T Cells Induce Pre-Metastatic Osteolytic Disease and Help Bone Metastases Establishment in a Mouse Model of Metastatic Breast Cancer
Figure 2
Pro-osteoclastogenic cytokine production by BM cells in response to tumor antigenic stimulation precedes metastatic colonization of the bones.
(A) Clonogenic metastatic assays were performed with cells obtained from the draining LNs and iliac BM of BALB/c mice orthotopically injected with 104 4T1 tumor cells. The number of metastatic clones was determined at different time points, using a 6-thioguanine resistant assay (left panel). Expression of cytokeratin 19 (CK19) was also determined in such samples by RT-PCR (right panel). 67NR or 4T1 tumor derived microvesicles (MV) were used as a control to ascertain that a positive RT-PCR indicates exclusively the presence of whole tumor cells in the target organ. 4T1 cells mRNA was obtained from in vitro cultures or in vivo tumors (+) and was used as a positive control in the RT-PCR assay. (B) Kinetics of pro-osteoclastogenic cytokine production in response to tumor soluble antigen (sAg) stimulation of BM cells obtained from iliac bones of mice bearing 4T1 or 67NR tumors. Naïve animals were used as controls. Supernatants of sAg stimulated iliac BM cells were harvested after 72hs and cytokine production was measured by ELISA. Data are expressed as the mean ± SD of five mice/group and are representative of at least three independent experiments. *p<0.05. (C) Osteoclastogenesis assays using naïve BM cells cultured in the presence of either recombinant M-CSF and RANKL, or M-CSF and supernatant of iliac BM cells derived from 4T1 tumor-bearing mice (11 d after tumor injection). Supernatant from iliac BM cells of naïve animals (BM Nv) stimulated or not with sAg was used as specific control. Cultures were also treated with r-OPG or α-IL17F as indicated. The number of TRAP+ multinucleated OC cells obtained in vitro was determined (left panel) and TRAP activity in such supernatants was measured by a colorimetric assay (middle panel). In the right panel, generation of functional OC cells in vitro was also determined using BD BioCoatTM OsteologicTM Bone Cell Culture System (BD Biosciences). The graphic represents the resorbed area on osteologic discs. All data are from at least two independent experiments (n=5 mice/group) and presented as mean ± SD. *p<0.05; **p<0.001.