Nanobody PET tracer enables GPA33 expression profiling for precision diagnosis of colorectal cancer

Colorectal cancer (CRC) remains a leading cause of cancer mortality, highlighting the need for precise molecular imaging tools targeting biomarkers like glycoprotein A33 (GPA33), which is highly expressed in over 95% of CRC but currently lacks an ideal non-invasive probe for rapid clinical translation. This study developed a GPA33-targeted nanobody tracer, ⁶⁸GaGa-NOTA-WWH347, for PET imaging of CRC by engineering and radiolabeling a specific nanobody WWH347 with Ga-68. Western blot confirmed higher GPA33 expression in LS174T cells versus HT29 controls. In vitro cell uptake assays demonstrated specific tracer accumulation in LS174T cells (10.27 ± 0.45% at 2 h) versus negative HT29 cells (1.13 ± 0.14%) or LS174T blocking groups (0.96 ± 0.55%). The novel tracer ⁶⁸GaGa-NOTAWWH347 enabled superior PET visualization of CRC tumors in subcutaneous, liver metastasis, and systemic metastasis models, clearly outperforming both ¹⁸FF-FDG and the non-specific probe ⁶⁸GaGa-NOTA-5D5. Subsequent biodistribution studies revealed significantly higher tracer uptake in subcutaneous LS174T tumors (10.45 ± 0.78%ID/g) compared to HT29 (3.29 ± 0.61%ID/g). In addition, animal models with liver metastases showed tracer uptake at 11.69 ± 2.69%ID/g, while 9.71 ± 1.01%ID/g in systemic metastatic lesions, enabling non-invasive visualization of GPA33 expression profiles on the whole-body level. These findings establish ⁶⁸GaGa-NOTA-WWH347 as a precision diagnostic tool that could optimize GPA33-targeted therapies by mapping expression patterns in CRC primary tumors and metastatic lesions through rapid, specific PET imaging.