TETRA BRANCHED NEUROTENSIN RESULTS AN EFFECTIVE TUMOR-TARGETING AGENT FOR SUPERFICIAL AND INFIL- TRATING BLADDER CANCER IN AN “IN VITRO” AND “EX VIVO” STUDY

Aim of the study The aim of this study is to confirm the possibility to use the Neurotensin-tetra-branched form (NT-4) as a targeting agent for tumor cells “in vitro” (Human bladder carcinoma HT1376 cell line) and “ex vivo” in 15 consecutive case-control bladder cancer patients. Materials and methods The sequence of NT (NT 1-13) was synthesized through a solid phase chemistry in a tetra-branched form (NT-4). Peptides syn- thesized in such an oligo-branched form offer the advantage of a multivalent binding and become extremely stable to proteolytic degradation. The “in vitro” phase consisted in the evaluation of binding and internalization of NT-4 TMR (Tetra metal rodamin) in human bladder carcinoma HT1376 cell lines. The “ex vivo” phase consisted in evaluating the fluorescence of fluorescein isothiocyanate (FITC) conjugated NT-4 molecules by confocal microscopy in transitional cell bladder cancer from 15 patients with either superficial or infiltrating bladder cancer (10 TURBTs plus healthy tissue biopsies and 5 radical cystectomies). Presence of tumor and healthy tissue was checked by standard staining procedure. Results Binding and internalization of fluorophore-conjugated branched NT were showed “in vitro”. At time 0 the NT-4 is present at the level of the cell membrane and at 1 hour is internalized in the cytoplasm. In the “ex vivo” phase, a significant difference between tumor and healthy tissue samples was observed in all the 15 specimens treated with NT-4. Both tumor cells from superficial transitional cell carcinoma and from infiltrating transitional cell carcinoma had a significantly higher staining compared to healthy transitional cells of the bladder. Indeed, the median of each sample in the green scale of the RGB system results significantly higher in the cancer group. Discussion Receptors for different endogenous regulatory peptides like NT are over-expressed on plasma membrane of human cancer cells and might be targeted as selective tumor markers. Peptides have a very short half-life but their synthesis in branched form could represent a general feasible method to improve peptide stability. NT-4 branched peptides seem to be promising theranostic molecules in bladder cancer, thanks to the endocavitary utilization, to its stability and capacity to bind receptors over-expressed in bladder cancer cell lines, which might allow a very precise cancer detection and selective therapy, by a simple switch of functional units. Conclusions NT-4, a protease-resistant tetra-branched form of NT, results an effective tumor-targeting agent. When NT-4 is conjugated to a fluo- rophore it is bound and internalized into the tumor cell cytoplasm “in vitro” in HT1376 cell lines and it can efficiently discriminate “ex vivo” between tumor (either superficial or infiltrating transitional cell bladder cancer) and healthy tissue.