Local radiotherapy might contribute to leukemia risk in breast cancer by recruitment of hematopoietic stem cells

Introduction: Secondary Acute Myeloid Leukemia (sAML) is the most common therapy induced malignancy affecting breasts cancer (BC) patients. Local breast radiation therapy (RT) is associated with a 3-fold increased risk in this iatrogenic complication; the mechanism of which remains unknown. Based on the assumption that AML is a stem cell-derived malignancy, we hypothesize that local RT contributes to sAML through the recruitment of Hematopoietic Stem Cells (HSC) to the site of radiation. Since BC patients are treated with fractionated RT, we propose that repeated RT causes a continuous recruitment of HSC to the site of RT, thereby increasing the risk of generating a leukemic stem cell. Hence, the objective of this study is to demonstrate that local RT causes recruitment of HSCs, induced by known chemokines and cytokines involved in this process.

Experimental Procedures:To investigate the effect of local RT on the migration of HSCs, Balb/C mice were irradiated on their left leg and injected 24h later with bone marrow cells (BMC) extracted from GFP+ Balb/c mice. Prior to injection, BMC were infected with a luciferase lentivirus. Migration of BMC was monitored in vivo using bioluminescence imaging (BLI). The presence of HSC among the recruited cells was analyzed by flow cytometry, using the Sca1, c-Kit, Thy1.1 and GFP markers. The expression of MMP2, 9 and SDF1 in the irradiated BM was assessed by quantitative real time PCR (qRT-PCR). The functional role of these chemokines and MMPs was evaluated using a blocking antibody for CXCR4, and a common inhibitor of MMP2 and 9.

Results: Our BLI results show differential accumulation of BMC to the irradiated leg, with maximum recruitment at 6 days post-injection (14 fold increase, compared to the right leg). Using flow cytometry, we show that 4 fold more donor-derived (GFP+) HSC are present in the left leg at 6 days, confirming preferential recruitment of HSC to the site of RT. The investigation by qRT-PCR of the chemokines relevant to this recruitment shows increased expression of SDF1, MMP2 and MMP9 in the irradiated leg by 16h post-RT (2.4, 14.5 and 2.9 fold, respectively). In accord with a functional role of these chemokines, complete inhibition of HSC recruitment was achieved by blockage of the MMP2 and 9 activity, while inhibition of CXCR reduced by 59% the number of HSC recruited to the site of RT.

Conclusion: These data suggest for the first time, that local RT has significant systemic effects, by recruiting HSC to the irradiated BM site; a process mediated by SDF1, MMP2 and MMP9. These data might also provide potential strategies, by which sAML could be prevented in BC patients.