<p class="MsoNormal" style="margin-bottom: 12.0pt; text-align: left; mso-line-height-alt: 14.0pt; layout-grid-mode: char; mso-layout-grid-align: none;" align="left">Evaluation of ¹⁷⁷Lu-PSMA-617 Based Targeted Radioligand Therapy with X-Ray Stimulated PSMA Relocation in the <em>PiggyBac</em> Reporter Gene Engineered Orthotopic Prostate Tumor Model

Because human prostate cancer (PCa) grows slowly, establishing PCa tumor models is often time-consuming and unpredictable, limiting the efficiency of preclinical theranostic development. To address this, we used a non-viral PiggyBac transposon system to introduce triple reporter genes into PSMA-expressing C4-2 cells, generating orthotopic and subcutaneous xenograft models that allow noninvasive, real-time monitoring of PCa progression and treatment response. Reporter-engineered C4-2 3R cells were produced by co-transfecting constructs encoding the reporter cassette and PB transposase, followed by enrichment using fluorescence microscopy and FACS, and implanted orthotopically or subcutaneously into mice. Tumor growth and response to a single 2 Gy X-ray dose followed by 14.8 MBq 177Lu-PSMA-617, or to each monotherapy, were monitored weekly using IVIS imaging and confirmed by tumor dissection and H&amp;E staining; PSMA expression was assessed by western blot and 18F-PSMA-1007 PET/CT. C4-2 3R cells successfully expressed mRFP, luc2, and HSV1-tk, generating detectable orthotopic bioluminescence within one week and persisting for at least five weeks, whereas subcutaneous implantation produced only transient luc2 signals with no tumor formation. X-ray exposure did not increase total PSMA levels but induced PSMA relocation to the cell membrane. Combined external beam radiotherapy (EBRT) and 177Lu-PSMA-617 treatment produced the highest 18F-PSMA-1007 uptake and strongest tumor suppression, with minimal residual tumor mass compared to single-treatment or control groups. Overall, the C4-2 3R reporter model enables faster, reliable monitoring of slow-growing PCa tumors and provides an effective platform for evaluating PSMA-targeted therapies with or without the combination of EBRT.