Anatomically Programmable and Optically Tunable 3D-Bioprinted Tumor-Mimicking Phantoms for Quantitative Nanofluorophore Benchmarking

Surgical resection remains a primary treatment for solid tumors, with fluorescence-guided surgery offering improved intraoperative visualization and precision. However, conventional murine models provide limited control over anatomical and optical variables critical to evaluating fluorescent imaging agents. Here, we developed 3D-bioprinted, anatomically tailored and optically tunable tumor-mimicking phantoms (TMPs) as a reproducible platform for quantitative nanofluorophore evaluation under tissue-relevant surgical conditions. These TMPs recreate subcutaneous, epithelial, and intraperitoneal tumor presentations while enabling controlled variation of lesion geometry, spatial distribution, tissue pigmentation, cellular composition, and nanofluorophore concentration. Semiconducting polymer nanofluorophores (SPNs) with distinct emission profiles were incorporated as model imaging agents and evaluated using an in vivo imaging system (IVIS). The engineered phantoms enabled quantitative assessment of wavelength-dependent fluorescence, lesion visualization and delineation across diverse geometries and tissue backgrounds, multiplexed imaging of spatially distinct lesions, and simulation of fluorescence-guided tumor resection. Constructs incorporating GFP-expressing cancer cells further demonstrated simultaneous visualization of cellular and nanoprobe-derived fluorescence. Collectively, these findings establish anatomically programmable TMPs as a versatile, reproducible framework for benchmarking fluorescent imaging agents, systematically interrogating anatomical and optical determinants of imaging performance, and advancing fluorescence-guided surgical strategies before in vivo evaluation and clinical translation.