Research Projects

Background: Retinoblastoma (RB) is a common intraocular tumor in children, highly treatable if detected early but fatal if untreated. Advanced RB treatment relies on chemotherapy, yet many cases still require enucleation due to recurrence or poor response. Uncontrolled tumors risk systemic metastasis with no cure. While clinical indicators predict outcomes, they are not absolute. Tumor genotype can provide critical information for assessing tumor burden, monitoring progression, predicting risk, and guiding treatment decisions, including specific therapy development. However, in RB, the lack of biopsy option limits the ability to sample tumor tissue, thereby hindering accurate prognosis prediction. Thus, there is an unmet need for alternative biopsy methods and reliable tumor prognostic markers. Hypothesis: We hypothesize that tear-derived small extracellular vesicles (sEVs) and their DNA could serve as a non-invasive tool for RB prognosis. sEVs, nanosized particles secreted by all cells, including tumor cells, carry biomolecules reflecting the properties of their parent cells. These vesicles can be isolated from various bodily fluids, with tears offering a concentrated, non-invasive source. In addition to the established diagnostic and prognostic abilities in the majority of ocular disorders, it was recently noted that sEVs from tears had practical application in metastatic breast cancer where specific microRNAs related to the breast cancer were detected, emphasizing the promise of sEVs from tears in diagnosing RB as well. Aim: We aim to investigate whether the quantity and phenotype of sEVs can differentiate RB from non-RB individuals and whether exosomal DNA derived from tears can accurately reflect the tumor genomic profile. Experimental Design: We will collect tears, serum, PBMCs, and tumor tissues from RB patients undergoing enucleation, as well as tears and serum from patients undergoing chemotherapy. sEVs will be quantified using nanoparticle tracking analysis, and their phenotype will be analyzed via cytoFLEX flow cytometry with specific EVs markers, including CD81, CD9, TSG-101, CD133 and CD63. Comparisons will be made between pre- and post-chemotherapy samples and samples from individuals with unrelated eye conditions (e.g., congenital nasolacrimal duct obstruction, strabismus and congenital cataracts), with 10-15 individuals per group. For the evaluation of tear-derived sEV DNA as a source of tumor DNA, we will perform side-by-side genomic comparisons of tumor DNA and tear-derived sEV DNA (n=6) using whole-genome sequencing. Serum and aqueous humor -derived sEVs DNA will be analyzed as an additional source of RB DNA. DNA from PBMCs of the same individuals will be used to filter germline variants. Significance: This study could revolutionize RB care by using tear-derived sEVs and their DNA as a non-invasive tool for predicting therapy response and recurrence, addressing current limitations in tumor genotyping and guiding treatment.