E‑Urea-K-Based PSMA Imaging Served as an Alternativein Assessing Tumor Neovascularization via Targeting CD31

To reveal the natural correlation between prostate-specific membrane antigen (PSMA) imaging and tumor neovascularization in prostate cancer and further explore E-urea-K-based PSMA-targeted (EK-PSMA) imaging as a potential indicator of tumor neovascularization, the 22Rv1 mouse models were established and underwent ^99mTc-HYNIC-ALUG SPECT/CT. Pearson correlation analysis was applied to assess the relationship between tumor tracer uptake and tumor characteristics, including size, glucose metabolism, and cell phenotypes (e.g., Ki-67, VEGF, CD31, and PSMA). Then, molecular docking further identified the key factors of EK-PSMA imaging, specifically related to tumor neovascularization. Finally, animal models with positive and negative PSMA expression (22Rv1, LNCaP, U87, SAOS-2, A549, and ACHN) were subjected to antibody-targeted blockade to verify the role of these key factors in EK-PSMA imaging. The Pearson’s <i>r</i> values of tracer uptake correlated with CD31 and tumor size were 0.82 and 0.99, respectively (<i>P</i> < 0.05), and the correlations of tracer uptake with SUV_max, SUV_mean, Ki-67, VEGF, and PSMA expressions were 0.47, 0.20, 0.69, −0.65, and 0.20, respectively (all <i>P</i> > 0.05). Molecular docking confirmed the affinity of E-urea-K to PSMA (two sites, binding scores, −5.4 kcal/mol and −6.0 kcal/mol) and CD31 (one site, binding score, −5.1 kcal/mol). The blockade of the CD31 antibody partially reduced the ^99mTc-HYNIC-ALUG uptake in five other types of tumors (paired <i>t</i> test, <i>P</i> = 0.0478). The Pearson’s <i>r</i> value of CD31 staining and tracer uptake prior to the antibody blockade was 0.84 (<i>P</i> < 0.05). Additionally, when removing the PSMA-positive models (22Rv1 and LNCaP), the Pearson’s <i>r</i> value of CD31 staining and tracer uptake prior to the antibody blockade was 0.99 (<i>P</i> < 0.05). Thus, CD31 was found to be a mutual target of EK-PSMA imaging; therefore, EK-PSMA imaging provides a viable assessment option for tumor neovascularization, especially for PSMA-negative tumors.