Abstract P5-04-13: Antibody-based therapy targeting integrin a5 is an effective strategy to treat experimental breast cancer bone metastasis

Abstract

Abstract Background: Integrin a5β1 is a specific fibronectin receptor that is often upregulated in breast cancer cells undergoing epithelial-to-mesenchymal transition. Preclinical evidence showed that α5β1-fibronectin interaction promotes in vitro the survival of growth-arrested breast cancer cells in the bone marrow. This is in line with the observation that disseminated tumor cells (DTCs) in the bone marrow, that are the earliest sign of development of metastatic disease in patients, express α5β1 integrin. However, the role of α5β1 in bone marrow micrometastasis formation remains unknown. Here, we investigated its role in experimental breast cancer bone metastasis, using an antibody-based strategy targeting the α5 subunit. Methods: Integrin α5 mRNA expression levels were quantified by qRT-PCR, using radically resected primary tumors of 427 breast cancer patients. Comparison between Kaplan-Meier curves was performed using the log-rank test. The Cox model was used for multivariate analysis. For experimental metastasis, female BALB/c immunodeficient mice were treated with a chimeric IgG4 monoclonal antibody that specifically binds to human integrin subunit α5 (M200;15mg/kg) or the vehicle. Treatment was performed 3 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. On day 28 after tumor cell inoculation, animals were sacrificed and histomorphometric analysis of metastatic legs was performed. Alternatively, animals were culled on day 7 after tumor cell inoculation, and the bone marrow was flushed for DTC colony assay. In-vitro cell-based assays were conducted to explore the functions of α5β1 on adhesion, invasion and survival. Statistical analysis was carried out by performing a Mann-Whitney U test. Results: Median durations of metastasis-free survival were 6.2 (high α5 expression) and 9.5 years (low α5 expression) (P = 0.0008). Additionally, compared with low expression, a high α5 expression was associated with shorter bone metastasis-free survival, both in univariate (P = 0.024) and multivariate analysis (HR = 1.65,95%CI = 1.02 to 2.67; P = 0.04).The treatment of tumor-bearing animals with M200 antibody statistically 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 with 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 with vehicle. Additionally, the number of DTC colonies in the bone marrow from M200-treated mice was dramatically decreased compared with vehicle (P<0.001). In vitro, M200 antibody did not affect tumor cell survival. By contrast, it specifically inhibited fibronectin-mediated tumor cell adhesion and invasion. Conclusion: Our results suggest that α5β1 integrin expression in breast cancer cells facilitates bone marrow micrometastasis formation and the subsequent development of osteolytic lesions. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-13.