Background: Immunotherapeutic approaches for cutaneous squamous cell carcinoma (cSCC) remain limited to programmed cell death protein 1 (PD-1) blockade. Although genomics studies have characterized key driver mutations in cSCC, preclinical models that faithfully recapitulate both the genetic landscape and immune microenvironment of the human disease, that could drive the development of novel, effective therapies, are lacking. Methods: To address this need, we generated genetically engineered mouse models harboring inducible p53 wmR172H expression and Cdkn2a deletion in stratified epithelia. These mice spontaneously developed well-differentiated and spindle cell cSCCs, whose histopathological and immunological features mimicked those observed in patients. We established syngeneic cell lines and characterized their genomic and transcriptomic profiles through whole exome sequencing and RNA sequencing. We used these syngeneic tumor models to test the effect of STING agonist, PD-1, VISTA and CTLA-4 blockade applied at different schedules and combinations. Finally, immune checkpoint molecules detected in the syngeneic models were validated in human cSCC tissue arrays. Results: Whole exome sequencing confirmed alterations overlapping with human cSCC, including mutations in Notch , interferon signaling, and cytokine pathways. Transcriptomic analysis revealed the upregulation of immunoregulatory genes, including Cd274 , Lgals9 , and Il33 , suggesting a suppressive tumor immune microenvironment (TIME). Preclinical therapeutic evaluation in this model demonstrated that PD-1 blockade and the treatment with a STING agonist elicited partial responses. The combination of STING agonist with checkpoint inhibition targeting either PD-1/CTLA-4 or PD-1/VISTA significantly enhanced antitumor immunity in tumors resistant to anti-PD-1 monotherapy. Conclusions: These results establish a robust, genetically defined, immunocompetent platform for modeling cSCC and evaluating novel immunotherapeutic strategies for treating patients with this disease. Our findings also identify VISTA and CTLA-4 as promising immune checkpoints to target in cSCC and support clinical evaluation of this combination of immune checkpoint blockade to overcome immune checkpoint inhibitor resistance and improve clinical outcomes.

Anxiety disorders are the most prevalent mental health conditions worldwide, yet current treatments remain suboptimal, with benzodiazepines carrying risks of tolerance and dependence. These limitations motivate the search for novel anxiolytics. Tropical marine macroalgae represents a promising source of neuroactive metabolites. Here, we investigate the anxiolytic potential of Stypopodium zonale using a neuroproteomics-based approach in Drosophila melanogaster. Crude organic extracts were prepared via ultrasonic-assisted extraction and administered acutely to adult flies for six hours. Proteins from fly heads were quantified and analyzed using LC-MS/MS, revealing 66 significantly differentially abundant proteins (fold change ≥ |1.5|, p ≤ 0.05), 72.7% of which were less abundant in the extract-treated group. Principal Component Analysis demonstrated clear separation between control and experimental samples. Ingenuity Pathway Analysis (IPA) mapped 33 of the differentially abundant proteins to human orthologs and identified significant predicted inhibition of the Protein Kinase A (PKA) signaling pathway. An IPA Interaction Network enabled the construction of a preliminary working model, suggesting that the extract may antagonize Drosophila’s Dop1R2 (DAMB). Overall, this study integrates natural product drug discovery with neuroproteomics in an invertebrate model system, providing a foundation for future behavioral validation and isolation of bioactive compounds from S. zonale.

Anxiety disorders are the most prevalent mental health conditions worldwide, yet current treatments remain suboptimal, with benzodiazepines carrying risks of tolerance and dependence. These limitations motivate the search for novel anxiolytics. Tropical marine macroalgae represent a promising source of neuroactive metabolites. Here, we investigate the anxiolytic potential of Stypopodium zonale using a neuroproteomics-based approach in Drosophila melanogaster. Crude organic extracts were prepared via ultrasonic-assisted extraction and administered acutely to adult flies for six hours. Proteins from fly heads were quantified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 66 significantly differentially abundant proteins (fold change ≥ |1.5|, p ≤ 0.05), 72.7% of which were less abundant in the extract-treated group. Principal component analysis demonstrated clear separation between control and experimental samples. Ingenuity Pathway Analysis (IPA) mapped 33 of the differentially abundant proteins to human orthologs and identified significant predicted inhibition of the Protein Kinase A (PKA) signaling pathway. An IPA Interaction Network enabled the construction of a preliminary working model, suggesting that the extract may antagonize Drosophila’s Dop1R2 (DAMB). Overall, this study integrates natural product drug discovery with neuroproteomics in an invertebrate model system, providing a foundation for future behavioral validation and isolation of bioactive compounds from S. zonale.