Defining the differential effects of Zoledronic acid under pre and post-menopausal concentrations of oestradiol on anti-cancer immunity

Background: Late-stage breast cancer often recurs in bone. Neoadjuvant Zoledronic Acid (Zol) combined with standard of care can reduce bone metastases in pre- and post- menopausal women while prolonging life in post-menopausal women. However, pre-menopausal women experience worse iDFS and increased soft tissue tumour recurrence. Zol and oestrogen can both affect bone homeostasis and immune cells. Therefore, we hypothesise that oestrogen may inhibit anti-tumour activities of Zol in pre-menopausal women through immune regulatory pathways, and that combination therapy with Avelumab and Zol could improve treatment efficacy. Methods: Menopausal conditions were modelled in mice by ovariectomy followed by 10 pM/L, 84 pM/L and 300pM/L oestradiol. Metastases were induced by intracardiac injection of mouse mammary cancer E0771 and 4T1 cells before treatment with 100ug/kg/week Zol or control. Effects on tumours, bone, immune cells and hormones were assessed by IVIS imaging, uCT, flowcytometry, NanoString and ELISA. In same model, mice were treated with 400μg/kg every 2-days Avelumab in combination with Zol. Finding: Ovariectomy followed by oestrogen administration (n=15 per group) reliably recreated pre- peri and post- menopausal serum concentrations, while trabecular bone volume was significantly decreased under postmenopausal oestradiol (p<0.0001). Non-bone metastasis in pre-menopausal mice increased following Zol treatment, mirroring clinical findings. Data from flowcytometry and Nanostring analysis suggest that Zol and oestradiol exert deferential effects on immune populations, with oestrogen increasing M2 macrophage populations while decreasing B cells (P= 0.0091), and Zol increasing M1 (P=0.0374) populations, increasing T cells and decreasing PD-1 expression in the bone. Combination therapy with Avelumab and Zol significantly reduced metastasis to bone (P=0.0011) and lung (P=0.0004), irrespective of oestradiol concentrations, while increasing T cell activity. Conclusions: Oestradiol inhibits the anti-metastatic effect of Zol by inducing a pro-tumour immune microenvironment. Novel combination therapy with the anti PD-L1 Avelumab removes this inhibition and prevents bone and soft tissue metastasis.