HER-2/neu overexpression increases the viable hypoxic cell population within solid tumors without causing changes in tumor vascularization

Abstract

The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6HER-2) into SCID-Rag2M mice. LCC6HER-2 tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6Vector). Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6HER-2 versus LCC6Vector tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6 HER-2 tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content ≥ 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6HER-2 than in LCC6Vector tumors (16.41 ± 8.1% and 5.96 ± 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser536) nuclear factor-κB p65 were significantly elevated in LCC6HER-2 tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1α protein levels was observed in LCC6HER-2 compared with LCC6Vector tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6HER-2 tumors, no significant differences were found (P > 0.05) between LCC6 HER-2 and LCC6Vector vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.