Zoledronic acid repolarize tumour associated macrophages towards anti-HCC responses both In vitro and In vivo

Interaction of tumour cells with Myeloid derived suppressive cells (MDSC), especially tumour associated macrophages (TAMs) favours their proliferation, survival and metastasis. TAM exhibit features of M2 like macrophages and are present in all stages of HCC. Our aim is to repolarize TAMs from tumor promoting M2-like into a tumor-antagonistic M1-like macrophage. In this respect, our data, point to TAMs as the most important immune target of Zoledronic acid-mediated antitumour activity and that pharmacological skewing of TAMs polarization from M2 to a full M1 phenotype is an important element in solving the therapeutic conundrum of early treatment in primary liver cancer.

Mouse bone marrow derived (BMD) macrophages and the RAW macrophage cell lines were cultured with either LPS and INF-γ, or IL-4 and IL-13 to generate M1 and M2 macrophages, respectively. Viability was assessed by the MTT assay. Polarized macrophages were exposed to increasing concentrations of zoledronic acid. Gene expression level of M1 markers (TNF-alpha, IL-1beta, iNOS, TIMP1) and M2 markers (ARG1, VEGF, YM1, MRC1, MMP9) were quantified by qPCR. IL-12 (M1) and IL-10 and VEGF (M2) secretion were measured by ELISA. M1/M2 macrophages and immune cells were quantified in sections of murine HCC (DEN/Mdr2KO model) using macrophage polarization markers and ZA was given to HCC bearing mice to assess its anti-tumor efficacy in vivo.

Zoledronic acid significantly increased transcript levels of M1 Markers (iNOS, TNF-alpha, IL-1β, TIMP1) in BMDM and RAW M2 polarized cells. This was paralleled by a significant upregulation of IL-12 and downregulation of IL-10 and VEGF protein production. In vivo in HCC bearing mice, FACS analysis demonstrated ZA boost innate and/or adaptive antitumour immune responses by means of IL-12, NKT stimulation and depletion of Foxp3⁺ Treg cells or MDSC. Immunohistology of ZA treated murine HCC liver sections showed a dramatic upregulation of M1>M2 macrophages thereby a significant reduction in HCC growth.

In summary, zoledronic acid exhibit potent macrophage repolarizing activity (M2 towards M1) in vitro and in vivo and thereby limit the angiogenesis (MMP9, VEGF) and provide novel insight for the connection between innate and adaptive immune responses in anti-tumour immunotherapy.