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Rhabdomyosarcoma is a soft tissue cancer that arises in skeletal muscle due to mutations in myogenic progenitors that lead to ineffective differentiation and malignant transformation. The transcription factors Pax3 and Pax7 and their downstream target genes are tightly linked with the fusion positive alveolar subtype, whereas the RAS pathway is usually involved in the embryonal, fusion negative variant. Here, we analyse the role of Pax3 in a fusion negative context, by linking alterations in gene expression in pax3a/pax3b double mutant zebrafish with tumour progression in kRAS-induced rhabdomyosarcoma tumours. Several genes in the RAS/MAPK signalling pathway were significantly down-regulated in pax3a/pax3b double mutant zebrafish. Progression of rhabdomyosarcoma tumours was also delayed in the pax3a/pax3b double mutant zebrafish indicating that Pax3 transcription factors have an unappreciated role in mediating malignancy in fusion negative rhabdomyosarcoma.

Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.

Rhabdomyosarcoma is a highly aggressive soft tissue cancer that predominantly affects children and adolescents. Current treatment outcomes are poor, highlighting the urgent need for potent therapeutic alternatives. Preclinical research on photodynamic therapy (PDT) continues to gain attention as a promising and minimally invasive treatment strategy. Recently, PDT using the heavy-atom-free photosensitizer dibenzothioxanthene imide (DBI), which targets cancer-associated G-quadruplex (G4) DNA, has demonstrated high efficacy at nanomolar concentrations. In here, transgenic zebrafish with rhabdomyosarcoma tumors were utilized to evaluate the therapeutic potential of DBI treatment. We demonstrate that photoactivated DBI efficiently induce localized tumor necrosis, resulting in significant rhabdomyosarcoma regression compared to untreated controls. In fact, in comparison to the healthy cells surrounding the tumor, a high level of G4s was detected, as visualized by a G4-specific antibody. Notably, muscle and nerve cells within the treated tumor area were particularly affected, further underscoring its potency. These findings position DBI as a promising candidate for PDT in the treatment of rhabdomyosarcoma, offering selective G4-targeting capabilities and delivering robust therapeutic outcomes in in vivo models.