Development of imaging methods capable of furnishing tumor-specific morphological, functional, and molecular information is paramount for early diagnosis, staging, and treatment of breast cancer. Ultrasound (US) and optoacoustic (OA) imaging methods exhibit excellent traits for tumor imaging in terms of fast imaging speed, ease of use, excellent contrast, and lack of ionizing radiation. Here, we demonstrate simultaneous tomographic whole body imaging of optical absorption, US reflectivity, and speed of sound (SoS) in living mice. In vivo studies of 4T1 breast cancer xenografts models revealed synergistic and complementary value of the hybrid imaging approach for characterizing mammary tumors. While neovasculature surrounding the tumor areas were observed based on the vascular anatomy contrast provided by the OA data, the tumor boundaries could be discerned by segmenting hypoechoic structures in pulse-echo US images. Tumor delineation was further facilitated by enhancing the contrast and spatial resolution of the SoS maps with a full-wave inversion method. The malignant lesions could thus be distinguished from other hypoechoic regions based on the average SoS values. The reported findings corroborate the strong potential of the hybrid imaging approach for advancing cancer research in small animal models and fostering development of new clinical diagnostic approaches.

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