CONICET | Buscador de Institutos y Recursos Humanos

Bone marrow (BM) mesenchymal stem cells play an important role on bone formation as being the major source of osteoprogenitor cells, maintaining bone remodeling. Moreover these stem cells may differentiate into BM stroma cells. It is well known, that BM stroma cells and osteoblasts can regulate proliferation and differentiation of hematopoietic cells during the life span. In previous in vitro studies we found that BM microenvironment represented by mesenchymal stroma cells (MSC, majority fibroblasts) from chemonaive advanced breast cancer patients (BCP), had a diminished proliferative and confluence capacity. A defective proliferative capacity of the mesenchymal stem cell may represent a previous uncharacterized mechanism in bone disease. Furthermore, breast tumors carry risks of developing bone metastasis and these metastases had been characterized as osteolytic or osteoblastic which can cause bone pain, pathological fractures and hypercalcemia as cardinal symptoms. Some authors found that some breast cancer cell lines release soluble factors that inhibit osteoblastic proliferation and/ or induce apoptosis of these cells or of the BM -MSC. Based on our previous results, we decided to study the osteogenic potential of BM-MSC from chemonaive advanced BCP. The analysis of the osteogenic potential was performed on 7 BM chemonaive advanced BCP (without osteoporosis or bone metastasis) and 7 normal control (NC). MSC derived from low density 3rd passage were cultured for 21 days in osteogenic medium. Optimal osteogenic differentiation analysis was determined by osteoblastic morphology and formation of mineralized extracellular matrix, determined by Von Kossa technique. The results were evaluated by the following parameters: osteogenic clusters or colonies, % of cells showing a distinct morphology to the fibroblast (% of differentiation) and we also characterized the cells different to fibroblasts into a pattern of differentiation from grade 0 to 3, 0=fibroblast like, 1=in the way to osteoblast, 2=osteoblast, 3=osteocyte. Also, osteocalcin monoclonal antibody for immunocytochemistry was performed. All NC cultures developed clusters and/ or osteogenic like colonies while only 3/7 BCP cultures did. BCP cultures showed a 15±7% of cells from Petri dish with characteristics different to the fibroblasts morphology compared with the 24±4% showed by NC. The 100% NC cultures responded to the osteogenic medium achieving morphologies with characteristics of the osteogenic linage. Grade 1 was achieved by 43% of these NC cultures and the other 57% (4/7) a grade 2-3. In contrast, only the 57% of the BCP cultures responded to the osteogenic medium by changing the fibroblastic morphology. From the ones that responded, the 75% reached grade 1 and only 25% grade 2 or 3. In relation to the mineralization matrix, NC cultures showed homogeneous distribution of calcium as well as an adequate mineralization of the entire examined surface. The BCP cultures with osteoblastic/osteocytic cells had a scattered anarchic distribution as well as a poor mineralized matrix, without evidence of uniform and homogeneous covering of the entire field. The anti-osteocalcin antibody showed a weak expression on both NC and BCP cell cultures in our culture conditions, for this reason they could not be used to obtain important information relative to the different grades of osteogenic differentiation. In conclusion these results showed an inadequate osteogenic potential of BM-MSC from BCP compared to NC. Therefore, the in-vitro study of osteogenic differentiation of BCP-MSC may be considered as a prognostic factor because its alteration could be the first sign in future bone disease.

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