PD-L1 localized to immunosuppressive small extracellular vesicles (sEV PD-L1) contributes to tumor progression and is associated with resistance to immune-checkpoint blockade (ICB) therapy. Here, by establishing a screening strategy with a combination of tissue microarray (TMA), IHC staining, and measurement of circulating sEV PD-L1, we found that the endosomal sorting complex required for transport (ESCRT) member protein hepatocyte growth factor–regulated tyrosine kinase substrate (HRS) was the key regulator of circulating sEV PD-L1 in head and neck squamous cell carcinoma (HNSCC) patients. Increased HRS expression was found in tumor tissues and positively correlated with elevated circulating sEV PD-L1 in patients with HNSCC. The expression of HRS was also negatively correlated to the infiltration of CD8+ T cells. Knockdown of HRS markedly reduced PD-L1 expression in HNSCC cell–derived sEVs, and these sEVs from HRS knockdown cells showed decreased immunosuppressive effects on CD8+ T cells. Knockout of HRS inhibited tumor growth in immunocompetent mice together with PD-1 blockade. Moreover, a higher HRS expression was associated with a lower response rate to anti–PD-1 therapy in patients with HNSCC. In summary, our study reveals HRS, the core component of ESCRT-0, regulates sEV PD-L1 secretion, and is associated with the response to ICB therapy in patients with HNSCC, suggesting HRS is a promising target to improve cancer immunotherapy.

Purpose: Neoadjuvant anti–PD-1 immunotherapy combined with chemotherapy has shown promising pathologic responses in various cancers, including oral squamous cell carcinoma (OSCC). However, the pathologic response of lymph node (LN) metastases remains poorly understood. This study aims to systematically evaluate the pathologic response rates (pRR) of LN metastases in patients with OSCC and identify potential targets to improve therapeutic outcomes. Patients and Methods: We assessed the pRRs of LN metastases and matched primary tumors (PT) in patients with OSCC enrolled in a randomized, two-arm, phase II clinical trial (NCT04649476). Single-cell and spatial transcriptomics and multiplex IHC were performed to analyze the tumor microenvironment and identify potential therapeutic targets in LN metastases. A neoadjuvant orthotopic OSCC mouse model was established to evaluate the therapeutic potential of these targets. Results: We observed significant heterogeneity in pathologic regression of LN metastases, with lower pRRs compared with PTs. pRRs in LN metastases were correlated with overall and disease-free survival in patients with OSCC. We identified an abundance of macrophages in LN metastases exhibiting an unfolded protein response and activated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling. These macrophages contributed to an extracellular matrix–enriched microenvironment through interactions with fibroblasts, which hindered T cell–mediated cytotoxicity. Pharmacologic inhibition of the PERK pathway significantly enhanced anti–PD-1 therapy in LN metastases and PTs in the mouse model. Conclusions: Our study confirms that the pathologic response of LN metastases in patients with OSCC undergoing neoadjuvant immunotherapy or immunochemotherapy is inferior to that of PTs. It suggests that targeting the PERK pathway in macrophages could be a potential strategy to enhance treatment outcomes.

Oral squamous cell carcinoma (OSCC) is the most prevalent subtype of head and neck tumors, highly prone to lymph node metastasis. This study aims to examine the expression pattern of Ras-related protein Rab-27A (RAB27A) and explore its potential implications in OSCC. The expression of RAB27A was assessed through immunohistochemical analysis utilizing tissue microarrays. In vitro experiments were conducted using RAB27A-knockdown cells to investigate its impact on OSCC tumor cells. Additionally, transcriptome sequencing was performed to elucidate potential underlying mechanisms. RAB27A was significantly overexpressed in OSCC, and particularly in metastatic lymph nodes. It was positively correlated with the clinical progression and poor survival prognosis. Silencing RAB27A notably decreased the proliferation, migration, and invasion abilities of OSCC cells in vitro. A Gene Ontology (GO) enrichment analysis indicated a strong association between RAB27A and the epidermal growth factor receptor (EGFR) signaling pathway. Further investigations revealed that RAB27A regulated the palmitoylation of EGFR via zinc finger DHHC-type containing 13 (ZDHHC13). These findings provide insights into OSCC progression and highlight RAB27A as a potential therapeutic target for combating this aggressive cancer.

The ESCRT (endosomal sorting complex required for transport) machinery is essential for various cellular processes, yet its role in head and neck squamous cell carcinoma (HNSCC) is poorly understood. We utilized The Cancer Genome Atlas (TCGA) datasets to analyze the expression of ESCRT genes. Bulk RNA-sequencing data and HNSCC tissue microarrays (TMAs) were used to evaluate VPS25 expression and its clinical significance. Single-cell RNA sequencing of tumor tissues and VPS25 knockdown experiments in CAL27 cells were used to investigate its biological functions. Immunohistochemistry, spatial transcriptomics, and immunotherapy datasets highlighted the involvement of VPS25 in immune suppression and its potential as a predictive biomarker. The results demonstrated significant VPS25 overexpression in HNSCC tissues, which correlated with poor clinical outcomes. It promoted tumor cell proliferation and migration while reducing immune cell infiltration in the tumor microenvironment (TME). Additionally, by upregulating PVR expression in tumor cells, VPS25 activated the immunosuppressive PVR-TIGIT signaling axis, thereby facilitating immune evasion. Furthermore, VPS25 emerged as a potential biomarker for predicting immunotherapy response. These findings highlight VPS25 as a pivotal regulator of tumor progression and immune evasion in HNSCC and a promising target for therapeutic strategies.

Background: Immune checkpoint blockade targeting programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) has revolutionized cancer therapy. However, its efficacy is frequently limited by primary and acquired resistance. While inflammatory signals transiently upregulate PD-L1 transcription, post-translational regulation is crucial for its sustained expression in chronically stressed tumors. Whether and how tumor-intrinsic stress-response pathways control PD-L1 stability to promote immune evasion remains incompletely understood. Methods: Using human oral squamous cell carcinoma (OSCC) specimens, syngeneic mouse models, single-cell RNA sequencing, and genetic and pharmacological perturbations, we dissected the role of the unfolded protein response effector ATF4 in regulating PD-L1 stability and antitumor immunity. Its therapeutic potential was further evaluated in immunocompetent mice treated with anti-PD-1 therapy. Results: We identified ATF4 as a tumor-intrinsic driver of immune evasion. In malignant cells, ATF4 induced reactive oxygen species (ROS), which activated the AKT-mTOR pathway and suppressed autophagy, thereby stabilizing the PD-L1 protein independently of inflammatory cues. Genetic ablation of ATF4 or pharmacological inhibition of ROS-AKT-mTOR signaling restored autophagic flux, reduced PD-L1 levels, and enhanced CD8 ⁺ T-cell infiltration and function. When combined with PD-1 blockade, ATF4 targeting further suppressed tumor growth. Clinically, ATF4 expression was inversely correlated with CD8 ⁺ T-cell infiltration and autophagy markers, positively correlated with PD-L1 levels, and predicted a poor response to immunotherapy in patients with OSCC. Conclusions: Our findings establish ATF4 as a stress-responsive regulator of PD-L1 proteostasis, directly linking tumor-intrinsic stress adaptation to immune checkpoint stabilization and therapy resistance. Targeting the ATF4-ROS-AKT-mTOR axis may represent a promising strategy to overcome resistance to PD-1/PD-L1 blockade.

PD-L1 localized to immunosuppressive small extracellular vesicles (sEV PD-L1) contributes to tumor progression and is associated with resistance to immune-checkpoint blockade (ICB) therapy. Here, by establishing a screening strategy with a combination of tissue microarray (TMA), IHC staining, and measurement of circulating sEV PD-L1, we found that the endosomal sorting complex required for transport (ESCRT) member protein hepatocyte growth factor–regulated tyrosine kinase substrate (HRS) was the key regulator of circulating sEV PD-L1 in head and neck squamous cell carcinoma (HNSCC) patients. Increased HRS expression was found in tumor tissues and positively correlated with elevated circulating sEV PD-L1 in patients with HNSCC. The expression of HRS was also negatively correlated to the infiltration of CD8+ T cells. Knockdown of HRS markedly reduced PD-L1 expression in HNSCC cell–derived sEVs, and these sEVs from HRS knockdown cells showed decreased immunosuppressive effects on CD8+ T cells. Knockout of HRS inhibited tumor growth in immunocompetent mice together with PD-1 blockade. Moreover, a higher HRS expression was associated with a lower response rate to anti–PD-1 therapy in patients with HNSCC. In summary, our study reveals HRS, the core component of ESCRT-0, regulates sEV PD-L1 secretion, and is associated with the response to ICB therapy in patients with HNSCC, suggesting HRS is a promising target to improve cancer immunotherapy.