Bone Abstracts

Aims: Using an extensive bioinformatic approach we identified integrin α5 subunit as a novel potential target to treat bone dissemination from breast cancer. Aim of this study is to confirm the value of this target.

Methods: Integrin α5 mRNA expression levels were quantified by qRT-PCR, using radically resected primary tumors of 427 breast cancer patients. α5 expression at protein level by IHC on primary tumor was correlated, in an additional cohort consisting of 268 radically resected breast cancer patients, with presence of disseminated tumor cells (DTCs) in bone marrow at the time of surgery. BALB/c mice were injected in to tail vein with human MDA-MB-231 α5 silenced or scramble. Animals were killed on day 14 after tumor cell inoculation and lung and bone marrow from the hind limbs was flushed for DTC colony assay. Alternatively female BALB/c mice were treated with a chimeric monoclonal antibody that specifically binds to human integrin subunit α5 (M200) or with vehicle. Treatment was performed three times per week starting the day before intra-arterial inoculation with luciferase expressing human MDA-MB-231/B02 breast cancer cells, which selectively metastasize to bone. Vehicle and M200-treated mice were analyzed by radiography and bioluminescence. In an additional protocol, animals were culled on day 7 after tumor cell inoculation, and the bone marrow was flushed for DTC colony assay.

Results: Compared to low expression, an high α5 expression was associated with shorter bone metastasis-free survival, both in univariate (P=0.024) and multivariate analysis (P=0.04). Moreover we found significant (P=0.039) positive association between α5 protein expression on primary cancer and presence of DTCs in bone marrow. Taking advantage of the ability of the MDA-MB-231 cancer cell to metastatize both in bone and lung, we found that abrogating α5 gene function dramatically reduced the number of bone micrometastases (P=0.015) without affecting lung dissemination. In addition treatment of MDA-MB-231/B02 injected animals with M200 antibody significantly delayed the onset of skeletal lesions (P=0.02) and caused a 50% reduction in the extent of osteolytic lesions (P=0.038), compared to vehicle. This difference was accompanied with a sharp reduction of tumor burden (P=0.02), as determined by bioluminescence. Histomorphometric analysis of metastatic legs showed that M200 treatment decreased skeletal tumor burden (P=0.027) and increased the bone volume (P=0.02), compared to vehicle. Additionally, the number of DTC colonies from M200-treated mice was dramatically decreased compared with vehicle (P<0.001).

Conclusion: Our results suggest that α5 integrin expression in breast cancer cells facilitates bone marrow micrometastasis formation and the subsequent development of osteolytic lesions. vehicle (P<0.001). colonies in the bone marrow from M200-treated mice was dramatically decreased compared with vehicle (P<0.001).