Zoledronic Acid promotes tumoricidal immunity and supresses' tumour associated macrophages and myeloid derived suppressor cells in murine HCC

Background and Objectives:

HCC is associated with chronic inflammation leading to recruitment of bone marrow derived cells, mainly tumour associated macrophages (TAM, M2) and myeloid-cell derived suppressor cells (MDSC). Both cell types markedly suppress anti-HCC immune responses and are present in all stages of HCC. Zoledronic acid (ZA) is used in patients with osteoporosis to inhibit osteoclasts, i.e., bone macrophages with TAM-like properties. We hypothesized that ZA can polarize tumour promoting TAM and MDSC towards M1 macrophages with anti-tumour activity.

Methods:

Mouse bone marrow derived in vitro M1 or M2 polarized macrophages were exposed to increasing concentrations of ZA. Transcript levels of M1 markers (TNFα, IL-1β, iNOS, TIMP1) and M2 markers (Fizz1, ARG1, VEGF, YM1, MRC1, MMP-9) were determined by qPCR. IL-12 and SOCS3 (M1), IL-10 and VEGF (M2) secretion were measured by ELISA. Mice bearing a syngenic HCC (DEN/Mdr2KO model) received 3 doses of 100 µg ZA/kg BW or vehicle control intraperitoneally weekly for a period of one month from age month 5 – 6. After treatment, mice were sacrificed to measure hepatic tumor number and volume and livers were assessed by H&E histology, IHC and qPCR.

Results:

ZA dose-dependently decreased M2 (Fizz1, ARG1, STAT6, YM1, MRC1, MMP-9) and increased M1 macrophage (iNOS, TNF-alpha, IL-1beta, SOCS3) specific transcript levels without affecting cell viability in vitro. This was paralleled by a significant upregulation of IL-12 and dowregulation of IL-10 protein secretion into the cell supernatant. DEN/Mdr2KO mice with ZA reduced tumour number and volume by 33.3% and 68%, respectively vs. control group. Tumour cell proliferation as evaluated via Ki-67+ cells was significantly blunted by ZA. Staining of YM-1+ and glypican+ M2 and CD68+ total macrophages of ZA treated HCC liver sections showed a significant upregulation of M1>M2 macrophages and reduced numbers of TAM.

Conclusions:

1. ZA exhibits potent macrophage (and MDSC) repolarizing activity (M2 towards M1) in- vitro and in-vivo, thereby increasing anti-HCC immune responses and limiting angiogenesis. 2. Since ZA has a well-known clinical safety profile, it should be assessed alone or in combination with other anti-cancer agents in patients with HCC.