Rapamycin and Zoledronic Acid strongly retard growth of advanced murine hepatocellular carcinoma via skewing and activation of innate and adaptive immunity

Treatment options for hepatocellular carcinoma (HCC) remain limited. Activation of the cancer immune microenvironment opens novel therapeutic opportunities to control tumor progression and block metastatic spread. We assessed single and combined treatment of experimental HCC with Zoledronic Acid (ZA) and Rapamycin (RA), two drugs that display myeloid cell regulating potential.

Methodology:

Mdr2(Abcb4)-/- mice and fvb wildtype were injected intraperitoneally with diethyl-nitrosamine DEN (10 µg/g of bw) at the age of 5 days, followed by 0.05% phenobarbital in drinking water starting at the age of 3 weeks. Mice were treated with vehicle, (ZA thrice a week as IP injection 100 µg/kg), RA (trice a week as oral gavage 5 mg/body weight), or a combination of ZA and RA from age 5 – 6 months. After 6 months, tumor volume and number of nodules were counted. FACS analysis were done using antibodies to CD11b, CD11c, Ly6C, Ly6G, CD86, CD4, CD8, CD25, CD3, CD90.2, CD206, F4/80, MHC-II, NK1.1, Foxp3 and CD19. qPCR and IHC were performed.

Results:

Treatment with RA>ZA significantly reduced tumor growth. The combination of ZA and RA synergistically reduced the volume and number of HCC foci by 90 and 85% respectively (p < 0.0001). The combination significantly reduced the population of M0- (CD45⁺CD11b⁺LY6G^-F4/80⁺) and M2 macrophages (CD45⁺CD11b⁺LY6G^-F4/80⁺CD206⁺), and of myeloid derived suppresser cells (CD45⁺CD11c⁺LY6C^high), representing central tumor promoting myeloid cell populations, while Myeloid derived dendric cells (CD45⁺CD11c^high CD11b⁺ CD86⁺) that promote anti-cancer immunity were significantly upregulated. In parallel, total CD4+ T cells and especially CD4+CD25 regulatory T cells were significantly suppressed, while CD8+T cells were significantly increased in the combination treatment vs. the untreated group. Changes in immune cell populations were confirmed further by reduced transcript levels (CSF-1, VEGF, TGFβ1, Cox2, HIF1α, CCL2, CCL3, CCL5 and CCL17) and IHC (Ki-67-positive cancer cells were nearly undetectable, and IHC for CD68 and YM-1 confirmed a dramatic shift from tumour associated M2 to M1 macrophages in the treated group.

Conclusion:

1. Combination therapy of RAPA and ZA polarized synergistically the myeloid (and T cells) towards a robust anti-HCC response.