Integrative single-cell and bulk transcriptomics analysis reveals cellular heterogeneity, immune microenvironment dynamics, and prognostic signatures in oral squamous cell carcinoma.

Squamous cell carcinoma is the most common head and neck malignancy arising from the oral mucosa and the skin. The histologic and immunohistochemical features of oral squamous cell carcinoma (OSCC) and head and neck cutaneous squamous cell carcinoma (HNcSCC) are similar, making it difficult to identify the primary site in cases of metastases. With the advent of immunotherapy, reliable distinction of OSCC and HNcSCC at metastatic sites has important treatment and prognostic implications. Here, we investigate and compare the genomic landscape of OSCC and HNcSCC to identify diagnostically useful biomarkers. Whole-genome sequencing data from 57 OSCC and 41 HNcSCC patients were obtained for tumor and matched normal samples. Tumor mutation burden (TMB), Catalogue of Somatic Mutations in Cancer (COSMIC) mutational signatures, frequent chromosomal alterations, somatic single nucleotide, and copy number variations were analyzed. The median TMB of 3.75 in primary OSCC was significantly lower (P < .001) than that of 147.51 mutations/Mb in primary HNcSCC. The COSMIC mutation signatures were significantly different (P < .001) between OSCC and HNcSCC. OSCC showed COSMIC single-base substitution (SBS) mutation signature 1 and AID/APOBEC activity–associated signature 2 and/or 13. All except 1 HNcSCC from hair-bearing scalp showed UV damage–associated COSMIC SBS mutation signature 7. Both OSCC and HNcSCC demonstrated a predominance of tumor suppressor gene mutations, predominantly TP53. The most frequently mutated oncogenes were PIK3CA and MUC4 in OSCC and HNcSCC, respectively. The metastases of OSCC and HNcSCC demonstrated TMB and COSMIC SBS mutation signatures similar to their primary counterparts. The combination of high TMB and UV signature in a metastatic keratinizing squamous cell carcinoma suggests HNcSCC as the primary site and may also facilitate decisions regarding immunotherapy. HNcSCC and OSCC show distinct genomic profiles despite histologic and immunohistochemical similarities. Their genomic characteristics may underlie differences in behavior and guide treatment decisions in recurrent and metastatic settings.

Induction chemotherapy in oral squamous cell carcinoma is a controversial issue in clinical practice. To investigate the evolution of cancer cells and tumor microenvironment (TME) response to chemotherapy in oral squamous cell carcinoma, single-cell transcriptome analysis was performed in a post-chemotherapy squamous cell carcinoma located in oral cavity. The main cell types were identified based on gene expression patterns determined using dimensionality reduction and unsupervised cell clustering. Non-negative matrix factorization clustering of the gene expression of Cancer-associated fibroblasts (CAFs) and macrophages was performed. Kyoto Encyclopedia of Genes and Genomes pathway analyses and gene set enrichment analysis were performed to explore significant functional pathways. CellPhoneDB and NicheNet were used to detect the intercellular communication between cell types. CAFs were divided into “inflammatory CAFs,” “antigen-presenting CAFs” and “myofibroblastic CAFs.” Three classic subgroups of tumor-associated macrophages (TAMs) were detected, namely C1Q (+), FCN1 (+) and SPP1(+) TAMs. The inflammatory cytokine expression is elevated, and several molecular pathways, such as PI3K/Akt/mTORC1, TNF-α via NFκB, TGF-β, IL-6/JAK2/STAT3 and CXCL12/CXCR4 axis associated with epithelial-mesenchymal transition were enriched in TME. Also, CD74-MIF/COPA/APP interactions were expressed in TME of oral squamous cell carcinoma after chemotherapy. The results revealed the characteristics of TME in post-chemotherapy oral squamous cell carcinoma at single-cell transcriptome level, providing new insights and clues for further investigation.

Lymph node metastasis (LNM) is a critical prognostic factor for patients with oral squamous cell carcinoma (OSCC). While previous research has implicated partial epithelial-to-mesenchymal transition (p-EMT) of tumor cells and cancer-associated fibroblasts (CAFs), specifically myofibroblastic CAFs (myCAFs), in LNM, the underlying molecular mechanisms remain poorly understood. Furthermore, the lack of diagnostic and prognostic biomarkers related to LNM has impeded the improvement in clinical outcomes for patients with OSCC. Here, we conducted a comprehensive molecular analysis integrating original and publicly available OSCC data of the bulk genome and transcriptome analyses, single-cell transcriptome profiling, and spatial transcriptome analyses. We found that myCAFs were both quantitatively and functionally activated in LNM-positive samples and spatially colocalized with OSCC cells within the invasive tumor front (ITF), providing a niche that may facilitate LNM. We validated spatial colocalization of myCAFs and OSCC cells using 50 additional Immunohistochemistry (IHC) samples. Mechanistically, myCAFs provided increased extracellular matrix (ECM) signals, such as COLLAGEN, Fibronectin1(FN1), and LAMININ to OSCC cells. Consequently, OSCC cells upregulate ECM receptors such as αβ integrins (ITGA-B), syndecans (SDC), and CD44, leading to upregulating stemness-related genes in OSCC cells. Further analysis using CRISPRGeneEffect data from DepMap found that ITGB1, ITGA3, and CD44 are among the most essential genes for the survival and growth of OSCC cells. By analyzing the spatial transcriptomic data of matched primary tumor sites (HUH001-P1 and HUH001-P2) and metastatic site (HUH001-met) from the same patient, we identified the origins of metastatic cells in the primary tumor site. We further extracted a 23-gene signature from the metastatic origin in ITF, where OSCC and myCAFs colocalize. This spatially-resolved 23-gene signature predicted LNM status and poor overall survival (OS) in clinical OSCC patients. The prognostic significance of the 23-gene signature we extracted from the spatial analysis was also validated in separate clinical cohorts from TCGA study and two other microarray datasets. Our findings provide novel insight into the molecular crosstalk between myCAFs and OSCC cells that facilitates LNM and identifies clinically valuable biomarkers for better prognostication in patients with OSCC. Citation Format: Ken Furudate, Shuya Kasai, Tadashi Yoshizawa, Yuya Sasaki, Kohei Fujikura, Shintaro Goto, Ryohei Ito, Tomoyuki Tanaka, Hiroshi Kijima, Kosei Kubota, Ken Itoh, Wataru Kobayashi, Koichi Takahashi. Spatial colocalization and molecular crosstalk of myofibroblastic CAFs and tumor cells shape lymph node metastasis in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1155.

Oral squamous cell carcinoma (OSCC) is a malignant tumor characterized by both clinical and molecular heterogeneity. Its rising global incidence and mortality rates pose considerable clinical challenges. Accumulating evidence suggests that dysregulation of mitochondria-associated pan-cell death, a process integrating multiple mitochondrial-related cell death modalities, is closely linked to tumor initiation and progression. Nevertheless, the identification of key therapeutic targets among mitochondria-associated pan-cell death-related genes (MAPGs) in OSCC remains an unresolved issue, underscoring the need for further investigation. We systematically investigated the expression patterns, prognostic relevance, and immune microenvironmental features of mitochondria-associated pan-cell death-related genes (MAPGs) in OSCC by integrating data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) datasets (GSE42743, GSE41613, GSE75538, GSE164241, GSE172577, and GSM6339633), and the MitoCarta3.0 database. Using nine machine learning algorithms, we compared the importance and prognostic value of MAPGs and identified INHBA as the most pivotal MAPG. Its predictive power and functional relevance were further explored through enrichment analysis. We validated INHBA mRNA expression levels in clinical OSCC samples using quantitative real-time PCR (qRT‑PCR). Pan-cancer analysis was performed to assess the potential of INHBA as a therapeutic target across multiple cancer types. Single-cell RNA sequencing, spatial transcriptomics, and cell-cell communication analyses were integrated to elucidate the spatial distribution of INHBA in OSCC and its crosstalk with the immune microenvironment. Finally, drug sensitivity profiling was conducted using the GDSC and PRISM databases. A total of 19 MAPGs with the greatest prognostic value were screened out. Among them, INHBA emerged as the most critical MAPG, which was overexpressed in HNSCC and strongly correlated with poor prognosis in OSCC patients (p < 0.05). Meanwhile, INHBA expression was elevated in cancer-associated fibroblasts (CAFs). Spatial transcriptomic analysis further revealed strong spatial colocalization between INHBA and CAF-localized regions. These results indicated that INHBA primarily functioned within CAFs, especially myCAFs, and may promote OSCC progression through modulation of the immune microenvironment. Elevated INHBA expression served as a prognostic indicator in OSCC, a finding consistent with its prognostic relevance in broader HNSCC analyses. Selumetinib and naltrexone showed high sensitivity in INHBA-high contexts. INHBA is a crucial MAPG that is upregulated in OSCC and mainly functions in CAFs. It likely affects the poor prognosis of OSCC by regulating the immune microenvironment. Selumetinib and naltrexone hold promise as innovative therapeutic agents, potentially opening up new treatment avenues for OSCC patients.

Lymph node metastasis (LNM) is a critical prognostic factor for patients with oral squamous cell carcinoma (OSCC). Previous research has implicated the partial epithelial-to-mesenchymal transition of tumor cells and myofibroblastic cancer-associated fibroblasts (myCAFs) in the LNM process. However, the underlying molecular mechanisms remain poorly understood. Here, we conducted a comprehensive molecular analysis integrating original and publicly available OSCC data from bulk genome and transcriptome, single-cell transcriptome, and spatial transcriptome analyses. We found that myCAFs were quantitatively and functionally activated in LNM-positive samples and spatially colocalized with OSCC cells within the invasive tumor front (ITF), providing a niche that may facilitate LNM. Immunohistochemical validation in 90 ITF samples confirmed significantly higher myCAF density in LNM-positive samples than in LNM-negative samples, and this density remained an independent predictor of LNM when adjusted for pathological grade and the pattern of invasion. In LNM-positive samples, myCAFs provided increased extracellular matrix (ECM) signals, upregulating stemness-related genes such as CD44 in OSCC cells. The functional importance of this myCAF-driven ECM-CD44 axis was further supported by our validation analysis of expanded, publicly available spatial transcriptome and experimental in vitro coculture data. We also extracted a spatially resolved, 23-gene signature from the metastatic ITF where OSCC and myCAFs colocalize. This signature predicted LNM status and poor overall survival in patients with OSCC. Our findings provide novel insight into the molecular myCAF/OSCC crosstalk that facilitates LNM and identify potential prognostic biomarkers and therapeutic targets for patients with OSCC.

CXCL12/CXCR4 pathway orchestrates CSC-like properties by CAF recruited tumor associated macrophage in OSCC

6045 Background: Spatial transcriptomics (ST) revealed conserved malignant leading edge (LE) and tumor core (TC) architectures in primary oral squamous cell carcinoma (OSCC) with potential for biomarker discovery. Spatial organization of tumor cells, as well as composition and prognostic significance of neighboring stromal cells in RM-HNSCC remain unknown. Methods: 21 tumor biopsy samples (14 baseline, 7 paired on-treatment) from 14 ICB-naive RM-HNSCC patients (pts) treated with pembrolizumab in INSPIRE (NCT02644369) were profiled using 10x Visium. Spatial organization was refined by scoring LE and TC gene sets identified in OSCC (Arora and Bose et al. Nat Comm 2023). Malignant (2,671 spots) and nonmalignant (8,177 spots) subclusters were annotated, with the latter classified into five cell subtypes using canonical markers: tumor-associated macrophages (TAMs) ( CD68 , CD14 , SCF1R ), regulatory stromal cells (reg) ( KRT17 , COL10A1 , SRBP1 ), plasma cells ( CD38 , IRF4 , PRDM1 ), T cells ( CD3D , CD3E , PTPRC ), and cancer-associated fibroblasts (CAFs) ( FAP , COL1A1 , PDGFRB ). Neighborhood analyses compared normalized counts of stromal cells adjacent to LE and TC, accounting for variations in cell density and sampling differences. A signature was built through k -means clustering of the five cell subtypes. Pts were stratified into high/low signature-score groups using the median cutoff and tested for association with progression-free survival (PFS). Results: Spatial organization revealed conserved malignant subclusters (C0 and C1) in 19/21 samples from 13 pts (11 non-responders). Top C0 genes were COL21A1 , S1PR3 , LIFR , and ZEB1 . Top C1 genes were KRT6B , KRT6C , KRTDAP , and LCN2 . Pathway analysis predicted activation of cell cycle and glycoprotein 6 in C0, and keratinization and neutrophil degranulation in C1. Comparative expression of OSCC-related gene sets revealed LE correlation with C0, and TC correlation with C1 (both p &lt; 0.0001); stronger overlap was seen with the latter highlighting TC as a more conserved feature in HNSCC. Among non-responders, dominant communication patterns in LE and TC included claudins, cadherins, WNT, and IL-6, linked to cell adhesion, migration/invasion, and immune evasion. Signature generated by neighborhood analysis was enriched in TAMs and T cells, depleted in CAFs and reg near LE and TC, while plasma cells were depleted near LE but enriched near TC. Pts with high signature scores (6/13) exhibited improved PFS compared to low scores (7/13), median PFS 6.0 months [95% CI, 2.3-NA] versus 1.9 months [95% CI, 1.8-NA] (p = 0.059). Conclusions: To our knowledge, this is the first report using ST analysis to characterize LE and TC architectures in RM-HNSCC, along with heterogeneous neighboring stromal cells with prognostic potential for ICB. Ongoing cohort expansion will elucidate the clinical significance of these findings.

ABSTRACTBetel nut chewing is a major etiological factor for oral squamous cell carcinoma (OSCC), yet its mechanistic underpinnings remain poorly defined. Here, we performed single‐cell RNA sequencing and spatial transcriptomics from six OSCC patients to comprehensively dissect the tumor microenvironment (TME) dynamics and cellular heterogeneity associated with betel nut‐induced oral mucosal carcinogenesis. We identify a fibrotic, immunosuppressive TME characterized by expanded cancer‐associated fibroblasts (CAFs) and B/plasma cells, alongside depletion of cytotoxic T/NK cells and macrophages. CAFs, particularly antigen‐presenting CAFs, are spatially enriched at the invasive front and drive epithelial plasticity and malignant transformation. Notably, we uncover a malignant epithelial subpopulation, LAMC2+ EpiC6, enriched for epithelial–mesenchymal transition (EMT) programs, angiogenesis, and metastasis‐associated pathways, which engaged in extensive crosstalk with CAFs and other nonmalignant components. Clinically, LAMC2 expression was significantly elevated in OSCC tissues from betel nut chewers, and arecoline treatment of OSCC cell lines induced LAMC2 upregulation, EMT, and enhanced migratory and invasive capacities in vitro. Collectively, our study delineates a malignant trajectory of epithelial cell progression, highlighting LAMC2+ EpiC6 as a key aggressive subpopulation orchestrated by EMT‐related transcriptional regulators and extracellular matrix remodeling. These findings offer mechanistic insights and identify potential therapeutic targets to disrupt tumor–stroma interplay and mitigate disease progression.

Background and Rationale: OSCC is the most frequent head and neck cancer. While it is mainly affecting patients with a smoking and/or alcohol history (SD), 10% to 15% of OSCC are diagnosed in patients with not known risk factors (NSND). We and others have shown that OSCC from NSND patients are characterized by an enrichment in interferon gamma (IFNγ) response and PD-1 signaling pathways together with a higher intratumor T-cell infiltrate. Herein, we seek to gain more insights into the immune and non-immune tumor microenvironment (TME) of OSCC in SD vs. NSND patients. Material and Methods: We made use of single-cell RNA seq (10x Genomics) from non-sorted and CD45 negative enriched cell suspensions of OSCC biopsies from 11 treatment naïve patients, human papillomavirus negative, including 6 SD and 5 NSND patients. Results: All samples were characterized by a strong immune cell infiltrate. Consistent with our previous results, the analysis of 51,629 immune cells confirmed that NSND patients’ samples displayed a significantly higher proportion of T cells (p = 0.005) compared to SD patients. NK cells were also increased, with 2% in SD patients versus 6% in NSND patients (p = 0.008). T cells strongly expressed IFNγ and remarkably, both myeloid cells and tumor cells from NSND patients had an increased enrichment in IFNγ response pathway in NSND as compared to SD patients. The TME of SD patients was characterized by the presence of 15% of plasma cells vs. 3% in NSND patients (p = 0.003). Thus, tumors from NSND versus SD patients represent two distinct groups of tumors characterized by a different immune TME. Moreover, tumors from SD patients displayed 2% vs. 4% Cancer Associated Fibroblasts (CAFs) (p = 0.01). In addition, among CAFs subpopulations, myofibroblast CAFs, defined as the subpopulation producing extracellular matrix proteins were found only in tumors from SD patients, suggesting that distinct CAFs subpopulations may shape the immune TME by affecting the recruitment and function of adaptive immune cells in SD versus NSND patients. Finally, we analyzed tumor cells using non-negative matrix factorization to find Meta Programs (MP), which are set of genes coordinately upregulated. We identified 5 MP associated with different biological functions: epithelial differentiation, stress, cell cycle (G2/M), partial epithelial-to-mesenchymal transition, and epithelial senescence. While the epithelial differentiation MP was found in all patients, the remaining ones were more frequently found in SD patients. Conclusion: Our results show that the TME of OSCC from NSND vs. SD differs in terms of T, NK, and plasma cells composition, CAFs, and tumor cells MP. Integration with spatial transcriptomics is ongoing. Those biological differences may represent an opportunity to refine the therapeutic approaches in the two populations of patients. Citation Format: Yannick Le Meitour, Béatrice Vanbervliet, Sonia Canjura-Rodriguez, Cyril Degletagne, Laurie Tonon, Lucas Michon, Jebrane Bouaoud, Philippe Zrounba, Aude Excoffier, Karène Mahtouk, Pierre Saintigny. Single-cell analysis of oral squamous cell carcinoma (OSCC) from smoker vs. non-smoker patients highlights two groups of tumors with distinct immune microenvironments. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4530.

Cancer-associated fibroblasts (CAFs) are important contributors to malignant progression in bladder cancer (BLCA), yet the mechanisms by which they promote tumor progression remain incompletely understood. In this study, we integrated patient-derived CAFs with bulk and single-cell transcriptomics, spatial transcriptomics, and multi-cohort clinical datasets to systematically define the molecular mechanisms underlying CAF-driven BLCA progression. Integrated analyses of bulk RNA sequencing, single-cell transcriptomics, spatial transcriptomics, immunohistochemistry, and multiplex immunofluorescence of paraffin-embedded tissues, together with PCR, immunoblotting, and immunofluorescence in patient-derived normal fibroblasts and CAFs, consistently demonstrated marked enrichment of NOTCH3 in CAFs in bladder cancer. Silencing of NOTCH3 attenuated extracellular matrix deposition, contractile capacity, intracellular ROS accumulation, and the ability of CAFs to support tumor growth. Mechanistically, TGF-β induced NOTCH3 transcription through SMAD2 binding to the NOTCH3 promoter. In turn, NOTCH3 recruited HSPA8 to promote K48-linked ubiquitination and proteasomal degradation of P62, thereby suppressing the P62-NRF2 antioxidant pathway and sustaining intracellular ROS accumulation. Restoration of P62 expression or pharmacological scavenging of ROS effectively reversed NOTCH3-driven myofibroblastic differentiation and stromal support of tumor growth. In vivo, CAF-specific deletion of NOTCH3 significantly inhibited tumor growth, reduced collagen deposition, and attenuated stromal remodeling in both subcutaneous and orthotopic bladder cancer models. Collectively, these findings identify CAF-intrinsic NOTCH3 as important regulator of myofibroblastic differentiation through integration of upstream TGF-β signaling with redox and proteostatic control, highlighting stromal NOTCH3 as a potential therapeutic target to limit malignant progression in BLCA.

It has been increasingly recognized that the tumour microenvironment is a critical factor involved in cancer progression. However, little is known about the clinical value of the stromal features in oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the clinical significance of cancer-associated fibroblasts (CAFs) and tumour-associated macrophages (TAMs) in OSCC. OSCC specimens were obtained from 60 patients who underwent surgery following 5-fluorouracil-based chemoradiotherapy. Paraffin-embedded sections obtained from biopsy specimens were immunohistochemically analysed. The associations among CAFs, TAMs and various clinicopathological features were examined, and the effects of CAFs and TAMs on the prognosis were evaluated. In the group with a high level of CAFs, the incidence of advanced pT- and pN-stage cases was significantly higher than that in the group with the low level. A high TAMs tumour expression was significantly correlated with a poor response to preoperative chemoradiotherapy. A Kaplan-Meier analysis revealed that higher numbers of CAFs and TAMs were significantly correlated with a poor prognosis. These findings suggest that TAMs are a potential biomarker for predicting the clinical response to 5-FU-based chemoradiotherapy, and the expression status of the CAFs and TAMs may be useful for making treatment decisions to improve the survival of OSCC patients.

Stromal cells are believed to affect cancer invasion and metastasis. The purpose of this study was to evaluate the distribution of cancer-associated fibroblasts (CAFs) and the incidence of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC), focusing on clinicopathological factors and patient prognosis, as well as cancer invasion. The study included 108 patients with OSCC. Anti-α-smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs. CAFs were divided into 4 grades on the basis of staining intensity: negative (0), scanty (1), focal (2), and abundant (3). The most intensive areas of macrophage concentration in each tumor invasive stroma were also evaluated. The cancer specimens were divided into Grade 0/1, Grade 2, and Grade 3 on the basis of CAF grade. In addition, they were divided into low- and high-grade groups on the basis of the number of CD68-positive and CD163-positive macrophages. The latter were significantly increased in the Grade 2 CAF group compared to the Grade 0/1 group (P = 0.009). Kaplan-Meier and multivariate survival analyses revealed that Grade 2 CAFs (P = 0.003) and high CD163-positive macrophage levels (P = 0.007) significantly correlated with a poor outcome in patients with OSCC, and that a high CD163-positive macrophage level was a significant and an independent prognostic factor (P = 0.045). Cancer-associated fibroblasts and CD163-positive macrophages may be potential prognostic predictors of OSCC.

Oral squamous cell carcinoma (OSCC) is a prevalent malignancy of the head and neck with rising global incidence. Despite advances in treatment modalities, OSCC prognosis remains diverse due to the complex molecular and cellular heterogeneity within tumours, as well as the heterogeneity in tumour microenvironment (TME). In this study, we utilized single‐cell RNA sequencing (scRNA‐seq) analysis to explore distinct subpopulations of tumour cells in OSCC tissues and their interaction with components in TME. We identified four major tumour cell subpopulations (C0, C1, C2 and C3) with unique molecular characteristics and functional features. Pathway enrichment analysis revealed that C0 primarily expressed genes involved in extracellular matrix interactions and C1 showed higher proliferation levels, suggesting that the two cell subpopulations exhibited tumour aggressiveness. Conversely, C2 and C3 displayed features associated with keratinization and cornified envelope formation. Accordingly, C0 and C1 subpopulations were associated with shorter overall and disease‐free survival times, while C2 and C3 were weakly correlated with longer survival. Genomic analysis showed that C1 demonstrated a positive correlation with tumour mutation burden. Furthermore, C0 exhibited resistant to cisplatin treatment, while C1 showed more sensitive to cisplatin treatment, indicating that C0 might exhibit more aggressive compared to C1. Additionally, C0 had a higher level of communication with fibroblasts and endothelial cells in TME via integrin‐MAPK signalling, suggesting that the function of C0 was maintained by that pathway. In summary, this study provided critical insights into the molecular and cellular heterogeneity of OSCC, with potential implications for prognosis prediction and personalized therapeutic approaches.

Objectives: Integrins, the coordinator of extracellular and intracellular signaling, are often found to be aberrant in tumors and can reshape the tumor microenvironment. Although previous studies showed that integrin beta 2 (ITGB2) is important for host defense, its expression profile and role in tumors, especially in cancer associated fibroblasts (CAFs) are still unknown.Methods: Immunofluorescence stain and fluorescence activated cell sorting were used to analyze the ITGB2 expression profile in oral squamous cell carcinoma (OSCC). RT-PCR and western blot were used to compare ITGB2 expression in normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs). Clinical data and function-based experiments were used to investigate the promoting tumor growth ability of ITGB2 expressing CAFs. Enhanced glycolysis activity was identified by using bioinformatics analyses and GC/MS assays. MCT1 knockdown OSCC cell lines were constructed to explore the pro-proliferative mechanisms of ITGB2 expressing CAFs in multiple in vitro and in vivo assays.Results: We found that CAFs exhibited significantly higher ITGB2 expression than the matched NFs. In addition, higher ITGB2 expression in CAFs was correlated with higher TNM stages and more Ki67+ tumor cells, indicating its ability to promote OSCC proliferation. Further, co-culture assay demonstrated that ITGB2-mediated lactate release in CAFs promoted OSCC cell proliferation. Mechanically, ITGB2 regulated PI3K/AKT/mTOR pathways to enhance glycolysis activity in CAFs. Accordingly, lactate derived from ITGB2-expressing CAFs was absorbed and metabolized in OSCC to generate NADH, which was then oxidized in the mitochondrial oxidative phosphorylation system (OXPHOS) to produce ATP. Notably, inhibiting the OXPHOS system with metformin delayed the proliferative capacity of OSCC cells cultured in the ITGB2-expressing CAFs medium.Conclusions: Our study uncovered the ITGB2high pro-tumoral CAFs that activated the PI3K/AKT/mTOR axis to promote tumor proliferation in OSCC by NADH oxidation in the mitochondrial oxidative phosphorylation system.

Oral squamous cell carcinoma (OSCC) exhibits significant cellular heterogeneity and metabolic reprogramming that influence tumor progression and therapeutic responses. However, the molecular mechanisms underlying these processes remain poorly understood. We performed an integrated analysis of single-cell RNA sequencing and spatial transcriptomics analysis on OSCC samples to characterize cellular heterogeneity and identify key regulatory factors. Summary-data-based Mendelian randomization (SMR) analysis was conducted to establish causal links between gene expression and OSCC risk. Functional enrichment, protein-protein interaction (PPI) network analysis, molecular docking, and co-immunoprecipitation (Co-IP) were employed to elucidate molecular mechanisms. Single-cell analysis of 50,667 cells identified nine distinct cell populations with epithelial cells showing the highest glutamine metabolism activity. SMR analysis revealed glutathione S-transferase omega 2 (GSTO2) as a genetically validated protective factor for OSCC risk. GSTO2-positive epithelial cells exhibited enhanced metabolic reprogramming, particularly in fatty acid degradation and amino acid catabolism pathways. Spatial transcriptomics demonstrated that GSTO2-positive cells were spatially organized and showed enhanced communication with immune cells. Protein-protein interaction analysis identified interferon-stimulated genes (MX1, OAS1, UBE2L6) and immunoproteasome subunits (PSMB8, PSMB9) as core regulatory hubs. Molecular docking confirmed direct binding interactions between GSTO2 and these hub proteins, with MX1-GSTO2 showing the strongest binding affinity (-10.7kcal/mol). Co-immunoprecipitation experiments validated the physical interaction between GSTO2 and MX1. This study indicates that GSTO2 defines an epithelial subset characterized by enhanced metabolic activity and immune interactions, highlighting its potential as a biomarker and therapeutic target in OSCC.