Science and Technology Production

EASL Congress 2025 - Liposomal doxorubicin and atezolizumab promote EMT regression by acting on the tumor microenvironment: preclinical in vitro and in vivo studies

Congreso

Authorship:

Ilaria Zanotto ; Daniela Gabbia ; Antonella Grigoletto ; Margherita Toffanin ; CALIENNI, MARIA NATALIA ; Gianfranco Pasut ; Sara De Martin

Date:

2025

Publishing House and Editing Place:

EASL

Summary *

Background and Aims: Hepatocellular carcinoma (HCC) is a highly aggressive liver cancer whose onset and progression is driven by immune-escape. HCC is characterized by the upregulation of the checkpoint inhibitor PD-L1, that helps immune evasion. The combination of the PD-L1 inhibitor atezolizumab and bevacizumab is the gold standard for advanced HCC treatment. Nevertheless, HCC management remains a significant challenge, highlighting the need for novel therapeutic approaches. The aim of this study is to investigate the anticancer and immunomodulatory role of atezolizumab targeted liposomal doxorubicin (Stealth Immunoliposomes, SIL), using as control its untargeted counterpart (Stealth Liposomes, SL) in vitro and in vivo models of HCC. Method: The effect of SL and SIL was evaluated on HepG2 and Huh7 cells treated with TNF-alpha to obtain a model of EMT, by analyzing the expression of PD-L1, and the EMT markers by ICC and qPCR. The role of the two formulations on Akt-1 and p65 protein and gene expression was also assessed. In vivo efficacy was assessed in a syngeneic mouse model of HCC, obtained by injecting subcutaneously Hepa1-6 cells in C57BL/6J immunocompetent mice. The mice were then treated with vehicle (control), SIL, SL, Atezolizumab, or SL+Atezolizumab. Treatments were administered intraperitoneally (5 mg/kg every 3 days for 6 doses). Tumor volumes were measured and collected at sacrifice to evaluate Cytotoxic T Lymphocytes, M1 and M2 macrophages by flow cytometry. Results: In the EMT in vitro model, SIL reduced PD-L1 protein (p<0.001) and gene expression (p<0.05), and increased E-cadherin (p<0.05), decreased Vimentin (p<0.001), and Beta-catenin (p<0.05) protein expression. An increasing effect on p65 protein expression was observed after SIL treatment only in the EMT model obtained with Huh7 cells (p<0.05), suggesting that the modulation of NFkBeta pathway occurs differently in the two cell lines. SIL decreased protein and gene expression of Akt-1 in both HepG2 (p<0.0001 and p<0.01 respectively) and Huh7 (p<0.0001 and p<0.001 respectively) cells, indicating that this formulation reduced EMT by acting on the Akt-1 pathway. In vivo, mice treated with Atezolizumab + SL was the most effective treatment in reducing tumor growth (p<0.05 vs controls and atezolizumab-treated mice). Flow cytometry analysis revealed higher number of Cytotoxic T cells and M1/M2 ratio in the tumor microenvironment of these mice (p<0.05 vs controls). Conclusion: In vitro, SIL treatment, eside exerting an immunomodulatory role in the tumor microenvironment, reduces tumor cell invasiveness by acting on the EMT. Atezolizumab + SL treatment reduces cancer growth in vivo, increases macrophage polarization towards an antitumoral phenotype and the immune response against cancer cells, suggesting that the combination of immunotherapy and chemotherapy might be a suitable strategy in HCC management. Information provided by the agent in SIGEVA

Key Words

DOXORUBICINHEPATOCELLULAR CARCINOMAATEZOLIZUMABIMMUNOLIPOSOMES