APPENDIX - SUPPLEMENTAL INFORMATION

Suppl. Figure 1. Ca2+ responses elicited by different GPCR agonists in bone-metastatic cancer cells. PC3-ML prostate cancer cells were consecutively exposed to ATP (10M) and CCh (100M). These two agonists generated similar Ca2+responses in individual cells, indicating that HP and LP kinetics are not related to the type of GPCR stimulated. The traces shown are representative of 3 separate experiments analyzing 128 single cells.Analogous results were obtained using MDA-MB-231 breast cancer cells.

Suppl. Figure 2. Bone-metastatic cells in culture can transition between HP and LP Ca2+ responses over time. Cancer cells were exposed to ATP (10M, five minutes) for three consecutive times over a period of twenty-four hours. Cells were plated on photo-etched glass cover slips (Bellco, Vineland, NJ), imaged after the first agonist stimulation and then returned to their normal culture conditions. Based on the points of reference provided by the etching, the position and orientation of the same cells were re-established before the following agonist stimulation. Cells were imaged using phase contrast microscopy (A), and fluorescence microscopy upon loading with the Ca2+sensitive probe Fura-2 (B); a representative cell produced a Ca2+response with HP features upon the first ATP pulse, an intermediate (transitional) response upon the following agonist stimulation and finally responded in a LP fashion to a third ATP pulse, (C). Similar results were obtained analyzing 23 PC3-ML prostate cancer cells and 45 MDA-231 breast cancer cells in 14 total experiments.

Suppl. Figure 3. Ca2+ responses observed in bone-metastatic cancer cells co-cultured with different normal human bone cells.Ca2+ responses to ATP (10M, five minutes) were analyzed in PC3-ML prostate cancer cells co-cultured with human bone stromal cells (BSC), bone mesenchymal cells (MSC) and immortalized fetal osteoblasts (FOB). Only FOBs were able to significantly increase the percentage of LP Ca2+responses in bone-metastatic PC3-ML cells. MSC showed only a partial effect, decreasing the percentage of HP responses but leaving LP responses unaltered. These results were obtained from 9 experiments analyzing 413 PC3-ML cells co-cultured with hFOBs, 4 experiments analyzing 192 PC3-ML cells co-cultured with BSC and 4 experiments analyzing 178 PC3-ML cells co-cultured with MSC. (*p = 0.0074; ∆ p = 0.0123 versus control cells).

Suppl. Figure 4. Time of onset and reversion for the increase in LP Ca2+responses induced in bone-metastatic cancer cells by osteoblasts.

PC3-ML prostate cancer cells were co-cultured with normal human osteoblasts from 1 to 7 days stimulated with ATP (10M, five minutes) and analyzed for their Ca2+ responses. The percentage of LP Ca2+ responses increased significantly at the sixth day of co-culture, although a trend towards this outcome was already evident at earlier time-points (A). When osteoblasts were removed from the co-culture but the conditioned medium was retained, the percentage of LPCa2+ responses reverted to pre-conditioning values within 48 hours (B); however, the removal of both osteoblasts and conditioned medium from the co-culture induced the same reversion in just nine hours (C). The number of experiments conducted for each set of data were as follows: Time of onset: 63 experiments; Osteoblasts removal: 8 experiments; Osteoblasts and conditioned medium removal: 14 experiments. (* = Panel A: day 6 p=0.0027, day 7 p=0.0004 – Panel B: 48 hours p =0.01 – Panel C: 9 hours p=0.023, 24 hours p=0.027)

Suppl. Figure 5.Inhibition of histone deacetylase affects Ca2+ signaling in cancer cells similarly to osteoblasts. Bone-metastatic cancer cells were treated with 100nM Trichostatin-A for twenty-four hours and then exposed to ATP (100M for 5 minutes). In these conditions, PC3-ML cells showed changes in the relative distribution of HP and LP Ca2+ responses that were comparable to cells co-cultured with osteoblasts for seven days (see fig.1). Removal of the histone deacetylase inhibitor resulted in reversal of the effect within twenty-four hours. Results shown are from 8 separate experiments using PC3-ML cells (*p= 0.005; **p= 0.036 versus control cells). Analogous results were obtained from 10 separate experiments analyzing MDA-231 cells.

Suppl. Table 1. Prostate and breast cancer cells lacking bone-metastatic potential co-cultured with human osteoblasts. Normal osteoblasts from human donors (NHOs) or immortalized human fetal osteoblasts (hFOB) were co-cultured with PC3-N, DU-145 or MDA-468 cancer cells.

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