{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,16]],"date-time":"2026-08-16T04:26:37Z","timestamp":1786854397348,"version":"build-2736575974"},"reference-count":51,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100011892","name":"bilateral project of Chang-Gung Memorial Hospital, Chang Gung University and National Yang Ming Chiao Tung University","doi-asserted-by":"publisher","award":["CGMH-NYCU-113-CORPG8P0221"],"award-info":[{"award-number":["CGMH-NYCU-113-CORPG8P0221"]}],"id":[{"id":"10.13039\/501100011892","id-type":"DOI","asserted-by":"publisher"}]},{"award":["CGMH-NYCU-113-CORPG8P0221"],"award-info":[{"award-number":["CGMH-NYCU-113-CORPG8P0221"]}],"id":[{"id":"https:\/\/ror.org\/00se2k293","id-type":"ROR","asserted-by":"publisher"}]},{"name":"National Science and Technology Council","award":["NSTC 114-2314-B-A49-065-MY3"],"award-info":[{"award-number":["NSTC 114-2314-B-A49-065-MY3"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: Because human prostate cancer (PCa) typically exhibits slow tumor growth, establishing reliable PCa tumor models is often time-consuming and unpredictable, thereby limiting the efficiency of preclinical theranostic research. To overcome this limitation, this study employed 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. Methods: Reporter-engineered C4-2 3R cells were generated by co-transfecting constructs encoding the reporter cassette and PB transposase, followed by enrichment through fluorescence microscopy and fluorescence-activated cell sorting (FACS) and implantation orthotopically or subcutaneously into mice. Tumor growth and treatment response to a single 2 Gy X-ray dose followed by 14.8 MBq of 177Lu-PSMA-617, or to each monotherapy, were monitored weekly using an IVIS imaging system. Imaging findings were validated by tumor dissection and hematoxylin and eosin (H&amp;E) staining, while PSMA expression was assessed by Western blotting and 18F-PSMA-1007 PET\/CT. Results: C4-2 3R cells stably expressed the triple reporter genes (mRFP, luc2, and HSV1-tk), generating detectable orthotopic bioluminescence within one week and persisting for at least five weeks. In contrast, subcutaneous implantation generated only transient luc2 signals with no tumor formation. X-ray exposure did not increase total PSMA levels but induced the redistribution of PSMA to the cell membrane. Combined external beam radiotherapy (EBRT) and 177Lu-PSMA-617 treatment produced higher 18F-PSMA-1007 uptake and stronger tumor suppression, with minimal residual tumor mass, as compared to single-treatment or control groups. Conclusions: This preliminary investigation suggests that the C4-2 3R model provides a practical and trackable tool for investigating slow-growing PCa tumors and evaluating PSMA-targeted therapies, either alone or in combination with EBRT.<\/jats:p>","DOI":"10.3390\/pharmaceutics18081009","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T15:48:36Z","timestamp":1786722516000},"page":"1009","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Preliminary Evaluation of 177Lu-PSMA-617-Based Targeted Radioligand Therapy with X-Ray Stimulated PSMA Relocation Using the PiggyBac Reporter-Gene-Engineered Orthotopic Prostate Tumor Model"],"prefix":"10.3390","volume":"18","author":[{"given":"Yen-Ta","family":"Chen","sequence":"first","affiliation":[{"name":"Division of Urology, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung 833401, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-0529-2933","authenticated-orcid":false,"given":"Ke-Hsin","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming Chiao Tung University, Taipei 112304, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8217-1692","authenticated-orcid":false,"given":"Chun-Yi","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming Chiao Tung University, Taipei 112304, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0340-7557","authenticated-orcid":false,"given":"Yi-Jang","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming Chiao Tung University, Taipei 112304, Taiwan"},{"name":"Cancer and Immunology Research Center, National Yang Ming Chiao Tung University, Taipei Branch, Taipei 112304, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1038\/s41572-020-00243-0","article-title":"Prostate cancer","volume":"7","author":"Rebello","year":"2021","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_2","first-page":"1809","article-title":"LNCaP model of human prostatic carcinoma","volume":"43","author":"Horoszewicz","year":"1983","journal-title":"Cancer Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"582","DOI":"10.1016\/j.drudis.2019.12.010","article-title":"Reporter gene imaging and its role in imaging-based drug development","volume":"25","author":"Shaikh","year":"2020","journal-title":"Drug Discov. Today"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"27","DOI":"10.4103\/2277-9175.98152","article-title":"Viral and nonviral delivery systems for gene delivery","volume":"1","author":"Nayerossadat","year":"2012","journal-title":"Adv. Biomed. Res."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Taghdiri, M., and Mussolino, C. (2024). Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25137333"},{"key":"ref_6","first-page":"102010","article-title":"The effect of enhancers on the lentiviral transduction efficiency in the human RPE cells: Insights for advancing retinal gene therapies","volume":"42","author":"Najafi","year":"2025","journal-title":"Biochem. Biophys. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Lin, P., Correa, D., Lin, Y., and Caplan, A.I. (2011). Polybrene inhibits human mesenchymal stem cell proliferation during lentiviral transduction. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0023891"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1007\/s10439-012-0678-1","article-title":"Physical non-viral gene delivery methods for tissue engineering","volume":"41","author":"Mellott","year":"2013","journal-title":"Ann. Biomed. Eng."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4118","DOI":"10.2174\/0929867322666151001121601","article-title":"Non-viral Delivery Systems for the Application in p53 Cancer Gene Therapy","volume":"22","author":"Wang","year":"2015","journal-title":"Curr. Med. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1089\/hum.2017.004","article-title":"Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery of Sleeping Beauty Transposons: Implications for Non-Viral Gene Therapy of Systemic Disease","volume":"28","author":"Aronovich","year":"2017","journal-title":"Hum. Gene Ther."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1007\/s10709-009-9391-x","article-title":"Gene therapy vectors: The prospects and potentials of the cut-and-paste transposons","volume":"138","author":"Chalmers","year":"2010","journal-title":"Genetica"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"14981","DOI":"10.1073\/pnas.0607282103","article-title":"The piggyBac transposon holds promise for human gene therapy","volume":"103","author":"Feschotte","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1038\/sj.mt.6300028","article-title":"PiggyBac transposon-mediated gene transfer in human cells","volume":"15","author":"Wilson","year":"2007","journal-title":"Mol. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e399","DOI":"10.1038\/cddis.2012.136","article-title":"Remnant living cells that escape cell loss in late-stage tumors exhibit cancer stem cell-like characteristics","volume":"3","author":"Chen","year":"2012","journal-title":"Cell Death Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1089\/ars.2023.0310","article-title":"The Emergence of Tumor-Initiating Cells in an Advanced Hypopharyngeal Tumor Model Exhibits Enhanced Angiogenesis and Nuclear Factor Erythroid 2-Related Factor 2-Associated Antioxidant Effects","volume":"41","author":"Lin","year":"2024","journal-title":"Antioxid. Redox Signal."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1091","DOI":"10.1056\/NEJMoa2107322","article-title":"Lutetium-177-PSMA-617 for Metastatic Castration-Resistant Prostate Cancer","volume":"385","author":"Sartor","year":"2021","journal-title":"N. Engl. J. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e15734056276494","DOI":"10.2174\/0115734056276494231207101146","article-title":"Impact of 68Ga-PSMA PET\/CT on Survival and Management in Prostate Cancer","volume":"20","author":"Algin","year":"2024","journal-title":"Curr. Med. Imaging"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1852","DOI":"10.2967\/jnumed.125.270465","article-title":"Current Developments in Combining External-Beam Radiotherapy and 177Lu-Labeled PSMA Ligands for Prostate Cancer Treatment","volume":"66","author":"Teunissen","year":"2025","journal-title":"J. Nucl. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2560","DOI":"10.7150\/thno.93249","article-title":"Exploring the role of combined external beam radiotherapy and targeted radioligand therapy with [177Lu]Lu-PSMA-617 for prostate cancer\u2014From bench to bedside","volume":"14","author":"Arbuznikova","year":"2024","journal-title":"Theranostics"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3652","DOI":"10.1158\/1078-0432.CCR-21-4332","article-title":"Combining Targeted Radionuclide Therapy and Immune Checkpoint Inhibition for Cancer Treatment","volume":"28","author":"Kleinendorst","year":"2022","journal-title":"Clin. Cancer Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1339","DOI":"10.1007\/s00259-020-05057-6","article-title":"Extensive preclinical evaluation of lutetium-177-labeled PSMA-specific tracers for prostate cancer radionuclide therapy","volume":"48","author":"Ruigrok","year":"2021","journal-title":"Eur. J. Nucl. Med. Mol. Imaging"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1772","DOI":"10.2967\/jnumed.123.265433","article-title":"Tandem Isotope Therapy with 225Ac- and 177Lu-PSMA-617 in a Murine Model of Prostate Cancer","volume":"64","author":"Meyer","year":"2023","journal-title":"J. Nucl. Med."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Kristiansson, A., Orbom, A., Ahlstedt, J., Karlsson, H., Zedan, W., Gram, M., Akerstrom, B., Strand, S.E., Altai, M., and Strand, J. (2021). 177Lu-PSMA-617 Therapy in Mice, with or without the Antioxidant alpha(1)-Microglobulin (A1M), Including Kidney Damage Assessment Using (99m)Tc-MAG3 Imaging. Biomolecules, 11.","DOI":"10.3390\/biom11020263"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1007\/s40846-022-00703-w","article-title":"Use of piggyBac Transposon System Constructed Murine Breast Cancer Model for Reporter Gene Imaging and Characterization of Metastatic Tumor Cells","volume":"42","author":"Chen","year":"2022","journal-title":"J. Med. Biol. Eng."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1016\/j.cellbi.2009.01.017","article-title":"The piggyBac transposon is an integrating non-viral gene transfer vector that enhances the efficiency of GDEPT","volume":"33","author":"Kang","year":"2009","journal-title":"Cell Biol. Int."},{"key":"ref_26","first-page":"e50873","article-title":"An orthotopic murine model of human prostate cancer metastasis","volume":"79","author":"Pavese","year":"2013","journal-title":"J. Vis. Exp."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1186\/s13550-018-0451-z","article-title":"Preclinical evaluation of PSMA expression in response to androgen receptor blockade for theranostics in prostate cancer","volume":"8","author":"Luckerath","year":"2018","journal-title":"EJNMMI Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3268","DOI":"10.21037\/tcr-21-372","article-title":"Comparison of tumor angiogenesis in subcutaneous and orthotopic LNCaP mouse models using contrast-enhanced ultrasound imaging","volume":"10","author":"Liu","year":"2021","journal-title":"Transl. Cancer Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1002\/pros.23115","article-title":"Characterization of a novel metastatic prostate cancer cell line of LNCaP origin","volume":"76","author":"Castanares","year":"2016","journal-title":"Prostate"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1038\/aja.2009.91","article-title":"The radiation response of androgen-refractory prostate cancer cell line C4-2 derived from androgen-sensitive cell line LNCaP","volume":"12","author":"Xie","year":"2010","journal-title":"Asian J. Androl."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1002\/pros.2990220203","article-title":"Growth of an androgen-sensitive human prostate cancer cell line, LNCaP, in nude mice","volume":"22","author":"Lim","year":"1993","journal-title":"Prostate"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1164","DOI":"10.1002\/pros.20965","article-title":"The prostatic environment suppresses growth of androgen-independent prostate cancer xenografts: An effect influenced by testosterone","volume":"69","author":"Jennbacken","year":"2009","journal-title":"Prostate"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1526","DOI":"10.1002\/pros.23027","article-title":"Orthotopic tumorgrafts in nude mice: A new method to study human prostate cancer","volume":"75","author":"Saar","year":"2015","journal-title":"Prostate"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Liu, W., Zhu, Y., Ye, L., Zhu, Y., and Wang, Y. (2022). Establishment of an orthotopic prostate cancer xenograft mouse model using microscope-guided orthotopic injection of LNCaP cells into the dorsal lobe of the mouse prostate. BMC Cancer, 22.","DOI":"10.1186\/s12885-022-09266-0"},{"key":"ref_35","first-page":"138","article-title":"Optimizing Prostate Cancer Diagnosis: A Prospective, Randomized Comparison of 12-core vs. 20-core Biopsy for Detection Accuracy and Upgrading Risk","volume":"12","author":"Baba","year":"2025","journal-title":"J. Urol. Surg."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e951","DOI":"10.1016\/j.urology.2012.06.023","article-title":"Potential establishment of lung metastatic xenograft model of androgen receptor-positive and androgen-independent prostate cancer (C4-2B)","volume":"80","author":"Yamamichi","year":"2012","journal-title":"Urology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2954","DOI":"10.7150\/thno.24108","article-title":"Multimodality reporter gene imaging: Construction strategies and application","volume":"8","author":"Li","year":"2018","journal-title":"Theranostics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2612","DOI":"10.7150\/thno.42228","article-title":"Targeted alpha therapy in a systemic mouse model of prostate cancer\u2014A feasibility study","volume":"10","author":"Stuparu","year":"2020","journal-title":"Theranostics"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1016\/j.neulet.2015.06.011","article-title":"Polybrene: Observations on cochlear hair cell necrosis and minimal lentiviral transduction of cochlear hair cells","volume":"600","author":"Han","year":"2015","journal-title":"Neurosci. Lett."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1038\/ijos.2015.18","article-title":"PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells","volume":"7","author":"Inada","year":"2015","journal-title":"Int. J. Oral Sci."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Ali, F.Z. (2025). Prostate Cancer Imaging Beyond PSMA: Applications of GRPR, AR, and Amino Acid Tracers. Diagnostics, 15.","DOI":"10.3390\/diagnostics15212737"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3104","DOI":"10.1158\/1078-0432.CCR-21-4531","article-title":"Prostate-Specific Membrane Antigen Expression and Response to DNA Damaging Agents in Prostate Cancer","volume":"28","author":"Sheehan","year":"2022","journal-title":"Clin. Cancer Res."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Zhang, C., Liu, J., Wu, J., Ranjan, K., Cui, X., Wang, X., Zhang, D., and Zhu, S. (2024). Key molecular DNA damage responses of human cells to radiation. Front. Cell Dev. Biol., 12.","DOI":"10.3389\/fcell.2024.1422520"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1042\/BJ20080413","article-title":"Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining","volume":"417","author":"Mahaney","year":"2009","journal-title":"Biochem. J."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1038\/s41585-024-00900-z","article-title":"Biological determinants of PSMA expression, regulation and heterogeneity in prostate cancer","volume":"22","author":"Bakht","year":"2025","journal-title":"Nat. Rev. Urol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1016\/j.canlet.2005.12.027","article-title":"Gene expression in the LNCaP human prostate cancer progression model: Progression associated expression in vitro corresponds to expression changes associated with prostate cancer progression in vivo","volume":"244","author":"Chen","year":"2006","journal-title":"Cancer Lett."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1016\/j.neo.2018.06.003","article-title":"A Novel Flavonoid Composition Targets Androgen Receptor Signaling and Inhibits Prostate Cancer Growth in Preclinical Models","volume":"20","author":"Mamouni","year":"2018","journal-title":"Neoplasia"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1038\/cgt.2013.34","article-title":"Bicalutamide-activated oncolytic adenovirus for the adjuvant therapy of high-risk prostate cancer","volume":"20","author":"Johnson","year":"2013","journal-title":"Cancer Gene Ther."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.canlet.2019.01.010","article-title":"Small molecule BKM1972 inhibits human prostate cancer growth and overcomes docetaxel resistance in intraosseous models","volume":"446","author":"Chen","year":"2019","journal-title":"Cancer Lett."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"955","DOI":"10.1089\/hum.2014.048","article-title":"Cellular immune response against firefly luciferase after sleeping beauty-mediated gene transfer in vivo","volume":"25","author":"Vezys","year":"2014","journal-title":"Hum. Gene Ther."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1177\/10915818221150790","article-title":"Non-Clinical Cell Therapy Development Using the NCG Mouse Model as a Test System","volume":"42","author":"Smutova","year":"2023","journal-title":"Int. J. Toxicol."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/18\/8\/1009\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,16]],"date-time":"2026-08-16T04:14:04Z","timestamp":1786853644000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/18\/8\/1009"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,14]]},"references-count":51,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["pharmaceutics18081009"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics18081009","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,14]]}}}