Trefoil factor family peptide 1 (TFF1) as a biomarker, its possible receptors, signaling pathways and prospective application strategies in retinoblastoma therapy

Abstract Retinoblastoma (RB) is the most common intraocular tumor in early childhood, originating from the juvenile retina and affecting approximately 7,500 patients each year. RB is most frequently caused by a biallelic loss of the tumor suppressor gene RB1. The first part of this study focuses on our latest research findings in the field of liquid biopsy (LB) and describes the protein "trefoil factor family 1" (TFF1) as an LB biomarker in aqueous humor (AH) for an advanced RB subtype. TFF1 is expressed in the eye only in tumor cells of the advanced RB subtype, demonstrating the potential diagnostic and prognostic value of TFF1. In addition, we were able to demonstrate the benefit of TFF1 for therapy monitoring, as decreasing TFF1 levels after treatment with chemotherapeutic agents correlated with the onset of therapeutic success. Previous studies of our group showed that overexpression of TFF1 in RB cells leads to upregulation of the “gastric inhibitory polypeptide receptor” (GIPR). The second part of this study, focuses on the role of GIPR in RB where our investigations showed that the expression of TFF1 and GIPR correlates in patient tumors. Further functional analysis in RB cells showed a significant decrease in cell viability, proliferation, and growth rates, with a concomitant increase in apoptosis after stable lentiviral GIPR overexpression, which was consistent with the effects after TFF1 overexpression. Further experiments in an alternative in vivo chicken chorioallantoic membrane (CAM) model showed that GIPR-overexpressing RB cells formed significantly smaller tumors than the control group. Furthermore, the effect of GIPR overexpression in RB cells could be reversed by the addition of a GIPR inhibitor. The addition of recombinant TFF1 showed no enhanced effect on GIPR overexpressing RB cells, suggesting that GIPR does not serve as a TFF1 receptor. Investigations into potential GIPR upstream and downstream mediators suggest the involvement of miR-542-5p and p53 in GIPR regulation and signal transduction. The third part of this study introduces new and optimized inhibitors of the “CXC chemokine receptor type 4” (CXCR4) which were tested on RB cells and compared with previously established inhibitors. The efficiency of the inhibitors of the CXCR4 receptor was investigated in various in vitro studies and in vivo CAM assays on RB cell lines. In particular, the more stable inhibitor #JM198 was shown to have an improved 9antagonistic effect compared to the CXCR4 inhibitor, resulting in a significantly improved reduction in tumor cell growth in RB cells. The fourth part of this study discusses novel drug delivery approaches using functionalized gold nanoparticles (GNPs) which were tested for the treatment of chemoresistant RB cells. For this purpose, GNPs were functionalized with hyaluronic acid (HA) for increased uptake and with atrial natriuretic peptide (ANP), known for its inhibitory effect on neoangiogenesis, and tested in various in vitro and in vivo experiments. In addition, different application strategies were tested in an orthotopic in vivo RB eye model of newborn rats. Treatment of chemoresistant RB cells with ANP- HA-GNPs in ovo resulted in a significant reduction of tumor growth and angiogenesis compared to control groups. This effect could be verified in the rat eye model, where a non-invasive application via eye drops was also tested, which resulted in a reduction of tumor growth in the rat eye. Overall, this study encompasses a multifaceted approach from the establishment of new biomarkers to the elucidation of molecular mechanisms and the development of targeted therapies with the aim of improving the treatment of RB, preserving patients' vision, and facilitating long-term monitoring of tumor progression.