Development of an immunocompetent cutaneous squamous cell carcinoma model identifies VISTA and CTLA-4 as targetable immune checkpoints.

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 p53wmR172H 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.