In a number of preclinical studies, multiparametric CEUS with VEGFR2-targeted microbubbles (MB) has been demonstrated to be a highly sensitive tool for monitoring the effects of therapy in a range of tumour entities (1-3). Consequently, VEGFR2-targeted CEUS may also be capable of providing combined functional and molecular imaging biomarkers for the non-invasive in vivo assessment of tumour angiogenesis and early therapy response in murine melanoma allografts under combined anti-PD-L1/anti-CTLA-4 immunotherapy.

Therefore, we hypothesised that CEUS with VEGFR2-targeted MB enables the functional and molecular in vivo monitoring of early immunotherapy effects in a murine melanoma allograft tumour model. We aimed to investigate whether the functional CEUS parameters of tumour perfusion and the molecular CEUS parameters of VEGFR2-targeted MB have potential as non-invasive in vivo imaging biomarkers of early therapy response with time point-matched multiparametric immunohistochemical validation as reference standard.