Glioblastoma (GB) is the most aggressive brain tumor, with high mortality and morbidity rates. The patients’ survival rate is approximately 12 months with current therapies, such as surgery, radiotherapy, chemotherapy, or target therapy. In addition, the assessment of therapy response and tumor progression by conventional imaging methods is a challenge for clinicians during patients’ follow-up. Thus, the discovery of new therapies and tests for monitoring patients is highly required in this disease. Radiolabeled peptides have gained prominence in Nuclear Medicine and Oncology since they can be used to treat patients with tumors with overexpression of specific receptors and to obtain images of tumors in vivo. Integrins are transmembrane proteins that are overexpressed in GB, play essential roles in cell survival and migration, and represent a potential target for GB tumors. Recently, an anti-integrin peptide modified by a conventional spacer (C6) and chelator (DOTA) radiolabeled with Ga-68 was produced by our group of researchers.

This study evaluated the anti-integrin peptide effects on cell survival and migration, binding and internalization, and gene expression as a first step toward targeted theranostics.

Anti-proliferative activity of the anti-integrin peptide (0.01 nM–100 µM) was tested in GB (U87, U118, U251, and Gl261) and non-tumoral (HaCaT) cells by comparing cell density at baseline and after 48h using sulforhodamine B staining (540 nm). Cell migration was assessed using the wound-healing assay after 24h of anti-integrin peptide treatment (0.1 µM–100 µM). Cell cycle and apoptosis assays were performed by flow cytometry. [68Ga]Ga-anti-integrin was labeled in sodium acetate buffer (pH 5.5, 95°C, 10 min) and purified via Sep-Pak C18. Radiochemical yield was assessed by iTLC-SG using methanol/ammonium acetate as the mobile phase. Binding and internalization assays were performed in GB cell lines at 30 and 60 min of incubation, with radioactivity quantified using a gamma counter. Expression levels of genes of interest were analyzed by quantitative PCR.

At the tested concentration range, the anti-integrin peptide did not affect proliferation, cell cycle phases, or apoptosis of GB and HaCaT cells. Treatment with the anti-integrin peptide had no effect on U118 cell migration at any of the tested concentrations, but it inhibited migration of U251 and Gl261 cells compared to untreated cells. Binding and internalization assays performed on U87, U118, U251, and Gl261 cells at 30 min revealed binding rates of 8.0%, 3.9%, 6.0%, and 8.6%, and internalization rates of 9.4%, 7.0%, 8.1%, and 10.2%, respectively. At 60 min, binding percentages were 7.8%, 3.9%, 4.7%, and 10.7%, while internalization values were 9.8%, 6.7%, 6.6%, and 9.6%, respectively. In U87 cells, the anti-integrin peptide treatment modulated the PI3K/AKT pathway, reducing the expression of ITGAV, AKT1, CCNE1, JUN, MDM2, and SRC genes and increasing CASP9 expression compared with non-treated cells.

In conclusion, we developed a novel [68Ga]-labeled anti-integrin peptide, that inhibits tumor cell migration and serves as vectors for radiopharmaceutical delivery in tumor, which can be seen as a promising theranostic strategy in GB.