Liposomal doxorubicin targeted with the Fab’ of atezolizumab exerts an immunomodulatory effect in in vitro models of hepatocellular carcinoma

Introduction PD-L1, one of the most studied immune checkpoints, represents a key pharmacological target for HCC therapy. Atezolizumab, a monoclonal antibody inhibiting PD-L1 and restoring T-cell–mediated antitumor activity, is approved for advanced HCC therapy in combination with bevacizumab. Liposomal doxorubicin (DXR) targeted with the Fab’ of atezolizumab (SIL treatment) reduced tumor growth in preclinical models of HCC. Aim unravel the effect of PD-L1 targeted liposomal DXR on the immune phenotype of tumor associated macrophages and liver cancer cells, and on their invasiveness, using untargeted liposomal DXR as a control (SL treatment), in 2D and 3D cellular HCC models. Materials and Methods The effect of SIL and SL treatments on PD-L1 expression was evaluated in HepG2 2D cultures and spheroids treated with INF-gamma to induce its overexpression. Both liposomal formulations were tested to assess the effect on macrophage polarization, clonogenicity and invasiveness on HepG2 spheroids co-cultured with THP-1 monocytic cells. The effect of SL and SIL on the epithelial-to-mesenchymal transition (EMT) of HepG2 cells was evaluated on the epithelial marker E‑cadherin and the mesenchymal marker Vimentin by immunocytochemistry. Results Only SIL significantly decreased the INF-gamma-induced PD-L1 overexpression in HepG2 2D cultures and spheroids (p<0.01 and p<0.001 respectively), showing immunomodulatory activity. In THP-1/HepG2 spheroids, SL and SIL decreased clonogenicity and invasiveness of HepG2 cells (p<0.0001) and pro-tumoral CD163-expressing macrophages (p<0.0001). Only SIL caused a significant downregulation of PD-L1 expression (p<0.01). Accordingly, they significantly increased E-cadherin expression and decreased Vimentin one, downregulated and upregulated by TNF-α, respectively, at variance to SL (p< 0.001). Furthermore, SIL downregulated PD-L1 expression also in the TNF-α model (p<0.001 vs SL). Conclusion PD-L1-targeted liposomal DXR enhances the cytotoxic effect of liposomal doxorubicin and plays an immunomodulatory activity by decreasing PD-L1 expression and prompting macrophage polarization towards an antitumoral phenotype, showing promising results in preclinical models of HCC.