Dual Blockade of CD47 and HER2 Re-sensitizes Resistant Breast Cancer Cells to Radiation Therapy

Abstract

Cancer cells with acquired resistance grow aggressively with a capacity to escape immune surveillance. Here, we aimed to report that CD47, a "do not eat” signal against Mφ-mediated phagocytosis, was concurrently up-regulated with HER2 in radio-resistant breast cancer cells and recurrent tumors, and co-expression of CD47 and HER2 is associated with a worse prognosis. In this study, we aimed to investigate the effect of radiation therapy in CD47 expression and whether NF-kB and/or HER2 is involved in the regulation of CD47 expression in breast cancer. Furthermore, we investigated the enhancement of in vivo phagocytosis in dual knock out cell lines (CD47-/- , HER2-/-) or after dual targeted therapy (anti-CD47, anti-Her2) post radiation. A panel of breast cancer cells, colon cancer cells, and glioblastoma were employed in this study. The cultured cells were irradiated by 5Gy, and the expression of CD47 was analyzed post-irradiation via immunohistochemistry and western blotting. DDAO-labeled wild type and radio resistant MCF7/C6 cells were co-cultured with mature human Mφ derived from the THP1 cell lines with or without CD47 antibody. The tumor aggressiveness phenotype was analyzed by mammosphere formation and matrigel invasion. NF-kB and HER2 in CD47 expression was studied by treating the cells with their respective inhibitors and/or siRNA-mediated depletion and testing the CD47 expression as well as macrophage-mediated phagocytosis of breast cancer cells. The use of anti-CD47 antibody as a strategy to inhibit “don’t eat me” signals was also analyzed by flow cytometry to look at macrophage-based phagocytosis. We generated a radio-resistant mouse breast tumor 4T1/C2 cell, and we used CRISPER technology to establish CD47 and HER2 knock out cell lines. The infiltration of inflammatory cells in the tumor were examined by CD11b+Mφ expression assay. The coordinate induction of CD47 and HER2 was achieved partly by a HER2-NF-κB loop. Antibody blockade of CD47 augmented Mφ-mediated phagocytosis and inhibited an aggressive phenotype. Dual antibody blockade or CRISPR-based dual gene suppression of CD47 and HER2 synergized radiation-mediated inhibition of clonogenicity and growth of orthotopic breast tumors, compared to blockading either receptor alone. These results suggest a network by which cancer cells enhance their aggressive behavior via CD47-mediated suppression of immune surveillance linked with HER2-initiated cell proliferation. Thus, dual blockade of CD47 and HER2 may enhance the eradication of resistant breast cancer cells during radiotherapy.