Osteoclastogenesis process in bone marrow of untreated advanced breast cancer patients

FERNÁNDEZ VALLONE VB; CHOI H; MARTINEZ LM; LABOVSKY V; BATAGELJ E; DIMASE F; FELDMAN L; BORDENAVE RH; CHASSEING NA

Philadelphia, PA, EEUU

Congreso; Metastasis and the Tumor Microenvironment; 2010

Most of advanced breast cancer patients (BCP) develop osteolytic bone metastasis as a result of the imbalance between osteogenesis, osteoclastogenesis and bone resorption processes. In previous studies we found in bone marrow  (BM) aspirates of   untreated BCP  (infiltrative ductal breast carcinoma, clinical stage III and IV, without bone and BM metastasis) a decrease of the cloning efficiency of BM-mesenchymal stem cells (MSC), measured as number of colony- forming unit-fibroblasts (CFU-F), and a decrease in its osteogenic differentiation capacity compared to healthy volunteers (HV).  It is well known that osteoblast and MSCs regulate osteoclastogenesis, so in the present study we evaluated the osteoclastogenic differentiation of BM-mononuclear cells (MNC) from untreated advanced BCP without bone disease. Moreover, we studied the effect of the conditioned medium (CM) of MNC cultures from BM-BCP over the osteoclastogenic differentiation of HV-peripheral blood monocytes (PB-Mo). Finally, we evaluated some stimulant (IL-6, IL-7, IL-8, IL-11, TNF-á, IL-17, PGE2, TGF-â, soluble-RANKL, M-CSF, IL-1â, and GM-CSF) and inhibitor factors (IL-4, IL-10, OPG and IFN-ã) of osteoclastogenesis in CM from CFU-F assays (7 and 14 days) from BCP and HV. Methodology: Osteoclastic Differentiation: a) BM-MNC of BCP and HV:  the cultures were incubated in alpha medium suplemented with 20% horse serum with or without Vitamin D3 (VitD3) (10-8M). After 5 days CM were harvested and cells were cultured in the same conditions previously described. b) PB-Mo of HV: Mo were cultured in 90% CM of BCP or HV and 10% of alpha medium supplemented with 10% FBS and M-CSF (25ng/ml). The count of the number of osteoclast cells (OC) was made at day 16. OC= cells with 3 or 5 nucleus, TRAP positive. Soluble factors were quantified by ELISA in CM of CFU-F cultures (days 7 and 14) from BM of BCP and HV. Results: we observed spontaneous osteoclastogenesis (SpOC) in BM of BCP, (OC)%=36.9±1.9 and non-responsiveness to VitD3, meanwhile SpOC was not observed in BM of HV, which did respond to VitD3 (OC%=9.8±0.6). Moreover, BCP-CM treatment induced osteoclastic differentiation of HV-PB-Mo in similar way to 25ng/ml of sRANKL (34.6±1.9 vs 32.6±2.5, respectively). OC from patients were of a higher size and number of nucleus/OC. In contrast, HV-CM treatment did not induced osteoclastic differentiation of HV-PB-Mo.  SpOC in BM could be related to the major mRANKL expression observed in MSC of BM from BCP vs HV (+++vs++). Finally, BCP presented significantly higher levels of GM-CSF (BCP=98±4 vs.  HV= <7.8 pg/ml; in 7 days CM from CFU-F) as osteoclastogenic factor and lower levels of OPG (BCP=177±40 vs. HV=1,422± 264; p<0.05) as inhibitor factor. We cannot discard other soluble factors present in BM-CM from BCP that could stimulate the differentiation of GM progenitors and PB-Mo. Conclusion: we consider important the evaluation of the osteoclastogenic potential of BM- hematopoietic progenitors as well as the level of OPG and GM-CSF as possible prognostic factors for future bone disorders that may favor the invasion of BC cells into bone.