2, #3 and #4) cells. B. PBMCs were primed with RANKL and M-CSF (primed pre-OC) for three days and then cultured with the addition of CM, obtained from wild type (MDA-MB-231 CM), scrambled shRNA (scrambled CM) and MMP-13 shRNA cells (clone #1) (MMP-13-shRNA CM) for further four days. The graph reports the mean ± SD of the number of multinucleated TRAP positive cells per field (20×). 10 fields for each experiment (n = 3) were examined. C. PBMCs were primed with M-CSF and RANKL for three days and then co-cultured with wild type, Scrambled and MMP-13 silenced MDA-MB-231 cells (clone #1) for further four days. Representative images of TRAP staining and their magnifications are reported. D. Recombinant human MMP-13 (10 ng/ml) was added alone or together with its specific inhibitor CL-82198 (10 μg/ml) to PBMCs primed with M-CSF and RANKL for three days. Representative images of TRAP staining of OC cultures at Day 7 are shown. The graph reports the mean ± SE of the number of multinucleated TRAP positive cells per field (20×). Five fields for each experiment (n = 3) were examined. *P < 0.05; NS, not significant. E. Western blotting analysis of galectin-3 in extracts of PBMCs cultured in the presence of RANKL and M-CSF for two, five or seven days (left panel) and of pre-OC cultured with CM as reported in (B) (right panel). F. Western blotting analysis for pro- and active MMP-9 forms in primed pre-OC treated as reported in (C) with silenced (sh#1 and sh#2) or Scrambled (scr#1 and scr#2) clones.

Fig 3: Proposed model for the role of MMP-13 in lytic bone metastases. MMP-13, secreted by breast tumour cells, after stimulation of ECM components or factors (IL-8, produced by inflammatory cells or osteoblasts, or PTHrP, secreted by tumour and stromal cells), could contribute to osteolysis cooperating with enzymes secreted by OCs (in particular MMP-9 present in the bone microenvironment) for matrix degradation. The effect on bone erosion could be enhanced by the indirect action of MMP-13 on the differentiation of pre-OCs and their activation. MMP-13 activates pro-MMP-9 which is known to recruit OCs and alone or together with MMP-9 cleaves galectin-3, thus blocking its inhibitory effect on osteoclastogenesis.

Fig 4: MMP-13 increases osteoclastogenic potential in vivo. WT and transfected cells were inoculated into the femurs of 6 weeks-old nude mice. Volume (A) and MMP-13 expression (B) of the tumours developed one month after the inoculation of WT, scrambled and shRNA MDA-MB-231 cell clones. Data in (A) are expressed as the mean ± SE (WT, n = 8; Scrambled, n = 11; MMP-13-shRNA, n = 12). C. Representative ultrasound images of WT, scrambled and shRNA #1 clones after one month from injection into nude mice. The basal skeletal condition is represented by PBS inoculation. Red and yellow dotted lines approximately label tumour mass and bone profile, respectively. D. Representative images and their magnification of CT analysis of skeletal lesions produced by MDA-MB-231 cell clones injected into nude mice. E. The graph shows the level of femur erosion, calculated as follows: (1- (length of the undamaged femur injected/length of untreated counterpart)) × 100. Results are the mean ± SE (WT, n = 8; Scrambled, n = 11; MMP-13-shRNA, n = 12). *P < 0.05; NS, not significant.

Fig 5: MMP-13 expression is associated with the presence of TRAP positive cells within bone marrow and tumour. A. Histology, immunohistochemical staining for MMP-13 and pan CK, and TRAP staining were performed on femurs injected with PBS, wild type, Scrambled and MMP-13 shRNA MDA-MB-231 cell clones. Representative images are shown for each conditions. The number of TRAP positive cells at tumour/bone interface (B) and in the bone marrow infiltrated by tumour cells (C) are reported. Six sections of 10 × fields of four mice for each treatment were examined. Data are expressed as mean ± SD. *P < 0.05. Scale bars = 100 μm.