CEUS with VEGFR2-targeted microbubbles for monitoring of early immunotherapy effects in a colorectal cancer model

Abstract Background To evaluate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for longitudinal monitoring of early treatment effects during combined anti-PD-L1/anti-CTLA-4 immunotherapy in a murine colorectal cancer model. Methods Murine colorectal cancer allografts (CT26) were established subcutaneously in 29 female Balb/c mice (therapy n = 15; control n = 14). Baseline CEUS with VEGFR2-targeted microbubbles was performed on day 7. The therapy group received intraperitoneal anti-PD-L1 and anti-CTLA-4 antibodies between days 7–15; controls received sham treatment. Follow-up CEUS was performed on days 14 and 21. Tumor perfusion (WiAUC) and VEGFR2 binding (SI 8min , SI 10min ) were quantified, and immunohistochemistry assessed CD8, Ki-67, TUNEL, CD31, and VEGFR2. Results At FU1, WiAUC was significantly lower in the therapy group compared with controls (4,029 ± 61 vs. 6,391 ± 412; p < 0.001), and remained significantly lower at FU2 (1,028 ± 27 vs. 2,049 ± 39; p < 0.001). VEGFR2-targeted microbubble binding was consistently lower under therapy. At FU1, SI 8min was 407 ± 11 vs. 626 ± 13 and SI 10min was 409 ± 10 vs. 634 ± 28 (both p < 0.001). At FU2, SI 8min was 106 ± 6 vs. 207 ± 4 and SI 10min was 107 ± 5 vs. 207 ± 4 (both p < 0.001). Immunohistochemistry confirmed higher apoptosis and tumor-infiltrating lymphocytes, and lower proliferation, microvascular density, and VEGFR2 expression in treated tumors (all p < 0.05). Conclusions VEGFR2-targeted CEUS enabled longitudinal monitoring of early response to dual anti-PD-L1/anti-CTLA-4 immunotherapy in colorectal cancer. Treated tumors showed lower perfusion and VEGFR2-targeted binding, paralleled by reduced vessel density and VEGFR2 expression as well as increased CD8 infiltration and apoptosis. These findings support VEGFR2-targeted CEUS as a promising noninvasive imaging biomarker for monitoring early immunotherapy-associated vascular changes.