Renal cancer early diagnosis using a novel B7-H3 targeted ultrasound contrast imaging

Aim: Cost-effective or accessible methods to detect kidney cancers, especially those with renal insufficiency, remain elusive. We propose ultrasound molecular imaging (UMI) using microbubbles (MBs) targeted to cancer-associated vascular endothelial (VE) markers as a diagnostic imaging tool for kidney cancer with high specificity. CD276 (B7-H3), an immune checkpoint (IC) marker and a member of the immunoglobulin superfamily, is overexpressed on the VE cells of different cancers including renal cancer. Methods: We have developed an engineered affibody (Aby) specifically targeting both mouse and human B7-H3 for imaging renal cancer in vivo using US. In this study, we biotinylated the Aby and linked to MBs that were functionalized with avidin to create B7-H3 targetable MBs (TMBs) or with a scrambled Aby not binding to B7-H3 as a control MBs (non-targeted MBs or NTMBs), yielding (10 x 10 8 MBs/mL). Balb/C mice (n=4) were implanted with renca kidney cancer cells at the lower flank for in vivo imaging of VE-B7-H3 expression in the tumor. We also performed immunofluorescence (IF) analysis of the tumor tissue for the presence of B7-H3 and CD31 on VE cells at the vessels in the tumor microenvironment (TME). Results: Mice were imaged after tumor growth reached to 0.8-1.1 mm diameter with UMI, after administration of 1-2 x10 7 TMBs or NTMBs in 200 µL of saline. Compared to control NTMBs, the UMI signal using B7-H3-targeted TMBs was significantly higher (P= 0.002). IF analysis confirmed a high correspondence between B7-H3 and the universal vascular CD31 marker. Conclusions: The developed TMBs were able to detect and image the in vivo expression of tumor-associated B7-H3 in the VE cells. The results support the feasibility of these B7-H3-TMBs in translating into human kidney cancer diagnosis using UMI.