Patients with oropharyngeal squamous cell carcinoma (OPSCC) caused by human papilloma virus (HPV) exhibit a better prognosis than those with HPV-negative OPSCC. This study investigated the distinct molecular pathways that delineate HPV-negative from HPV-positive OPSCC to identify biologically relevant therapeutic targets. Bulk mRNA from 23 HPV-negative and 39 HPV-positive OPSCC tumors (n = 62) was sequenced to uncover the transcriptomic profiles. Differential expression followed by gene set enrichment analysis was performed to outline the top enriched biological process in the HPV-negative compared with HPV-positive entity. INHBA, the highest overexpressed gene in the HPV-negative tumor, was knocked down. Functional assays (migration, proliferation, cell death, stemness) were conducted to confirm the target's oncogenic role. Correlation analyses to reveal its impact on the tumor microenvironment were performed. We revealed that epithelial-to-mesenchymal transition (EMT) is the most enriched process in HPV-negative compared with HPV-positive OPSCC, with INHBA (inhibin beta A subunit) being the top upregulated gene. INHBA knockdown downregulated the expression of EMT transcription factors and attenuated migration, proliferation, stemness, and cell death resistance of OPSCC cells. We uncovered that INHBA associates with a pro-tumor microenvironment by negatively correlating with antitumor CD8+ T and B cells while positively correlating with pro-tumor M1 macrophages. We identified three miRNAs that are putatively involved in repressing INHBA expression. Our results indicate that the upregulation of INHBA is tumor-promoting. We propose INHBA as an attractive therapeutic target for the treatment of INHBA-enriched tumors in patients with HPV-negative OPSCC to ameliorate prognosis. Significance: Patients with HPV-negative OPSCC have a poorer prognosis due to distinct molecular pathways. This study reveals significant transcriptomic differences between HPV-negative and HPV-positive OPSCC, identifying INHBA as a key upregulated gene in HPV-negative OPSCC's oncogenic pathways. INHBA is crucial in promoting EMT, cell proliferation, and an immunosuppressive tumor environment, suggesting its potential as a therapeutic target for HPV-negative OPSCC.

Immunoregulatory Arginase-1 (Arg-1) is present in the tumor microenvironment of solid tumors. Its association to clinicopathology and its prognostic impact are inconsistent among different tumor types and biological fluids. This study evaluated Arg-1 protein levels in tumors and the circulation of patients with head and neck squamous cell carcinoma (HNSCC) in relation to clinical stage and prognosis. Tumor Arg-1 expression was monitored via immunohistochemistry while plasma Arg-1 levels via ELISA in 37 HNSCC patients. Arg-1 presence in plasma-derived exosomes was assessed using Western blots in 20 HNSCC patients. High tumor Arg-1 expression correlated with favorable clinicopathology and longer recurrence-free survival (RFS), while high plasma Arg-1 levels were associated with unfavorable clinicopathology. All patients with low tumor and high plasma Arg-1 had nodal metastases and developed recurrence. This discrepancy was attributed to the presence of Arg-1-carrying exosomes. Arg-1 was found in plasma-derived exosomes from all HNSCC patients. High exosomal Arg-1 levels were associated with positive lymph nodes and short RFS. Circulating Arg-1⁺ exosomes represent a mechanism of active Arg-1 export from the tumor to the periphery. Exosomes reflected biologically relevant Arg-1 levels in metastatic HNSCC and emerged as potentially more accurate biomarkers of metastatic disease and RFS than tissue or plasma Arg-1 levels.

Background: Metastases are associated with poor survival in head and neck squamous cell carcinoma (HNSCC) patients and tumour-associated macrophages (TAMs) are important drivers in tumour progression and metastasis formation. Small extracellular vesicles (sEVs) are another important factor that contribute to systemic immunosuppression and pre-metastatic niche formation. Here, we investigate the effect of plasma sEVs from HNSCC patients on pre-metastatic niche formation, directly or through modulation of macrophages. Methods: Primary macrophages were incubated with sEVs from plasma of HNSCC patients or healthy donors (HD). RNA profiles and inflammatory properties of macrophages were evaluated. Direct and indirect effects of sEVs on chemotaxis, T cell activation, proliferation and epithelial-to-mesenchymal transition (EMT) of tumour cells were investigated. Results: sEVs of HNSCC patients and HD induced different RNA profiles in macrophages. sEVs induced apoptosis and inhibition of T cell activation, while tumour cells were attracted by sEV-treated macrophages, but not sEVs directly. Proliferation was inhibited by both, sEVs and supernatant of EV-treated macrophages in HNSCC. Additionally, EMT in tumour cells was reversed by HNSCC sEV-treated macrophages. Conclusion: sEVs from plasma of HNSCC patients transformed macrophages into metastasis-promoting TAMs and inhibited anti-tumour T cells, highlighting the potential of sEVs and TAMs as targets for therapeutic approaches.

Background: Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic disorder leading to frequent bleeding in several organs. As HHT diagnosis is demanding and depends on clinical criteria, liquid biopsy would be beneficial. Exosomes from biofluids are nano-sized vesicles for intercellular communication. Their cargo and characteristics represent biomarkers for many diseases. Here, exosomes of HHT patients were examined regarding their biosignature. Methods: Exosomes were isolated from the plasma of 20 HHT patients and 17 healthy donors (HDs). The total exosomal protein was quantified, and specific proteins were analyzed using Western blot and antibody arrays. Human umbilical vein endothelial cells (HUVECs) co-incubated with exosomes were functionally examined via immunofluorescence, proliferation, and scratch assay. Results: The levels of the angiogenesis-regulating protein Thrombospondin-1 were significantly higher in HHT compared to HD exosomes. Among HHT, but not HD exosomes, a negative correlation between total exosomal protein and soluble Endoglin (sENG) levels was found. Other exosomal proteins (ALK1, ALK5) and the particle concentration significantly correlated with disease severity parameters (total consultations/interventions, epistaxis severity score) in HHT patients. Functionally, HUVECs were able to internalize both HD and HHT exosomes, inducing a similar change in the F-Actin structure and a reduction in migration and proliferation. Conclusions: This study provided first insights into the protein cargo and function of HHT-derived exosomes. The data indicate changes in sENG secretion via exosomes and reveal exosomal Thrombospondin-1 as a potential biomarker for HHT. Several exosomal characteristics were pointed out as potential liquid biomarkers for disease severity, revealing a possible new way of diagnosis and prognosis of HHT.

Cell death upon INHBA knockdown. A, Cell death of native UDSCC1, Mock KD, INHBA KD1/KD2 and native UDSCC2 cells as assessed by Annexin V/7-AAD staining and flow cytometry. Results are shown as mean ± SD of the percentages of dead cells normalized to UDSCC1 (n = 3). Conditions were compared with native UDSCC1 using unpaired t test with *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; and ns, P > 0.05. B, Representative flow cytometry plots for each condition. Gates show early apoptotic in the lower right quadrant, late apoptotic in the upper right quadrant and necrotic cell populations in the upper left quadrant. t0-plots represent the baseline timepoint right before transfection.

Differential expression analysis output