Stable Reconstitution of Gasdermin-E in a Gasdermin-E-Deficient Head and Neck Squamous Cell Carcinoma Mouse Cell Line Enhances Pyroptosis-Mediated Release of Tumor Immunogenic Molecules

Abstract

<h3>Purpose/Objective(s)</h3> Gasdermin-E (GSDME) is known to potentiate immunogenic cell death in many tumor types after exposure to anti-cancer agents in a caspase-3 dependent manner. This GSDME-mediated release of immunostimulatory proteins, such as HMGB1, has been shown to increase tumor immunogenicity in mouse model systems of melanoma and colon cancer, resulting in reduced tumor load and lower distal metastasis. We sought to evaluate the role of GSDME-mediated cell death in human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) mouse cell lines. <h3>Materials/Methods</h3> To test whether HNSCC exhibits a GSDME-associated anti-tumor response, two HPV+ HNSCC mouse cell lines, MTEC and TC-1, were screened for GSDME expression by Western blot and then treated with apoptotic agents (raptinal, cisplatin, etoposide, and UV). Primary outcome measures were HMGB1 release (a potent immunostimulatory factor), percent propidium iodide (%PI) uptake (an indicator of plasma membrane damage and pyroptosis) and cell morphology. Using retroviral transduction, MTEC cells were reconstituted with GSDME, and single cell clones expressing comparable levels of recombinant mouse wild type (WT) GSDME and a caspase-3-uncleavable inactive mutant (Aspartate 270/Glutamate) D270A GSDME were selected. Control MTEC transduced with empty vector (EV) were also generated. Single cell clones were tested <i>in vitro</i> with apoptotic agents and evaluated for HMGB1 release, %PI uptake, and cell morphology. <h3>Results</h3> Western blot analysis demonstrated GSDME expression in TC-1 but not MTEC cells. Following treatment with apoptotic agents, TC-1 cells demonstrated HMGB1 release and increased %PI uptake as well as a distinct secondary necrotic phenotype characterized by ballooning of the plasma membrane on morphological analysis. This was not seen in MTEC cells. To examine whether the absence of GSDME in MTEC cells correlated with reduced release of immune-stimulatory molecules, MTEC cells stably reconstituted with GSDME were treated with apoptotic agents. Unlike MTEC cells expressing the uncleavable D270A GSDME or EV control, MTEC cells expressing WT GSDME demonstrated a significant release of HMGB1 and increased %PI uptake. Morphological analysis demonstrated significant ballooning of the plasma membrane only in the WT GSDME MTEC cells. <h3>Conclusion</h3> Treatment with apoptotic agents induces a GSDME-dependent pyroptotic cell death in MTEC and TC-1 cells as evidenced by HMGB1 release, increased %PI uptake, and cell morphology. As pyroptotic cell death of cancer cells induces anti-tumor immunity through the release of HMGB1 and other immunostimulatory factors, promoting GSDME-dependent pyroptosis might be a strategy to overcome treatment resistance in some HNSCC.