Unlocking the potential of HHLA2: identifying functional immune infiltrating cells in the tumor microenvironment and predicting clinical outcomes in laryngeal squamous cell carcinoma.

BACKGROUND: HHLA2 (human endogenous retrovirus-H long terminal repeat-associating protein 2) represents a recently identified member of the B7 immune checkpoint family, characterized by limited expression in normal tissues but notable overexpression in various cancer types. Nevertheless, the precise function and interaction with immune cells remain poorly understood, particularly in laryngeal squamous cell carcinoma (LSCC). This investigation endeavored to elucidate the biological significance of HHLA2 within the tumor microenvironment of human LSCC tissues and delineate the clinical relevance and functional roles of HHLA2 in LSCC pathogenesis. METHODS: Through multiplexed immunohistochemistry analyses conducted on tissue microarrays sourced from LSCC patients (n = 72), the analysis was executed to assess the expression levels of HHLA2, density and spatial patterns of CD68+HLA-DR+CD163- (M1 macrophages), CTLA-4+CD4+FoxP3+ (CTLA-4+Treg cells), CTLA-4+CD4+FoxP3- (CTLA-4+Tcon cells), exhausted CD8+T cells, and terminally exhausted CD8+T cells in LSCC tissues. Survival analysis was conducted to evaluate the prognostic significance of HHLA2 and these immune checkpoints or immune cell populations, employing COX regression analysis to identify independent prognostic factors. RESULTS: Kaplan-Meier (K-M) survival curves revealed a significant association between HHLA2 expression and overall survival (OS) in LSCC. Elevated levels of HHLA2 were linked to reduced patient survival, indicating its potential as a prognostic marker (HR: 3.230, 95%CI 0.9205-11.34, P = 0.0067). Notably, increased infiltration of CD68+ cells (total macrophages), STING+CD68+HLA-DR+CD163- (STING+M1 macrophages), CTLA-4+CD4+FoxP3+, CTLA-4+CD4+FoxP3-, PD-1+LAG-3+CD8+T cells, and PD-1+LAG-3+TIM-3+CD8+T cells strongly linked to poorer survival outcomes (P < 0.05). A discernible trend was observed between the levels of these immune cell populations, STING+CD68+ (STING+ total macrophages), CD68+HLA-DR+CD163-, STING+CD68+CD163+HLA-DR- (STING+M2 macrophages), PD-1+LAG-3-CD8+T cells, PD-1+TIM-3+CD8+T cells, and PD-1+LAG-3+TIM-3-CD8+T cells and prognosis. Importantly, multivariate COX analysis identified HHLA2 as an independent predictive factor for OS in LSCC patients (HR = 3.86, 95% CI 1.08-13.80, P = 0.038). This underscored the potential of HHLA2 as a critical marker for predicting patient outcomes in LSCC. CONCLUSIONS: HHLA2 emerged as a detrimental prognostic biomarker for assessing OS in LSCC patients. Relative to other immune checkpoints, HHLA2 exhibited heightened predictive efficacy for the prognosis of LSCC patients.

Sculpting the tumour microenvironment by combining radiotherapy and ATR inhibition for curative-intent adjuvant immunotherapy.

The combination of radiotherapy/chemoradiotherapy and immune checkpoint blockade can result in poor outcomes in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). Here, we show that combining ATR inhibition (ATRi) with radiotherapy (RT) increases the frequency of activated NKG2A+PD-1+ T cells in animal models of HNSCC. Compared with the ATRi/RT treatment regimen alone, the addition of simultaneous NKG2A and PD-L1 blockade to ATRi/RT, in the adjuvant, post-radiotherapy setting induces a robust antitumour response driven by higher infiltration and activation of cytotoxic T cells in the tumour microenvironment. The efficacy of this combination relies on CD40/CD40L costimulation and infiltration of activated, proliferating memory CD8+ and CD4+ T cells with persistent or new T cell receptor (TCR) signalling, respectively. We also observe increased richness in the TCR repertoire and emergence of numerous and large TCR clonotypes that cluster based on antigen specificity in response to NKG2A/PD-L1/ATRi/RT. Collectively, our data point towards potential combination approaches for the treatment of HNSCC.

Clinical, genomic and immune microenvironmental determinants of nivolumab response in head and neck squamous cell carcinoma.

Background: In view of improving biomarkers predicting the efficacy of immunotherapy for head and neck squamous cell carcinoma (R/M HNSCC), this multicenter retrospective study aimed to identify clinical, tumor microenvironmental, and genomic factors that are related to therapeutic response to the anti- Programmed cell death protein 1 (PD-1) antibody, nivolumab, in patients with R/M HNSCC. Methods: The study compared 53 responders and 47 non-responders, analyzing formalin-fixed paraffin-embedded samples using 14-marker multiplex immunohistochemistry and targeted gene sequencing. Results: Of 100 patients included, responders had significantly lower smoking and alcohol index, higher incidence of immune related adverse events, and higher PD-1 ligand (PD-L1) expression in immune cells as well as PD-L1 combined positive score (CPS) than non-responders. The frequency of natural killer cells was associated with nivolumab response in patients with prior cetuximab use, but not in cetuximab-naïve status. Age-stratified analysis showed nivolumab response was linked to high CPS and lymphoid-inflamed profiles in patients aged ≥ 65. In contrast, lower NLR in peripheral blood counts was associated with response in patients aged < 65. Notably, TP53 mutation-positive group had lower CPS and T cell densities, suggesting an immune-excluded microenvironment. Patients with altered tumor suppressor gene pathways, including TP53, CDKN2A, and SMAD4 mutations, had lower CPS, higher smoking index, and were associated with poor responses. Conclusion: Nivolumab treatment efficacy in HNSCC is influenced by a combination of clinical factors, age, prior treatment, immune environmental characteristics, and gene mutation profiles.

Multi-omics analysis of overexpressed tumor-associated proteins: gene expression, immunopeptide presentation, and antibody response in oropharyngeal squamous cell carcinoma, with a focus on cancer-testis antigens.

Introduction: The human leukocyte antigen complex (HLA) is essential for inducing specific immune responses to cancer by presenting tumor-associated peptides (TAP) to T cells. Overexpressed tumor associated antigens, mainly cancer-testis antigens (CTA), are outlined as essential targets for immunotherapy in oropharyngeal squamous cell carcinoma (OPSCC). This study assessed the degree to which presentation, gene expression, and antibody response (AR) of TAP, mainly CTA, are correlated in OPSCC patients to evaluate their potential as immunotherapy targets. Materials and methods: Snap-frozen tumor (NLigand/RNA=40), healthy mucosa (NRNA=6), and healthy tonsils (NLigand=5) samples were obtained. RNA-Seq was performed using Illumina HiSeq 2500/NovaSeq 6000 and whole exome sequencing (WES) utilizing NextSeq500. HLA ligands were isolated from tumor tissue using immunoaffinity purification, UHPLC, and analyzed by tandem MS. Antibodies were measured in serum (NAb=27) utilizing the KREX™ CT262 protein array. Data analysis focused on 312 proteins (KREX™ CT262 panel + overexpressed self-proteins). Results: 183 and 94 of HLA class I and II TAP were identified by comparative profiling with healthy tonsils. Genes from 26 TAP were overexpressed in tumors compared to healthy mucosa (LFC>1; FDR<0.05). Low concordance (r=0.25; p<0.0001) was found between upregulated mRNA and class I TAP. The specific mode of correlation of TAP was found to be dependent on clinical parameters. A lack of correlation was observed both between mRNA and class II TAP, as well as between class II tumor-unique TAP (TAP-U) presentation and antibody response (AR) levels. Discussion: This study demonstrates that focusing exclusively on gene transcript levels fails to capture the full extent of TAP presentation in OPSCC. Furthermore, our findings reveal that although CTA are presented at relatively low levels, a few CTA TAP-U show potential as targets for immunotherapy.

Highlighting immune features of the tumor ecosystem and prognostic value of Tfh and Th17 cell infiltration in head and neck squamous cell carcinoma by single-cell RNA-seq.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) typically present with a complex anatomical distribution, often accompanied by insidious symptoms. This combination contributes to its high incidence and poor prognosis. It is now understood that the immune features of cellular components within the tumor ecosystem and their complex interactions are critical factors influencing both tumor progression and the effective immune response. METHODS: We obtained single-cell RNA sequencing data of 26,496 cells from three tumor tissues and five normal tissues and performed subsequent analyses. Immunohistochemical staining on tumor sections was used to validate the presence of malignant cells. Additionally, we included bulk RNA sequencing data from 502 HNSCC patients. Kaplan-Meier analysis and the log-rank test were employed to assess predictors of patient outcomes. RESULTS: We identified three epithelial subclusters exhibiting immune-related features. These subclusters promoted the infiltration of T cells, dendritic cells, and monocytes into the tumor microenvironment. Additionally, cancer-associated fibroblasts displayed tumor-promoting and angiogenesis characteristics, contrasting with the predominant antigen-presenting and inflammatory roles observed in fibroblasts from normal tissues. Furthermore, tumor endothelial subsets exhibited a double-sided effect, promoting tumor progression and enhancing the effectiveness of immune response. Finally, follicular helper T cells and T helper 17 cells were found to be significantly correlated with improved outcomes in HNSCC patients. These CD4+ T cell subpopulations could promote the anti-tumor immune response by recruiting and activating B and T cells. CONCLUSION: Our findings provide deeper insights into the immune features of the tumor ecosystem and reveal the prognostic significance of follicular helper T cells and T helper 17 cells. These findings may pave the way for the development of therapeutic approaches.

Hypoxia-related signature to risk stratify patients for the benefit of immune checkpoint inhibitors therapy in head and neck squamous cell carcinoma: An experimental study.

BACKGROUND: Increasing evidence has shown that hypoxia is a biomarker of tumor proliferation and metastasis. This research aimed to identify a hypoxia-associated gene prognostic index (HAGPI) in head and neck squamous cell carcinoma (HNSCC) and based on HAGPI-defined subgroups to predict prognosis and response to immune checkpoint inhibitors therapy. METHODS: RNA-sequencing transcriptomic data for patients with HNSCC were downloaded from The Cancer Genome Atlas (TCGA). Protein-protein interaction network analysis was performed to select hypoxia-related hub genes. Univariate and multivariate cox regression analyses were used to identify hub genes to develop the HAGPI. Afterward expression data were imported into CIBERSORT to evaluate the relative proportion of 22 immune cells and compared the relative proportions of immune cells between the 2 HAGPI subgroups. The relationship between immunopheno score (IPS) and HAGPI was validated for immune checkpoint inhibitors (ICIs) response in TCGA cohorts. RESULTS: The HAGPI was constructed based on HS3ST1, HK1, PGK1, STC2, SERPINE1, PKLR genes. In high-HAGPI patients, the primary and secondary endpoint events in TCGA and GEO cohorts were significantly lower than low-HAGPI groups (P < .05). HAGPI-high patients exhibited a poorer prognosis than HAGPI-low patients did. The abundance of M2 macrophages and NK cell were significantly enhanced in the high-HAGPI while T cells regulatory and T cells CD8, were markedly elevated in the low-HAGPI. Meanwhile, patients in the low-HAGPI patients had higher levels of immunosuppressant expression and less aggressive phenotypes. Furthermore, IPS analysis showed that the low-HAGPI group with higher IPS represented a more immunogenic phenotype. CONCLUSION: The current study developed and verified a HAPGI model that can be considered as an independent prognostic biomarker and elucidated the tumor immune microenvironment of HNSCC.

Fe3O4 Nanoparticles That Modulate the Polarisation of Tumor-Associated Macrophages Synergize with Photothermal Therapy and Immunotherapy (PD-1/PD-L1 Inhibitors) to Enhance Anti-Tumor Therapy.

Introduction: Traditional surgical resection, radiotherapy, and chemotherapy have been the treatment options for patients with head and neck squamous cell carcinoma (HNSCC) over the past few decades. Nevertheless, the five-year survival rate for patients has remained essentially unchanged, and research into treatments has been relatively stagnant. The combined application of photothermal therapy (PTT) and immunotherapy for treating HNSCC has considerable potential. Methods: Live-dead cell staining and CCK-8 assays proved that Fe3O4 nanoparticles are biocompatible in vitro. In vitro, cellular experiments utilized flow cytometry and immunofluorescence staining to verify the effect of Fe3O4 nanoparticles on the polarisation of tumor-associated macrophages. In vivo, animal experiments were conducted to assess the inhibitory effect of Fe3O4 nanoparticles on tumor proliferation under the photothermal effect in conjunction with BMS-1. Tumour tissue sections were stained to observe the effects of apoptosis and the inhibition of tumor cell proliferation. The histological damage to animal organs was analyzed by hematoxylin and eosin (H&E) staining. Results: The stable photothermal properties of Fe3O4 nanoparticles were validated by in vitro cellular and in vivo animal experiments. Fe3O4 photothermal action not only directly triggered immunogenic cell death (ICD) and enhanced the immunogenicity of the tumor microenvironment but also regulated the expression of tumor-associated macrophages (TAMs), up-regulating CD86 and down-regulating CD206 to inhibit tumor growth. The PD-1/PD-L1 inhibitor promoted tumor suppression, and reduced tumor recurrence and metastasis. In vivo studies demonstrated that the photothermal action exhibited a synergistic effect when combined with immunotherapy, resulting in significant suppression of primary tumors and an extension of survival. Conclusion: In this study, we applied Fe3O4 photothermolysis in a biomedical context, combining photothermolysis with immunotherapy, exploring a novel pathway for treating HNSCC and providing a new strategy for effectively treating HNSCC.

Identification of tumor stemness and immunity related prognostic factors and sensitive drugs in head and neck squamous cell carcinoma.

The presence of cancer stem cells (CSCs) contributes significantly to treatment resistance in various cancers, including head and neck squamous cell carcinoma (HNSCC). Despite this, the relationship between cancer stemness and immunity remains poorly understood. In this study, we aimed to identify potential immunotherapeutic targets and sensitive drugs for CSCs in HNSCC. Using data from public databases, we analyzed expression patterns and prognostic values in HNSCC. The stemness index was calculated using the single-sample gene set enrichment analysis (ssgsea) algorithm, and weighted gene co-expression network analysis (WGCNA) was employed to screen for key stemness-related modules. Consensus clustering was then used to group samples for further analysis, and prognosis-related key genes were identified through regression analysis. Our results showed that tumor samples from HNSCC exhibited higher stemness indices compared to normal samples. WGCNA identified a module highly correlated with stemness, comprising 187 genes, which were significantly enriched in protein digestion and absorption pathways. Furthermore, we identified sensitive drugs targeting prognostic genes associated with tumor stemness. Notably, two genes, HLF and CCL11, were found to be highly associated with both stemness and immunity. In conclusion, our study identifies a stemness-related gene signature and promising drug candidates for CSCs of HNSCC. Additionally, HLF and CCL11, which are associated with both stemness and immunity, represent potential targets for immunotherapy in HNSCC.

Immune cell topography of head and neck cancer.

BACKGROUND: Approximately 50% of head and neck squamous cell carcinomas (HNSCC) recur after treatment with curative intent. Immune checkpoint inhibitors are treatment options for recurrent/metastatic HNSCC; however, less than 20% of patients respond. To increase this response rate, it is fundamental to increase our understanding of the spatial tumor immune microenvironment (TIME). METHODS: In total, 53 HNSCC specimens were included. Using a seven-color multiplex immunohistochemistry panel we identified tumor cells, CD163+macrophages, B cells, CD8+T cells, CD4+T helper cells and regulatory T cells (Tregs) in treatment-naive surgical resection specimens (n=29) and biopsies (n=18). To further characterize tumor-infiltrating CD8+T cells, we stained surgical resection specimens (n=12) with a five-color tumor-resident panel including CD103, Ki67, CD8 and pan-cytokeratin. Secretome analysis was performed on matched tumor suspensions (n=11) to measure protein levels. RESULTS: Based on CD8+T cell infiltrates, we identified four different immunotypes: fully infiltrated, stroma-restricted, immune-excluded, and immune-desert. We found higher cytokine levels in fully infiltrated tumors compared with other immunotypes. While the highest immune infiltrates were observed in the invasive margin for all immune cells, CD163+macrophages and Tregs had the highest tendency to infiltrate the tumor center. Within the tumor center, especially B cells stayed at the tumor stroma, whereas CD163+macrophages, followed by T cells, were more often localized within tumor fields. Also, B cells were found further away from other cells and often formed aggregates while T cells and CD163+macrophages tended to be more closely located to each other. Across resection specimens from various anatomical sites within the head and neck, oral cavity tumors exhibited the highest densities of Tregs. Moreover, the distance from B cells and T cells to tumor cells was shortest in oral cavity squamous cell carcinoma (OCSCC), suggesting more interaction between lymphocytes and tumor cells. Also, the fraction of T cells within 10 µm of CD163+macrophages was lowest in OCSCC, indicating fewer myeloid/T-cell suppressive interactions in OCSCC. CONCLUSIONS: We comprehensively described the TIME of HNSCC using a unique data set of resection specimens. We discovered that the composition, as well as the relative localization of immune cells in the TIME, differed in distinct anatomical sites of the head and neck.

Lysine Methyltransferase 2D Regulates Immune Response and Metastasis in Head and Neck Cancer.

BACKGROUND/AIM: The most frequently altered epigenetic modifier in head and neck squamous carcinoma (HNSC) is the histone methyltransferase KMT2D. KMT2D catalyzes methylation of histone H3K4 resulting in open chromatin and the activation of target genes. Tumor-associated macrophages (TAMs) promote cancer growth by causing T lymphocyte exhaustion. C-C motif chemokine ligand 2 (CCL2) is a potent TAM chemotactic factor. In HNSC, TAMs have been associated with unfavorable patient outcomes and metastasis. The aim of this study was to determine the role of KMT2D in HNSC using genetically engineered in vivo models. MATERIALS AND METHODS: KMT2D protein expression was correlated with lymph node metastasis in human HNSC using immunohistochemistry. Genetically engineered KMT2D and CCL2 knockout models of HNSC were created in vivo. HNSC was characterized using qRT-PCR, histopathology, and immunohistochemistry/immunofluorescence microscopy. We also analyzed the effects of KMT2D expression on the proliferation and migration of human HNSC lines. The regulation of the CCL2 gene by KMT2D was characterized using chromatin immunoprecipitation-sequencing assay of transposase accessible chromatin-sequencing, and chromatin conformation capture-sequencing. RESULTS: Human HNSC cases with high KMT2D expression exhibited significantly increased lymph node metastasis. Reduced KMT2D expression in our genetically engineered model correlated with reduced lymph node metastasis, longer latency, and slow tumor growth. CCL2 expression was decreased in KMT2D deficient HNSC, which correlated with a reduced TAM gene expression signature. Genomic experiments demonstrated that KMT2D directly targeted the CCL2 gene. A new genetically engineered in vivo model of CCL2-null HNSC was created, recapitulating the KMT2D deficient phenotype and showing a decreased T lymphocyte exhaustion signature. CONCLUSION: KMT2D regulates CCL2-mediated immune response and metastasis in HNSC.

Differential tumor immune microenvironment coupled with tumor progression or tumor eradication in HPV-antigen expressing squamous cell carcinoma (SCC) models.

Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.

Spatial dynamics of tertiary lymphoid aggregates in head and neck cancer: insights into immunotherapy response.

BACKGROUND: Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) generally has a poor prognosis for patients with limited treatment options. While incorporating immune checkpoint inhibitors (ICIs) has now become the standard of care, the efficacy is variable, with only a subset of patients responding. The complexity of the tumor microenvironment (TME) and the role of tertiary lymphoid structures (TLS) have emerged as critical determinants for immunotherapeutic response. METHODS: In this study, we analyzed two independently collected R/M HNSCC patient tissue cohorts to better understand the role of TLS in response to ICIs. Utilizing a multi-omics approach, we first performed targeted proteomic profiling using the Nanostring GeoMx Digital Spatial Profiler to quantify immune-related protein expression with spatial resolution. This was further characterized by spatially resolved whole transcriptome profiling of TLSs and germinal centers (GCs). Deeper single-cell resolved proteomic profiling of the TLSs was performed using the Akoya Biosciences Phenocycler Fusion platform. RESULTS: Our proteomic analysis revealed the presence of T lymphocyte markers, including CD3, CD45, and CD8, expressing cells and upregulation of immune checkpoint marker PD-L1 within tumor compartments of patients responsive to ICIs, indicative of 'hot tumor' phenotypes. We also observed the presence of antigen-presenting cells marked by expression of CD40, CD68, CD11c, and CD163 with upregulation of antigen-presentation marker HLA-DR, in patients responding to ICIs. Transcriptome analysis of TLS and GCs uncovered a marked elevation in the expression of genes related to immune modulation, diverse immune cell recruitment, and a potent interferon response within the TLS structure. Notably, the distribution of TLS-tumor distance was found to be significantly different across response groups (H = 9.28, p = 0.026). The proximity of TLSs to tumor cells was found to be a critical indicator of ICI response, implying that patients with TLSs located further from tumor cells have worse outcomes. CONCLUSION: The study underscores the multifaceted role of TLSs in modulating the immunogenic landscape of the TME in R/M HNSCC, likely influencing the efficacy of ICIs. Spatially resolved multi-omics approaches offer valuable insights into potential biomarkers for ICI response and highlight the importance of profiling the TME complexity when developing therapeutic strategies and patient stratification.

The immunomodulatory ballet of tumour-derived extracellular vesicles and neutrophils orchestrating the dynamic CD73/PD-L1 pathway in cancer.

Head and neck squamous cell carcinoma (HNSCC) is a global cancer burden with a 5-year overall survival rate of around 50%, stagnant for decades. A tumour-induced immunosuppressive microenvironment contributes to HNSCC progression, with the adenosine (ADO) pathway and an upregulated expression of inhibitory immune checkpoint regulators playing a key role in this context. The correlation between high neutrophil-to-lymphocyte ratio (NLR) with advanced tumour staging suggests involvement of neutrophils (NØ) in cancer progression. Interestingly, we associated a high NLR with an increased intracellular PD-L1 localization in primary HNSCC samples, potentially mediating more aggressive tumour characteristics and therefore synergistically favouring tumour progression. Still, further research is needed to harness this knowledge for effective treatments and overcome resistance. Since it is hypothesized that the tumour microenvironment (TME) may be influenced by small extracellular vesicles (sEVs) secreted by tumours (TEX), this study aims to investigate the impact of HNSCC-derived TEX on NØ and blockade of ADO receptors as a potential strategy to reverse the pro-tumour phenotype of NØ. UMSCC47-TEX exhibited CD73 enzymatic activity involved in ADO signalling, as well as the immune checkpoint inhibitor PD-L1. Data revealed that TEX induce chemotaxis of NØ and the sustained interaction promotes a shift into a pro-tumour phenotype, dependent on ADO receptors (P1R), increasing CD170high subpopulation, CD73 and PD-L1 expression, followed by an immunosuppressive secretome. Blocking A3R reduced CD73 and PD-L1 expression. Co-culture experiments with HNSCC cells demonstrated that TEX-modulated NØ increase the CD73/PD-L1 axis, through Cyclin D-CDK4/6 signalling. To support these findings, the CAM model with primary tumour was treated with NØ supernatant. Moreover, these NØ promoted an increase in migration, invasion, and reduced cell death. Targeting P1R on NØ, particularly A3R, exhibited potential therapeutic strategy to counteract immunosuppression in HNSCC. Understanding the TEX-mediated crosstalk between tumours and NØ offers insights into immunomodulation for improving cancer therapies.

[The expression and prognosis prediction of CFL-1 in head and neck squamous cell carcinoma].

PURPOSE: The purpose of this study was to explore the expression, prognostic value and immune correlation of Cofilin 1 protein(CFL-1) in head and neck squamous cell carcinoma(HNSCC). METHODS: The expression and prognostic value of CFL-1 in head and neck squamous cell carcinoma(HNSCC) was explored in the cancer genome map database (TCGA) and gene expression total databases (GEO), and the potential immune pathway of CFL-1 in HNSCC was revealed by GESA and cibersoft analysis. The data were statistically analyzed using SPSS 26.0 software package. RESULTS: CFL-1 was significantly up-regulated in HNSCC tissue. The expression level of CFL-1 was significantly correlated with the overall survival status of HNSCC. High expression of CFL-1 was significantly associated with a lower overall survival rate. In addition, multivariate Cox survival analysis showed that CFL-1 expression was independent predictors of poor prognosis of HNSCC. GESA and cibersoft analysis showed that the imbalance of CFL-1 expression affected multiple signal pathways and infiltration of immune cells. CONCLUSIONS: CFL-1 is highly expressed in HNSCC and is significantly associated with poor prognosis of NHSCC. It is a potential prognostic marker of HNSCC.

The role of CD73 in predicting the response to immunotherapy in head and neck cancer patients.

Immunotherapy has a crucial role in the treatment of recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). However, only a small percentage of patients achieve long-term benefit in terms of overall response and survival. It was shown that HNSCC has an immunosuppressive microenvironment due to high levels of regulatory T cells and immunosuppressive molecules, such as LAG3 and CD73. The aim of our study was to investigate if the expression of CD73 by neoplastic and immune cells could affect the efficacy of anti-PD-1 immunotherapy. We reviewed data from 50 patients with R/M HNSCC receiving first line immunotherapy with or without chemotherapy based on a combined positive score (CPS). CD73 expression by cancer and immune cells was evaluated on pre-treatment and the percentage of stained cells was recorded. We analysed the association between CD73 expression on neoplastic and immune cells and early progression (EP), defined as progression occurring within 3 months. In 88 % of patients the primary tumour site was in the oral cavity or larynx. All patients received pembrolizumab associated in 40 % of cases to chemotherapy. CD73 was positive in 82 % and 96 % of cases on neoplastic and immune cells, respectively. The median value of CD73 was 32 % for neoplastic cells and 10 % for the immune ones. We observed a significant association between the CD73 expression on neoplastic cells over the median value and EP disease. We didn't record a correlation between the expression of CD73 on immune cells and early progression. Our findings suggest that higher expression of CD73 on neoplastic cells could predict resistance to immunotherapy in patients with CPS positive R/M HNSCC. The addition of this biomarker to routine evaluation of CPS could help to select the patients primary resistant to anti-PD-1 immunotherapy.

Fibroblast activation protein (FAP) as a prognostic biomarker in multiple tumors and its therapeutic potential in head and neck squamous cell carcinoma.

Background: Fibroblast activation protein (FAP), a cell surface serine protease, plays roles in tumor invasion and immune regulation. However, there is currently no pan-cancer analysis of FAP. Objective: We aimed to assess the pan-cancer expression profile of FAP, its molecular function, and its potential role in head and neck squamous cell carcinoma (HNSC). Methods: We analyzed gene expression, survival status, immune infiltration, and molecular functional pathways of FAP in The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) tumors. Furthermore, to elucidate the role of FAP in HNSC, we performed proliferation, migration, and invasion assays post-FAP overexpression or knock-down. Results: FAP expression was elevated in nine tumor types and was associated with poor survival in eight of them. In the context of immune infiltration, FAP expression negatively correlated with CD8+ T-cell infiltration in five tumor types and positively with regulatory T-cell infiltration in four tumor types. Our enrichment analysis highlighted FAP's involvement in the PI3K-Akt signaling pathway. In HNSC cells, FAP overexpression activated the PI3K-Akt pathway, promoting tumor proliferation, migration, and invasion. Conversely, FAP knockdown showed inhibitory effects. Conclusion: Our study unveils the association of FAP with poor tumor prognosis across multiple cancers and highlights its potential as a therapeutic target in HNSC.

Ubiquitination-Related Gene Signature, Nomogram and Immune Features for Prognostic Prediction in Patients with Head and Neck Squamous Cell Carcinoma.

The objective of this research was to create a prognostic model focused on genes related to ubiquitination (UbRGs) for evaluating their clinical significance in head and neck squamous cell carcinoma (HNSCC) patients. The transcriptome expression data of UbRGs were obtained from The Cancer Genome Atlas (TCGA) database, and weighted gene co-expression network analysis (WGCNA) was used to identify specific UbRGs within survival-related hub modules. A multi-gene signature was formulated using LASSO Cox regression analysis. Furthermore, various analyses, including time-related receiver operating characteristics (ROCs), Kaplan-Meier, Cox regression, nomogram prediction, gene set enrichment, co-expression, immune, tumor mutation burden (TMB), and drug sensitivity, were conducted. Ultimately, a prognostic signature consisting of 11 gene pairs for HNSCC was established. The Kaplan-Meier curves indicated significantly improved overall survival (OS) in the low-risk group compared to the high-risk group (p < 0.001), suggesting its potential as an independent and dependable prognostic factor. Additionally, a nomogram with AUC values of 0.744, 0.852, and 0.861 at 1-, 3-, and 5-year intervals was developed. Infiltration of M2 macrophages was higher in the high-risk group, and the TMB was notably elevated compared to the low-risk group. Several chemotherapy drugs targeting UbRGs were recommended for low-risk and high-risk patients, respectively. The prognostic signature derived from UbRGs can effectively predict prognosis and provide new personalized therapeutic targets for HNSCC.

The Complex Role of Mast Cells in Head and Neck Squamous Cell Carcinoma: A Systematic Review.

Background and Objectives: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous malignancy influenced by various genetic and environmental factors. Mast cells (MCs), typically associated with allergic responses, have recently emerged as key regulators of the HNSCC tumor microenvironment (TME). This systematic review explores the role of MCs in HNSCC pathogenesis and their potential as prognostic markers and therapeutic targets. Materials and Methods: A systematic search was conducted in the PubMed, Scopus and ClinicalTrials.gov databases until 31 December 2023, using "Mast cells" AND "Head and neck squamous cell carcinoma" as search terms. Studies in English which reported on MCs and HNSCC were included. Screening, data extraction and analysis followed PRISMA guidelines. No new experiments were conducted. Results: Out of 201 articles, 52 studies met the inclusion criteria, 43 of which were published between 2020 and 2023. A total of 28821 HNSCC and 9570 non-cancerous tissue samples had been examined. MC density and activation varied among normal tissues and HNSCC. Genetic alterations associated with MCs were identified, with specific gene expressions correlating with prognosis. Prognostic gene signatures associated with MC density were established. Conclusions: MCs have arisen as multifaceted TME modulators, impacting various aspects of HNSCC development and progression. Possible site-specific or HPV-related differences in MC density and activation should be further elucidated. Despite conflicting findings on their prognostic role, MCs represent promising targets for novel therapeutic strategies, necessitating further research and clinical validation for personalized HNSCC treatment.

Integrated analysis of methylation and transcriptome identifies a novel risk model for diagnosis, prognosis, and immune characteristics in head and neck squamous cell carcinoma.

BACKGROUND: DNA methylation is an important epigenetic modification that plays a crucial role in the development and progression of various tumors. However, the association between methylation­driven genes and diagnosis, prognosis, and immune characteristics of head and neck squamous cell carcinoma (HNSCC) remains unclear. METHODS: We obtained transcriptome, methylation, and clinical data from HNSCC patients in TCGA database, and used MethylMix algorithm to identify methylation-driven genes. A methylation driven gene-related risk model was constructed using Lasso regression analysis, and validated using data from GEO database. Immune infiltration and immune function analysis of the expression profiles were conducted using ssGSEA. Differences in immune checkpoint-related genes were analyzed, and the efficacy of immunotherapy was evaluated using TCIA database. Finally, a series of cell functional experiments were conducted to validate the results. RESULTS: Five methylation-driven genes were identified and utilized to construct a prognostic risk model. Based on the median risk score, all patients were categorized into high-risk and low-risk groups. The K-M analysis revealed that patients in the high-risk group have a worse prognosis. Additionally, the risk model demonstrated better prognostic predictive value as indicated by ROC analysis. GSEA enrichment analysis indicated that gene sets in the high and low-risk groups were primarily enriched in pathways associated with tumor immunity and metabolism. Our subsequent investigations showed that high-risk patients exhibited more immunosuppressive phenotypes, while low-risk patients were more likely to respond positively to immunotherapy. CONCLUSION: These findings of our research have the potential to improve patient stratification, guide treatment decisions, and advance the development of personalized therapies for HNSCC.

Differential Immune Checkpoint Protein Expression in HNSCC: The Role of HGF/MET Signaling.

Although inhibitors targeting the PD1/PD-L1 immune checkpoint are showing comparably good outcomes, a significant percentage of head and neck squamous cell carcinoma (HNSCC) patients do not respond to treatment. Apart from using different treatment strategies, another possibility would be to target other immune checkpoints operating in these non-responding tumors. To obtain an overview of which checkpoint ligands are expressed on HNSCC tumor cells and if these ligands are affected by HGF/MET signaling, we used mRNA sequencing and antibody-based techniques for identifying checkpoint ligands in six HNSCC tumor cell lines. Furthermore, we compared our results to mRNA sequencing data. From the checkpoint ligands we investigated, VISTA was expressed the highest at the RNA level and was also the most ubiquitously expressed. PD-L2 and B7-H3 were expressed comparably lower and were not present in all cell lines to the same extent. B7-H4, however, was only detectable in the Detroit 562 cell line. Concerning the effect of HGF on the ligand levels, PD-L2 expression was enhanced with HGF stimulation, whereas other checkpoint ligand levels decreased with stimulation. B7-H4 levels in the Detroit 562 cell line drastically decreased with HGF stimulation. This is of interest because both the checkpoint ligand and the growth factor are reported to be connected to epithelial-mesenchymal transition in the literature.