Metabolic Reprogramming and GM-CSF Secretion in Areca Nut-Activated Fibroblast Drives Oral Precancer Progression

Metabolic Reprogramming and GM-CSF Secretion in Areca Nut-Activated Fibroblast Drives Oral Precancer Progression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Metabolic Reprogramming and GM-CSF Secretion in Areca Nut-Activated Fibroblast Drives Oral Precancer Progression Yen-Yun Wang, Chang-Wei Su, Chih-Huang Tseng, Pang-Yu Chen, Hieu D.H. Nguyen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7567949/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oral cancer, marked by a rising global incidence and dismal prognosis, is associated with areca nut use, particularly in South and Southeast Asia. Yet, its influence on the oral tissue microenvironment is not well elucidated. This research aimed to elucidate the effects of areca nut extract (ANE) on oral carcinogenesis by modulating fibroblast behavior within the microenvironment. In this study, cell viability assay, transwell migration and invasion assays, and flow cytometry were employed to study cell behaviors. Cytokine array, ELISA, phospho-kinase array, Western blot analysis, extracellular O2 consumption, and glycolysis assays were conducted to assess cellular functionalities. In vivo experiments, complemented by immunohistochemical and immunofluorescent staining of oral lesions, were performed to corroborate in vitro observations. Our results showed that upregulation of α-SMA and FAP was observed in fibroblasts within oral pre-cancer and cancer lesions. ANE enhanced mitochondrial metabolism in fibroblasts and induced their transition into myofibroblasts. Additionally, ANE triggered epithelial-to-mesenchymal transition (EMT) and stimulated GM-CSF secretion in fibroblasts, thereby advancing the progression of oral pre-cancer. The conditioned medium from ANE-treated human gingival fibroblasts, along with recombinant GM-CSF, elevated EGFR phosphorylation and facilitated oncogenic transformation in DOK dysplastic oral keratinocytes. Moreover, in a hamster model, daily application of ANE on buccal pouches for 37 weeks replicated the in vitro findings for ANE-induced GM-CSF expression in fibroblasts and EGFR phosphorylation in mucosa epithelial cells. In conclusion, this investigation provides in vitro and in vivo evidence suggesting that ANE promotes EMT and GM-CSF secretion in fibroblasts, which activates EGFR and the malignant transformation of oral pre-cancer cells. Areca oral precancer and cancer lesions epithelial-to-mesenchymal transition tumor microenvironment cytokines carcinogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction According to the projection from GLOBOCAN, oral cancer incidence will rise by 50% from 2020 to 2040 [ 1 ]. Approximately 90% of them are classified as oral squamous cell carcinoma (OSCC) [ 2 ]. In South and Southeast Asia, chewing of areca (betel) nut and betel quid is a OSCC risk factor [ 3 , 4 ]. The International Agency for Research on Cancer has concluded their carcinogenic effect in humans ( https://www.who.int/news/item/07-08-2003-iarc-monographs-programme-finds-betel-quid-and-areca-nut-chewing-carcinogenic-to-humans ), with betel quid chewers facing a 28-fold higher oral cancer risk [ 5 ]. Notably, before OSCC develops, these substances often induce various pre-cancer lesions in the oral cavity [ 6 ]. In Taiwan, over 75% of OSCC patients chew betel quid [ 7 ], highlighting the urgent need for target strategies. Areca nut, the essential ingredient of betel quid, contains arecoline, arecaidine, guvacoline, and guvacine, with arecoline being the most abundant and predominant in saliva during and after chewing [ 8 ]. Both areca nut extract and arecoline contribute to DNA damage and cytopathological changes in oral epithelial cells [ 9 ] and mucosal fibroblasts [ 10 ]. Furthermore, ANE also activates immune cells, particularly macrophages, promoting precancer-to-cancer transition [ 11 ]. Other than epithelial cells, the oral tissue microenvironment includes fibroblasts, immune cells, endothelial cells, pericytes, mesenchymal cells, and adipocytes [ 12 ]. Understanding this microenvironment is important for preventing and treating oral pre-cancer and cancer. During oral cancer development, fibroblasts transform into myofibroblasts, also known as cancer-associated fibroblasts (CAFs), which interact with cancer cells, immune cells to drive tumor progression [ 13 ]. Though poorly defined, CAFs exhibit an activated myofibroblast phenotype and support tumor growth and metastasis [ 14 ]. However, the mechanisms of fibroblasts-to- myofibroblast transition in oral carcinogenesis remain largely unexplored. Although areca nut has been linked to oral cancer [ 15 ], and fibroblast transformation [ 16 ], its role in driving fibroblasts-to- myofibroblasts transition and oral pre-cancer progression remains unclear. This study aims to determine the effect of ANE on fibroblast transformation and its impact on malignant progression of oral pre-cancer cells through integrated clinical, in vitro, and in vivo approaches, aiming to provide innovative and targeted topical therapeutic strategies. Materials and Methods Cell culture and dissociation of primary fibroblasts The human gingiva fibroblast cells (HGF) were acquired from the American Type Culture Collection (ATCC) and were maintained in Fibroblast Basal Medium (ATCC) with fibroblast growth low serum (ATCC), whereas the oral premalignant cell line DOK was maintained in DMEM/F12 medium (Thermo Fisher Scientific). Primary fibroblasts were isolated from normal tissues and tumor tissues within an hour post-surgical. Tissues were washed in phosphate buffer saline (PBS), minced into 2–4 mm pieces, and digested using a tumor dissociation kit in a gentle MACS™ C-Tube (Miltenyi) for 60 minutes at 37°C in a gentle MACS Octo dissociator. Digests were filtered through 100-µm sterile cell strainers, followed by centrifugation at 300 × g for 10 minutes, and resuspended in complete Dulbecco's modified Eagle Medium F12 (DMEM F12). Patient samples Human tissue specimens were obtained from patients undergoing treatment at Kaohsiung Medical University Hospital (KMUH), Taiwan. This study received approval from the Institutional Review Board of Kaohsiung Medical University Hospital (Approval no. KMUHIRB-E(I)-20220312, KMUHIRB-F(I)-20220016, and KMUHIRB-F(I)-20180069). XTT cell viability assay Cells were plated into 96-well plates at the densities of 5×10 3 cells per well for HGF cells and 2×10 3 cells per well for DOK cells. For 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, the procedure followed a previous report [ 17 ]. Trans-well migration and invasion assays HGF cells, primary fibroblasts, and DOK cells were seeded in serum-free medium onto 24-well transwell inserts (8 µm pores; FALCON) at 1×10 4 cells and 5×10 4 cells/well, respectively. Migration and invasion abilities were assessed at 48 and 72 hours respectively, following a previous report [ 18 ]. Cell surface markers using flow cytometric Primary fibroblasts, HGF, and OECM-1 cells were harvested, washed with flow cytometer buffer, and fixed with 4% paraformaldehyde for 30 minutes. Subsequently, they were stained with antibodies targeting CD90 (Invitrogen, 11-0909-42), CD45 (Invitrogen, 11-0459-42), or CD326 (Invitrogen, 53-8326-42) for 30 minutes. Fluorescence intensity was analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter), followed by FlowJo software version 10 (Tree Star Inc.). Identification of ANE components using LC-MS Analysis of ANE components was conducted using a TSQ Quantum Ultra mass spectrometer (Thermo Scientific). A C18 column measuring 150 × 0.5 mm i.d., with 5 µm particle size (Agilent), was employed for component separation. The detailed procedure followed a previous report [ 17 ]. Cytokine array HGF cells were subjected to treatment with ANE at a concentration of 0.4 mg/mL for 72 hours. Subsequently, the conditioned medium was utilized for cytokine array analysis, employing Proteome Profiler Human XL Cytokine Array Kit (ARY002B, R&D Systems). Enzyme-linked immunosorbent assay (ELISA) To assess the cell secretome, HGF cells or primary fibroblasts were exposed to ANE for 72 hours and the conditioned medium was filtered for ELISA utilizing GM-CSF DuoSet® Immunoassay Development kit (ab17448, Abcam). Phospho-kinase array The Human Phospho-kinase antibody array (ARY003B, R&D Systems) was employed to explore potential signaling pathways underlying the effect of ANE treated HGF conditioned medium to DOK cells, with the detailed procedure following the manufacturer's instructions. Western blot The detailed procedure followed a previous report [ 17 ]. The primary antibodies used for Western blot included α-SMA (MA1-06110, Invitrogen), FAP (ab28244, Abcam), Vimentin (GTX100619, Genetex), Claudin-1 (#4933, Cell Signaling Technology), ZO-1 (#5406, Cell Signaling Technology), p-EGFR Tyr1086 (GTX100448, Genetex), EGFR (GTX100448, Genetex), and α-tubulin (GTX112141, Genetex) was used as an internal control. Immunohistochemistry For immunohistochemistry (IHC) analysis, slides underwent baking, de-waxing, and staining with avidin-biotin complexes following established protocols [ 19 ]. To quantify the staining - the immunoreactive score (IRS-score), the sum of the percentage of stained cells (0–4) multiplied by the staining intensity (0–3) - was employed to assess signal intensity. Scores were conducted independently by two experts under identical imaging conditions. Extracellular O 2 consumption assay and glycolysis assay HGF cells were seeded into 96-well plates with black walls and clear flat bottoms at a density of 2×10 4 cells per well and allowed to incubate overnight. For extracellular oxygen consumption assay and glycolysis assay, the detailed procedures followed a previous report [ 18 ]. Seahorse bioscience extracellular flux analyzer for oxygen consumption rate Seahorse XF Analyzers (Agilent) was applied for oxygen consumption rate (OCR) analyses. Cells were seeded in the 8 well Seahorse XF Cell Culture Microplates at density of 8×10 3 cells per well and cultured with 5% CO 2 at 37°C in a humidified incubator and the following procedure followed a previous report [ 18 ]. Hamster buccal pouch model Experiments involving animals were approved by the Institutional Animal Care and Utilization Committee of Kaohsiung Medical University, Kaohsiung, Taiwan (IACUC Approval no. 111219) and the study conformed to the ARRIVE Guidelines. According to 3Rs, the number of animals used in our experiments was minimized. The hamster buccal pouch model has been extensively utilized in vivo to investigate oral lesions, as demonstrated in our previous studies [ 20 – 22 ]. Six outbred male Syrian golden hamsters with an average weight of 100 g at the start of the experiments, were assigned into one of the two experimental groups: (1) a control group treated with double-distilled water (ddH 2 O), the solvent of ANE (n = 1), and (2) a treatment group receiving ANE (n = 5). Each treatment group was housed separately and cage number corresponded to group assignment. Briefly, the buccal pouch region of each hamster was painted daily with either ddH 2 O or ANE. At predetermined time pointes, the hamsters were euthanized by injecting Zoletil® (VIRBAC, French) before CO2 asphyxiation, and the buccal pouch tissues were collected, each tissue sample was evenly divided into four portions for subsequent analyses. Hematoxylin and Eosin (H&E) staining H & E staining was performed according to a reported protocol [ 18 ]. Immunofluorescence Multi-fluorescence immunohistochemistry (IF) was performed following previously described procedures [ 17 ]. The percentage of cells positively stained with multi-fluorescence was calculated as follows: (number of multi-fluorescent stained cells) / (number of all cells)] × 100%, derived from examination of five random fields. The primary antibodies used for patient tissue staining included α-SMA (ab32575, Abcam), GM-CSF (GTX31170, Genetex). The primary antibodies used for hamster tissue staining included α-SMA (MA1-0611, Invitrogen), GM-CSF (sc-32753, Santa Cruz), β-catenin (IR49-149, iReal) and ZO-1 (IR56-184, iReal). Statistical analysis Statistical analyses performed using Prism 9.5.0 software (GraphPad). The Student's t-test was used to compare two groups. One-way analysis of variance (ANOVA) with post-hoc Dunnett's test and Tukey’s test was used for multiple group comparisons. For in vitro studies, data are presented as the mean ± SD from three independent experiments. The results were considered statistically significant if the p value was less than 0.05. Results Elevated expression of α-SMA and FAP in fibroblasts during oral cancer progression Immunohistochemistry showed increased α-SMA and FAP expression in clinical specimens from oral pre-cancer to cancer patients. Patients with a history of betel quid chewing revealed progressively upregulated expression of α-SMA and FAP in both severe epithelial dysplasia (SED) and oral squamous cell carcinoma (SCC), in contrast to normal oral mucosa tissues (Fig. 1 A-B). To study the effect of areca nut extract (ANE) on fibroblasts, the cytotoxicity of ANE on HGF human gingival fibroblasts was assessed using XTT cell viability assay. At concentrations up to 0.4 mg/mL, ANE did not significantly affect cell viability after treatment for 48 hours (Fig. S1 A), similar to TGF-β treatment at 10 ng/mL (Fig. 1 C). Notably, LC-MS identified arecoline as the major ANE component (Fig. S1 B). ANE or arecoline treatment also enhanced cell migration ability in human gingival fibroblasts (HGF) (Fig. 1 D). Notably, ANE treatment led to higher expression of mesenchymal marker β-catenin but lower expression of epithelial marker ZO-1 in HGF cells (Fig. 1 E). Further study also showed that ANE increased cell migration ability in CD90-positive primary normal fibroblasts (NF), but not cancer associated fibroblasts (CAFs) (Fig. 1 F and Fig. S1 C-D). ANE increased GM-CSF secretion, mitochondrial metabolism and migration in HGF cells To explore the potential bioactive factors secreted by HGF cells following ANE treatment, a cytokine array was performed. GM-CSF exhibited the most notable increase among 105 proteins in ANE-treated HGF cells, compared to untreated cells (Fig. 2 A). This observation was further validated using ELISA kit (Fig. 2 B). Also, the increase of GM-CSF after ANE treatment was observed in primary fibroblasts (Fig. 2 C). To explore these in vitro findings in a clinical setting, immunofluorescent staining was conducted to examine the co-localization of α-SMA and GM-CSF. A significant increase in the α-SMA and GM-CSF co-localization was observed in tissue samples of severe epithelial dysplasia (SED) and squamous cell carcinoma (SCC) compared to the normal group (Fig. 2 D). Recent research has highlighted the significance of mitochondrial metabolism in fibroblast transformation [ 23 ]. Using CLARIOstar Plus and Seahorse XF Analyzer, ANE-treated HGF cells showed increased oxygen consumption rate (OCR) but decreased extracellular acidification rate (ECAR) (Fig. 3 A-C). Further transwell migration assay revealed that ANE-enhanced HGF migration was suppressed mitochondrial complexes I (metformin and rotenone) and III (antimycin A) inhibitors without affecting cell viability (Fig. 3 D and Fig. S2 A-D). Conditioned medium from ANE-treated HGF cells promoted migration, invasion, and mesenchymal phenotypes in DOK cells To explore the biological effects of ANE-treated HGF cells on DOK oral pre-cancer cells, conditioned medium (CM) from HGF cells was collected. CM treatment increased DOK cell migration and invasion (Fig. 4 A-B) and the expression of mesenchymal markers, Vimentin and Claudin-1, while the expression of epithelial marker ZO-1 was decreased (Fig. 4 C). Furthermore, morphologic change from epithelial to mesenchymal phenotype was noted in DOK cells after treatment with either ANE-treated HGF CM or GM-CSF recombinant protein (Fig. 4 D-E). ANE-induced GM-CSF expression in HGF cells promote EGFR activation and malignant transformation in DOK oral pre-cancer cells Phospho-kinase array was used to identify potential signaling pathways activated in dysplastic oral keratinocytes (DOK) by CM from ANE-treated HGF cells. Increased p-EGFR (Tyr1086) expression (Fig. 5 A) was noted, which was confirmed by Western blotting (Fig. 5 B). Similarly, increased p-EGFR (Tyr1086) expression in DOK cells was observed after treatment with CM from arecoline-treated HGF cells (Fig. 5 C) or with GM-CSF recombinant protein (Fig. 5 D). In addition, treatment of DOK cells with EGFR inhibitor Gefitinib led to a reduction in p-EGFR expression in DOK cells (Fig. S3B). Subsequent studies revealed that the GM-CSF-induced DOK migration and invasion abilities was mitigated when co-treated with Gefitinib (Fig. 5 E-F, Fig. S3 D-E) at concentrations that did not affect DOK cell viability (Fig. S3A). Immunohistochemical staining results demonstrated that p-EGFR expression were significantly higher in tissue samples from patients with severe epithelial dysplasia (SED) and squamous cell carcinoma (SCC) compared to p-EGFR expression in normal epithelial tissues of oral fibroma patients (Fig. 5 G). ANE-induced GM-CSF expression in HGF cells and EGFR activation in DOK cells were reproduced in hamster buccal pouch model A hamster buccal pouch model showed increased α-SMA + cells after 37 weeks of ANE application (Fig. 6 A). Consistent with the in vitro findings, double-fluorescence immunohistochemistry revealed higher α-SMA co-localization with GM-CSF and β-catenin, but lower with ZO-1, in the buccal tissues of the ANE-treated group (Fig. 6 B-D). Also, immunohistochemical staining result showed that p-EGFR expression was significantly higher in the buccal mucosa tissues of the ANE-treated group, compared to the untreated group (Fig. 6 E). Discussion Although there is accumulating evidence correlating areca nut consumption and oral cancer [ 24 ] and its effects on fibroblast transformation [ 16 ], the mechanisms underlying ANE-induced fibroblast transition and its contribution to oral pre-cancer malignant transformation remains underexplored. In this study, we conclude that ANE induces epithelial-mesenchymal transition and GM-CSF expression in fibroblasts to promote oral pre-cancer malignant transformation, which is illustrated in Fig. 7 . ANE promotes mitochondrial metabolism in HGF cells Mitochondrial respiration plays an important role in cell functioning [ 25 ]. ANE treatment enhanced oxygen consumption rate in HGF cells, including basal respiration, ATP production, respiration, and spare respiratory capacity (Fig. 3 ), suggesting ANE plays a dual maximal role in the promotion of oral pre-cancer malignant transformation, acting via both metabolic reprogramming in HGF and transition of microenvironment fibroblast to myofibroblast. Synergy between these pathways warrants further investigation, potentially allowing the identification of an upstream target that targets both pathways simultaneously. Combined with previous findings on tumor associated macrophages, ANE likely alters the tumor microenvironment via multiple cell types, enhancing epithelial-to-mesenchymal transition (EMT) in DOKs cells through multiple pathways including VCAM-1 and GM-CSF. To our knowledge, this is the first evidence showing that areca nut induces metabolic reprogramming towards mitochondrial biogenesis. Notably, a recent study demonstrated that adenosine drives anti-inflammatory effects in HGF cells through metabolic reprogramming towards mitochondrial biogenesis [ 26 ]. ANE-induced EMT and GM-CSF secretion in fibroblasts mediate oral pre-cancer progression This study explored EMT induction by ANE in oral pre-cancer progression. Arecoline, the major component of ANE, is able to induce EMT and enhance cancer development, and consequently, ANE-induced EMT is reported to be a major mechanism mediating malignant phenotypes that foster metastasis in cancer cells [ 27 , 28 ]. In agreement with previous reports, our study revealed that ANE induces EMT-like phenotype in HGF cells, characterized by morphology change and upregulation of Vimentin and Claudin-1 (Fig. 4 C). ANE can initiate various oncogenic pathways through cytokine secretion, including IL-8, IL-2 and GM-CSF [ 29 ]. GM-CSF, a key player in inflammation and EMT [ 30 ], was secreted by ANE-treated HGF cells, enhancing EMT, migration, and invasion abilities in DOK cells (Fig. 4 ), implicating GM-CSF as a crucial mediator in oral carcinogenesis. Conditioned medium from ANE-treated HGF cells and GM-CSF promote EGFR activation and malignant progression in DOK cells ANE activates various oncogenic pathways, including JNK, NF-κB, and MAPK, triggering initial inflammatory responses through growth factor and cytokine secretion [ 31 ]. In this study, ANE-treated HGF cells showed elevated GM-CSF levels (Fig. 2 A). GM-CSF is linked to tumor progression in various cancers: it can drive autocrine tumor growth in lung cancer [ 32 ], and is associated with metastasis and poor survival in breast cancer [ 33 ]. High GM-CSF expression is also observed in 56–93% of head and neck squamous cell carcinoma. [ 34 ] GM-CSF plays an essential role in head and neck cancer microenvironment [ 35 ], with elevated levels of GM-CSF, platelet-derived growth factor, and vascular endothelial growth factor correlating with invasion and poor prognosis [ 36 ]. Our phosphokinase array result (Fig. 5 A) and Western blot analyses (Figs. 5 B-D) showed p-EGFR upregulation in DOK cells treated with conditioned medium from ANE- or arecoline-treated HGF or GM-CSF recombinant protein. EGFR regulates cellular processes, including proliferation, migration and survival, and EGFR upregulation promotes cancer cell metastasis in a variety of cancer types. Additionally, EGFR activates GM-CSF promoter activity via c-Jun/TNF-α in keratinocytes [ 37 ]. Given GM-CSF’s role in promoting pre-cancer cell migration and invasion (Figs. 5 E-F), understanding the impact of GM-CSF on tumor progression and the activated signaling pathways is crucial for exploring novel therapeutic strategies for oral cancer. Areca nut extract selectively promoted cell migration in primary normal fibroblasts, not cancer-associated fibroblasts. Using primary normal fibroblasts (obtained from normal tissues at least 1cm away from oral cancer tissues) and cancer-associated fibroblasts (from oral cancer tissues), we found that ANE increased cell migration ability in normal fibroblasts but not cancer associated fibroblasts (Fig. 1 F and Figs. S1 C-D). As normal fibroblasts undergo EMT to become cancer-associated fibroblasts in tumors [ 38 ] and our data showed that ANE promoted EMT and migration activity in fibroblasts, this suggests that ANE selectively enhances migration-promoting activity in normal fibroblasts, not cancer-associated fibroblasts which have already gained EMT activity. Limitations and future work The mechanism of GM-CSF–mediated EGFR activation and enhanced migration/invasion in DOK cells remains unclear. While EGFR is a known target in oral cancer [ 39 ], its role in oral pre-cancer is underexplored and deserves further study. ANE alone did not induce lesions in the hamster model, but when combined with 0.5% DMBA, lesion frequency and p-EGFR expression increased [ 40 ] (Fig. S4). Nonetheless, cancer lesions occurred more frequent in DMBA + ANE group than DMBA alone group (Fig. S4A) and p-EGFR expression was relatively higher in DMBA + ANE group than DMBA alone group (Fig. S4B), consistent with in vitro data. Future studies using ANE with lower DMBA doses may clarify its independent carcinogenic effects. Investigating how different cell types in the tumor microenvironment drive DOK malignancy may identify shared upstream targets for oral cancer prevention. Conclusions These data add clarity to the accumulating evidence on the diverse role of ANE as a promotor of oral cancer in the tumor microenvironment. The current study demonstrates that areca nut exerts dual effects on fibroblasts, with metabolic reprogramming and tumor microenvironment changes - that result in epithelial-to-mesenchymal transition and GM-CSF secretion in fibroblasts which serves as a promoter in oral pre-cancer progression. Declarations Acknowledgements The authors thank the Center for Laboratory Animals in Kaohsiung Medical University for the animal care and Center for Research Resources and Development in Kaohsiung Medical University for providing the service of LC-MS. Author contributions Yen-Yun Wang: Contributed to conception, design, data acquisition and interpretation, drafted and critically revised the manuscript. Chang-Wei Su: Contributed to conception, data interpretation, and critically revised the manuscript. Chih-Huang Tseng: Contributed to conception, data analysis, and drafted manuscript. Pang-Yu Chen: Contributed to conception, design, data acquisition and analysis, and drafted manuscript. Hieu D.H. Nguyen: Contributed to conception, data interpretation, and drafted manuscript. Leong-Perng Chan: Contributed to conception, data acquisition, and critically revised the manuscript. Yuk-Kwan Chen: Contributed to conception, data acquisition and analysis, and drafted manuscript. Shih Sheng Jiang: Contributed to conception, data interpretation, and drafted manuscript. Steven Lo: Contributed to conception, and critically revised the manuscript. Shyng-Shiou F. Yuan: Contributed to conception, design, data analysis and interpretation, and drafted and critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work. Declaration of Interest statement The authors have no conflicts of interest to declare. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (Approval no. KMUHIRB-E(I)-20220312, KMUHIRB-F(I)-20220016, and KMUHIRB-F(I)-20180069). Patient informed consent was waived by the Institutional Review Board due to the retrospective nature of the study. All animal experiments were approved by the Institutional Animal Care and Utilization Committee of Kaohsiung Medical University, Kaohsiung, Taiwan (IACUC Approval no. 111219). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Funding This work was supported by grants from the National Science and Technology Council (NSTC 113-2314-B-037-045-, NSTC 112-2314-B-037-112-MY3) and the Center for Intelligent Drug Systems and Smart Biodevices (IDS 2 B) from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education, Taiwan. This work was also supported by grants from Kaohsiung Medical University Hospital (KMUH110-0R43, KMUH111-1R37, KMUH112-2R42, KMUH-DK(A)110001, KMUH-DK(A)112001, KMUH-DK(A)113001, KMUH-DK(B)114007-1, KMUH-DK(B)114007-2, KMU-TB114004, KMU-TB114009) and Kaohsiung Medical University (KMU-DK(A)111005, KMU-DK(A)112006, KMU-DK(A)113002, NYCUKMU-111-I002, NYCU-KMU-112-I005, NYCUKMU-113-I002, KMU-DK(B)114007-1), Taiwan. References H. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray, Cancer J. Clin. 71 , 209–249 (2021) A. Chamoli, A.S. Gosavi, U.P. Shirwadkar, K.V. Wangdale, S.K. Behera, N.K. 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Johnson, B. Burtness, C.R. Leemans, V.W.Y. Lui, J.E. Bauman, J.R. Grandis, Nat. Rev. Dis. Primers. 6 , 92 (2020). 10.1038/s41572-020-00224-3 Y.K. Chen, L.M. Lin, Expert Rev. Anticancer Ther. 10 , 1485–1496 (2010). 10.1586/era.10.108 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures.pptx uncroppedGelsandBlotsimages.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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6","display":"","copyAsset":false,"role":"figure","size":1863407,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7567949/v1/4596a4f618cc6523493ac7ba.png"},{"id":92745253,"identity":"83845a96-0164-4764-b630-38eb6bf64648","added_by":"auto","created_at":"2025-10-03 18:54:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":950407,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7567949/v1/47bbaad3e159c9fd8c47de8d.png"},{"id":93583692,"identity":"bb514ff2-11af-4451-b458-a5c579e5c8b9","added_by":"auto","created_at":"2025-10-15 10:54:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8342709,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7567949/v1/5f381ad1-9a78-44c4-b310-54a8fe4c6c10.pdf"},{"id":92745713,"identity":"80e20075-022b-45bc-a574-b97f4b1ff3ef","added_by":"auto","created_at":"2025-10-03 19:02:37","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10403776,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7567949/v1/bf6297dfd6c36e28812eaecd.pptx"},{"id":92745275,"identity":"a61ac98d-f42e-48c9-97f2-bb4211a8f5e9","added_by":"auto","created_at":"2025-10-03 18:54:39","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":50110349,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedGelsandBlotsimages.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7567949/v1/6bf59496efbbcea2e1bf5de4.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metabolic Reprogramming and GM-CSF Secretion in Areca Nut-Activated Fibroblast Drives Oral Precancer Progression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording to the projection from GLOBOCAN, oral cancer incidence will rise by 50% from 2020 to 2040 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Approximately 90% of them are classified as oral squamous cell carcinoma (OSCC) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In South and Southeast Asia, chewing of areca (betel) nut and betel quid is a OSCC risk factor [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The International Agency for Research on Cancer has concluded their carcinogenic effect in humans (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/news/item/07-08-2003-iarc-monographs-programme-finds-betel-quid-and-areca-nut-chewing-carcinogenic-to-humans\u003c/span\u003e\u003cspan address=\"https://www.who.int/news/item/07-08-2003-iarc-monographs-programme-finds-betel-quid-and-areca-nut-chewing-carcinogenic-to-humans\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with betel quid chewers facing a 28-fold higher oral cancer risk [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notably, before OSCC develops, these substances often induce various pre-cancer lesions in the oral cavity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn Taiwan, over 75% of OSCC patients chew betel quid [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], highlighting the urgent need for target strategies. Areca nut, the essential ingredient of betel quid, contains arecoline, arecaidine, guvacoline, and guvacine, with arecoline being the most abundant and predominant in saliva during and after chewing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Both areca nut extract and arecoline contribute to DNA damage and cytopathological changes in oral epithelial cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and mucosal fibroblasts [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, ANE also activates immune cells, particularly macrophages, promoting precancer-to-cancer transition [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOther than epithelial cells, the oral tissue microenvironment includes fibroblasts, immune cells, endothelial cells, pericytes, mesenchymal cells, and adipocytes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Understanding this microenvironment is important for preventing and treating oral pre-cancer and cancer. During oral cancer development, fibroblasts transform into myofibroblasts, also known as cancer-associated fibroblasts (CAFs), which interact with cancer cells, immune cells to drive tumor progression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Though poorly defined, CAFs exhibit an activated myofibroblast phenotype and support tumor growth and metastasis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the mechanisms of fibroblasts-to- myofibroblast transition in oral carcinogenesis remain largely unexplored.\u003c/p\u003e\u003cp\u003eAlthough areca nut has been linked to oral cancer [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and fibroblast transformation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], its role in driving fibroblasts-to- myofibroblasts transition and oral pre-cancer progression remains unclear. This study aims to determine the effect of ANE on fibroblast transformation and its impact on malignant progression of oral pre-cancer cells through integrated clinical, in vitro, and in vivo approaches, aiming to provide innovative and targeted topical therapeutic strategies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture and dissociation of primary fibroblasts\u003c/h2\u003e\u003cp\u003eThe human gingiva fibroblast cells (HGF) were acquired from the American Type Culture Collection (ATCC) and were maintained in Fibroblast Basal Medium (ATCC) with fibroblast growth low serum (ATCC), whereas the oral premalignant cell line DOK was maintained in DMEM/F12 medium (Thermo Fisher Scientific).\u003c/p\u003e\u003cp\u003ePrimary fibroblasts were isolated from normal tissues and tumor tissues within an hour post-surgical. Tissues were washed in phosphate buffer saline (PBS), minced into 2\u0026ndash;4 mm pieces, and digested using a tumor dissociation kit in a gentle MACS\u0026trade; C-Tube (Miltenyi) for 60 minutes at 37\u0026deg;C in a gentle MACS Octo dissociator. Digests were filtered through 100-\u0026micro;m sterile cell strainers, followed by centrifugation at 300 \u0026times; g for 10 minutes, and resuspended in complete Dulbecco's modified Eagle Medium F12 (DMEM F12).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePatient samples\u003c/h3\u003e\n\u003cp\u003eHuman tissue specimens were obtained from patients undergoing treatment at Kaohsiung Medical University Hospital (KMUH), Taiwan. This study received approval from the Institutional Review Board of Kaohsiung Medical University Hospital (Approval no. KMUHIRB-E(I)-20220312, KMUHIRB-F(I)-20220016, and KMUHIRB-F(I)-20180069).\u003c/p\u003e\n\u003ch3\u003eXTT cell viability assay\u003c/h3\u003e\n\u003cp\u003eCells were plated into 96-well plates at the densities of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well for HGF cells and 2\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well for DOK cells. For 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, the procedure followed a previous report [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eTrans-well migration and invasion assays\u003c/h3\u003e\n\u003cp\u003eHGF cells, primary fibroblasts, and DOK cells were seeded in serum-free medium onto 24-well transwell inserts (8 \u0026micro;m pores; FALCON) at 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells and 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well, respectively. Migration and invasion abilities were assessed at 48 and 72 hours respectively, following a previous report [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCell surface markers using flow cytometric\u003c/h3\u003e\n\u003cp\u003ePrimary fibroblasts, HGF, and OECM-1 cells were harvested, washed with flow cytometer buffer, and fixed with 4% paraformaldehyde for 30 minutes. Subsequently, they were stained with antibodies targeting CD90 (Invitrogen, 11-0909-42), CD45 (Invitrogen, 11-0459-42), or CD326 (Invitrogen, 53-8326-42) for 30 minutes. Fluorescence intensity was analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter), followed by FlowJo software version 10 (Tree Star Inc.).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of ANE components using LC-MS\u003c/h2\u003e\u003cp\u003eAnalysis of ANE components was conducted using a TSQ Quantum Ultra mass spectrometer (Thermo Scientific). A C18 column measuring 150 \u0026times; 0.5 mm i.d., with 5 \u0026micro;m particle size (Agilent), was employed for component separation. The detailed procedure followed a previous report [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCytokine array\u003c/h3\u003e\n\u003cp\u003eHGF cells were subjected to treatment with ANE at a concentration of 0.4 mg/mL for 72 hours. Subsequently, the conditioned medium was utilized for cytokine array analysis, employing Proteome Profiler Human XL Cytokine Array Kit (ARY002B, R\u0026amp;D Systems).\u003c/p\u003e\n\u003ch3\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h3\u003e\n\u003cp\u003eTo assess the cell secretome, HGF cells or primary fibroblasts were exposed to ANE for 72 hours and the conditioned medium was filtered for ELISA utilizing GM-CSF DuoSet\u0026reg; Immunoassay Development kit (ab17448, Abcam).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePhospho-kinase array\u003c/h2\u003e\u003cp\u003eThe Human Phospho-kinase antibody array (ARY003B, R\u0026amp;D Systems) was employed to explore potential signaling pathways underlying the effect of ANE treated HGF conditioned medium to DOK cells, with the detailed procedure following the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot\u003c/h2\u003e\u003cp\u003eThe detailed procedure followed a previous report [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The primary antibodies used for Western blot included α-SMA (MA1-06110, Invitrogen), FAP (ab28244, Abcam), Vimentin (GTX100619, Genetex), Claudin-1 (#4933, Cell Signaling Technology), ZO-1 (#5406, Cell Signaling Technology), p-EGFR Tyr1086 (GTX100448, Genetex), EGFR (GTX100448, Genetex), and α-tubulin (GTX112141, Genetex) was used as an internal control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eFor immunohistochemistry (IHC) analysis, slides underwent baking, de-waxing, and staining with avidin-biotin complexes following established protocols [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To quantify the staining - the immunoreactive score (IRS-score), the sum of the percentage of stained cells (0\u0026ndash;4) multiplied by the staining intensity (0\u0026ndash;3) - was employed to assess signal intensity. Scores were conducted independently by two experts under identical imaging conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eExtracellular O\u003csub\u003e2\u003c/sub\u003e consumption assay and glycolysis assay\u003c/h2\u003e\u003cp\u003eHGF cells were seeded into 96-well plates with black walls and clear flat bottoms at a density of 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well and allowed to incubate overnight. For extracellular oxygen consumption assay and glycolysis assay, the detailed procedures followed a previous report [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eSeahorse bioscience extracellular flux analyzer for oxygen consumption rate\u003c/h2\u003e\u003cp\u003eSeahorse XF Analyzers (Agilent) was applied for oxygen consumption rate (OCR) analyses. Cells were seeded in the 8 well Seahorse XF Cell Culture Microplates at density of 8\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well and cultured with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C in a humidified incubator and the following procedure followed a previous report [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eHamster buccal pouch model\u003c/h2\u003e\u003cp\u003e Experiments involving animals were approved by the Institutional Animal Care and Utilization Committee of Kaohsiung Medical University, Kaohsiung, Taiwan (IACUC Approval no. 111219) and the study conformed to the ARRIVE Guidelines. According to 3Rs, the number of animals used in our experiments was minimized. The hamster buccal pouch model has been extensively utilized in vivo to investigate oral lesions, as demonstrated in our previous studies [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Six outbred male Syrian golden hamsters with an average weight of 100 g at the start of the experiments, were assigned into one of the two experimental groups: (1) a control group treated with double-distilled water (ddH\u003csub\u003e2\u003c/sub\u003eO), the solvent of ANE (n\u0026thinsp;=\u0026thinsp;1), and (2) a treatment group receiving ANE (n\u0026thinsp;=\u0026thinsp;5). Each treatment group was housed separately and cage number corresponded to group assignment. Briefly, the buccal pouch region of each hamster was painted daily with either ddH\u003csub\u003e2\u003c/sub\u003eO or ANE. At predetermined time pointes, the hamsters were euthanized by injecting Zoletil\u0026reg; (VIRBAC, French) before CO2 asphyxiation, and the buccal pouch tissues were collected, each tissue sample was evenly divided into four portions for subsequent analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eHematoxylin and Eosin (H\u0026amp;E) staining\u003c/h2\u003e\u003cp\u003eH \u0026amp; E staining was performed according to a reported protocol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eMulti-fluorescence immunohistochemistry (IF) was performed following previously described procedures [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The percentage of cells positively stained with multi-fluorescence was calculated as follows: (number of multi-fluorescent stained cells) / (number of all cells)] \u0026times; 100%, derived from examination of five random fields. The primary antibodies used for patient tissue staining included α-SMA (ab32575, Abcam), GM-CSF (GTX31170, Genetex). The primary antibodies used for hamster tissue staining included α-SMA (MA1-0611, Invitrogen), GM-CSF (sc-32753, Santa Cruz), β-catenin (IR49-149, iReal) and ZO-1 (IR56-184, iReal).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses performed using Prism 9.5.0 software (GraphPad). The Student's t-test was used to compare two groups. One-way analysis of variance (ANOVA) with post-hoc Dunnett's test and Tukey\u0026rsquo;s test was used for multiple group comparisons. For in vitro studies, data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent experiments. The results were considered statistically significant if the \u003cem\u003ep\u003c/em\u003e value was less than 0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eElevated expression of α-SMA and FAP in fibroblasts during oral cancer progression\u003c/h2\u003e\u003cp\u003eImmunohistochemistry showed increased α-SMA and FAP expression in clinical specimens from oral pre-cancer to cancer patients. Patients with a history of betel quid chewing revealed progressively upregulated expression of α-SMA and FAP in both severe epithelial dysplasia (SED) and oral squamous cell carcinoma (SCC), in contrast to normal oral mucosa tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). To study the effect of areca nut extract (ANE) on fibroblasts, the cytotoxicity of ANE on HGF human gingival fibroblasts was assessed using XTT cell viability assay. At concentrations up to 0.4 mg/mL, ANE did not significantly affect cell viability after treatment for 48 hours (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), similar to TGF-β treatment at 10 ng/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Notably, LC-MS identified arecoline as the major ANE component (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). ANE or arecoline treatment also enhanced cell migration ability in human gingival fibroblasts (HGF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Notably, ANE treatment led to higher expression of mesenchymal marker β-catenin but lower expression of epithelial marker ZO-1 in HGF cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Further study also showed that ANE increased cell migration ability in CD90-positive primary normal fibroblasts (NF), but not cancer associated fibroblasts (CAFs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e C-D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eANE increased GM-CSF secretion, mitochondrial metabolism and migration in HGF cells\u003c/h2\u003e\u003cp\u003eTo explore the potential bioactive factors secreted by HGF cells following ANE treatment, a cytokine array was performed. GM-CSF exhibited the most notable increase among 105 proteins in ANE-treated HGF cells, compared to untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This observation was further validated using ELISA kit (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Also, the increase of GM-CSF after ANE treatment was observed in primary fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To explore these in vitro findings in a clinical setting, immunofluorescent staining was conducted to examine the co-localization of α-SMA and GM-CSF. A significant increase in the α-SMA and GM-CSF co-localization was observed in tissue samples of severe epithelial dysplasia (SED) and squamous cell carcinoma (SCC) compared to the normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRecent research has highlighted the significance of mitochondrial metabolism in fibroblast transformation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Using CLARIOstar Plus and Seahorse XF Analyzer, ANE-treated HGF cells showed increased oxygen consumption rate (OCR) but decreased extracellular acidification rate (ECAR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Further transwell migration assay revealed that ANE-enhanced HGF migration was suppressed mitochondrial complexes I (metformin and rotenone) and III (antimycin A) inhibitors without affecting cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e A-D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eConditioned medium from ANE-treated HGF cells promoted migration, invasion, and mesenchymal phenotypes in DOK cells\u003c/h2\u003e\u003cp\u003eTo explore the biological effects of ANE-treated HGF cells on DOK oral pre-cancer cells, conditioned medium (CM) from HGF cells was collected. CM treatment increased DOK cell migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B) and the expression of mesenchymal markers, Vimentin and Claudin-1, while the expression of epithelial marker ZO-1 was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, morphologic change from epithelial to mesenchymal phenotype was noted in DOK cells after treatment with either ANE-treated HGF CM or GM-CSF recombinant protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eANE-induced GM-CSF expression in HGF cells promote EGFR activation and malignant transformation in DOK oral pre-cancer cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePhospho-kinase array was used to identify potential signaling pathways activated in dysplastic oral keratinocytes (DOK) by CM from ANE-treated HGF cells. Increased p-EGFR (Tyr1086) expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) was noted, which was confirmed by Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Similarly, increased p-EGFR (Tyr1086) expression in DOK cells was observed after treatment with CM from arecoline-treated HGF cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) or with GM-CSF recombinant protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In addition, treatment of DOK cells with EGFR inhibitor Gefitinib led to a reduction in p-EGFR expression in DOK cells (Fig. S3B). Subsequent studies revealed that the GM-CSF-induced DOK migration and invasion abilities was mitigated when co-treated with Gefitinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F, Fig. S3 D-E) at concentrations that did not affect DOK cell viability (Fig. S3A). Immunohistochemical staining results demonstrated that p-EGFR expression were significantly higher in tissue samples from patients with severe epithelial dysplasia (SED) and squamous cell carcinoma (SCC) compared to p-EGFR expression in normal epithelial tissues of oral fibroma patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eANE-induced GM-CSF expression in HGF cells and EGFR activation in DOK cells were reproduced in hamster buccal pouch model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA hamster buccal pouch model showed increased α-SMA\u003csup\u003e+\u003c/sup\u003e cells after 37 weeks of ANE application (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Consistent with the in vitro findings, double-fluorescence immunohistochemistry revealed higher α-SMA co-localization with GM-CSF and β-catenin, but lower with ZO-1, in the buccal tissues of the ANE-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). Also, immunohistochemical staining result showed that p-EGFR expression was significantly higher in the buccal mucosa tissues of the ANE-treated group, compared to the untreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough there is accumulating evidence correlating areca nut consumption and oral cancer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and its effects on fibroblast transformation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the mechanisms underlying ANE-induced fibroblast transition and its contribution to oral pre-cancer malignant transformation remains underexplored. In this study, we conclude that ANE induces epithelial-mesenchymal transition and GM-CSF expression in fibroblasts to promote oral pre-cancer malignant transformation, which is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003eANE promotes mitochondrial metabolism in HGF cells\u003c/h2\u003e\u003cp\u003eMitochondrial respiration plays an important role in cell functioning [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. ANE treatment enhanced oxygen consumption rate in HGF cells, including basal respiration, ATP production, respiration, and spare respiratory capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting ANE plays a dual maximal role in the promotion of oral pre-cancer malignant transformation, acting via both metabolic reprogramming in HGF and transition of microenvironment fibroblast to myofibroblast. Synergy between these pathways warrants further investigation, potentially allowing the identification of an upstream target that targets both pathways simultaneously. Combined with previous findings on tumor associated macrophages, ANE likely alters the tumor microenvironment via multiple cell types, enhancing epithelial-to-mesenchymal transition (EMT) in DOKs cells through multiple pathways including VCAM-1 and GM-CSF. To our knowledge, this is the first evidence showing that areca nut induces metabolic reprogramming towards mitochondrial biogenesis. Notably, a recent study demonstrated that adenosine drives anti-inflammatory effects in HGF cells through metabolic reprogramming towards mitochondrial biogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003eANE-induced EMT and GM-CSF secretion in fibroblasts mediate oral pre-cancer progression\u003c/h2\u003e\u003cp\u003eThis study explored EMT induction by ANE in oral pre-cancer progression. Arecoline, the major component of ANE, is able to induce EMT and enhance cancer development, and consequently, ANE-induced EMT is reported to be a major mechanism mediating malignant phenotypes that foster metastasis in cancer cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In agreement with previous reports, our study revealed that ANE induces EMT-like phenotype in HGF cells, characterized by morphology change and upregulation of Vimentin and Claudin-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). ANE can initiate various oncogenic pathways through cytokine secretion, including IL-8, IL-2 and GM-CSF [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. GM-CSF, a key player in inflammation and EMT [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], was secreted by ANE-treated HGF cells, enhancing EMT, migration, and invasion abilities in DOK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), implicating GM-CSF as a crucial mediator in oral carcinogenesis.\u003c/p\u003e\u003cp\u003e\u003cem\u003eConditioned medium from ANE-treated HGF cells and GM-CSF promote EGFR activation and malignant progression in DOK cells\u003c/em\u003e\u003c/p\u003e\u003cp\u003eANE activates various oncogenic pathways, including JNK, NF-κB, and MAPK, triggering initial inflammatory responses through growth factor and cytokine secretion [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, ANE-treated HGF cells showed elevated GM-CSF levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). GM-CSF is linked to tumor progression in various cancers: it can drive autocrine tumor growth in lung cancer [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and is associated with metastasis and poor survival in breast cancer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. High GM-CSF expression is also observed in 56\u0026ndash;93% of head and neck squamous cell carcinoma. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eGM-CSF plays an essential role in head and neck cancer microenvironment [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], with elevated levels of GM-CSF, platelet-derived growth factor, and vascular endothelial growth factor correlating with invasion and poor prognosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our phosphokinase array result (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and Western blot analyses (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D) showed p-EGFR upregulation in DOK cells treated with conditioned medium from ANE- or arecoline-treated HGF or GM-CSF recombinant protein. EGFR regulates cellular processes, including proliferation, migration and survival, and EGFR upregulation promotes cancer cell metastasis in a variety of cancer types. Additionally, EGFR activates GM-CSF promoter activity via c-Jun/TNF-α in keratinocytes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Given GM-CSF\u0026rsquo;s role in promoting pre-cancer cell migration and invasion (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F), understanding the impact of GM-CSF on tumor progression and the activated signaling pathways is crucial for exploring novel therapeutic strategies for oral cancer.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAreca nut extract selectively promoted cell migration in primary normal fibroblasts, not cancer-associated fibroblasts.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eUsing primary normal fibroblasts (obtained from normal tissues at least 1cm away from oral cancer tissues) and cancer-associated fibroblasts (from oral cancer tissues), we found that ANE increased cell migration ability in normal fibroblasts but not cancer associated fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and Figs. S1 C-D). As normal fibroblasts undergo EMT to become cancer-associated fibroblasts in tumors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and our data showed that ANE promoted EMT and migration activity in fibroblasts, this suggests that ANE selectively enhances migration-promoting activity in normal fibroblasts, not cancer-associated fibroblasts which have already gained EMT activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003eLimitations and future work\u003c/h2\u003e\u003cp\u003eThe mechanism of GM-CSF\u0026ndash;mediated EGFR activation and enhanced migration/invasion in DOK cells remains unclear. While EGFR is a known target in oral cancer [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], its role in oral pre-cancer is underexplored and deserves further study. ANE alone did not induce lesions in the hamster model, but when combined with 0.5% DMBA, lesion frequency and p-EGFR expression increased [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] (Fig. S4). Nonetheless, cancer lesions occurred more frequent in DMBA\u0026thinsp;+\u0026thinsp;ANE group than DMBA alone group (Fig. S4A) and p-EGFR expression was relatively higher in DMBA\u0026thinsp;+\u0026thinsp;ANE group than DMBA alone group (Fig. S4B), consistent with in vitro data. Future studies using ANE with lower DMBA doses may clarify its independent carcinogenic effects. Investigating how different cell types in the tumor microenvironment drive DOK malignancy may identify shared upstream targets for oral cancer prevention.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThese data add clarity to the accumulating evidence on the diverse role of ANE as a promotor of oral cancer in the tumor microenvironment. The current study demonstrates that areca nut exerts dual effects on fibroblasts, with metabolic reprogramming and tumor microenvironment changes - that result in epithelial-to-mesenchymal transition and GM-CSF secretion in fibroblasts which serves as a promoter in oral pre-cancer progression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Center for Laboratory Animals in Kaohsiung Medical University for the animal care and Center for Research Resources and Development in Kaohsiung Medical University for providing the service of LC-MS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYen-Yun Wang: Contributed to conception, design, data acquisition and interpretation, drafted and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003eChang-Wei Su: Contributed to conception, data interpretation, and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003eChih-Huang Tseng: \u0026nbsp;Contributed to conception, data analysis, and drafted manuscript.\u003c/p\u003e\n\u003cp\u003ePang-Yu Chen: Contributed to conception, design, data acquisition and analysis, and drafted manuscript.\u003c/p\u003e\n\u003cp\u003eHieu D.H. Nguyen: Contributed to conception, data interpretation, and drafted manuscript.\u003c/p\u003e\n\u003cp\u003eLeong-Perng Chan: Contributed to conception, data acquisition, and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003eYuk-Kwan Chen: Contributed to conception, data acquisition and analysis, and drafted manuscript.\u003c/p\u003e\n\u003cp\u003eShih Sheng Jiang: Contributed to conception, data interpretation, and drafted manuscript.\u003c/p\u003e\n\u003cp\u003eSteven Lo: Contributed to conception, and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003eShyng-Shiou F. Yuan: Contributed to conception, design, data analysis and interpretation, and drafted and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors gave their final approval and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (Approval no. KMUHIRB-E(I)-20220312, KMUHIRB-F(I)-20220016, and KMUHIRB-F(I)-20180069). Patient informed consent was waived by the Institutional Review Board due to the retrospective nature of the study. All animal experiments were approved by the Institutional Animal Care and Utilization Committee of Kaohsiung Medical University, Kaohsiung, Taiwan (IACUC Approval no. 111219).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Science and Technology Council (NSTC 113-2314-B-037-045-, NSTC 112-2314-B-037-112-MY3) and the Center for Intelligent Drug Systems and Smart Biodevices (IDS\u003csup\u003e2\u003c/sup\u003eB) from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education, Taiwan. This work was also supported by grants from Kaohsiung Medical University Hospital (KMUH110-0R43, KMUH111-1R37, KMUH112-2R42, KMUH-DK(A)110001, KMUH-DK(A)112001, KMUH-DK(A)113001, KMUH-DK(B)114007-1, KMUH-DK(B)114007-2, KMU-TB114004,\u0026nbsp;KMU-TB114009) and Kaohsiung Medical University (KMU-DK(A)111005, KMU-DK(A)112006, KMU-DK(A)113002, NYCUKMU-111-I002, NYCU-KMU-112-I005, NYCUKMU-113-I002,\u0026nbsp;KMU-DK(B)114007-1), Taiwan.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eH. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray, Cancer J. Clin. \u003cb\u003e71\u003c/b\u003e, 209\u0026ndash;249 (2021)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eA. Chamoli, A.S. Gosavi, U.P. Shirwadkar, K.V. Wangdale, S.K. Behera, N.K. Kurrey, K. Kalia, A. 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Yet, its influence on the oral tissue microenvironment is not well elucidated. This research aimed to elucidate the effects of areca nut extract (ANE) on oral carcinogenesis by modulating fibroblast behavior within the microenvironment. In this study, cell viability assay, transwell migration and invasion assays, and flow cytometry were employed to study cell behaviors. Cytokine array, ELISA, phospho-kinase array, Western blot analysis, extracellular O2 consumption, and glycolysis assays were conducted to assess cellular functionalities. In vivo experiments, complemented by immunohistochemical and immunofluorescent staining of oral lesions, were performed to corroborate in vitro observations. Our results showed that upregulation of α-SMA and FAP was observed in fibroblasts within oral pre-cancer and cancer lesions. ANE enhanced mitochondrial metabolism in fibroblasts and induced their transition into myofibroblasts. Additionally, ANE triggered epithelial-to-mesenchymal transition (EMT) and stimulated GM-CSF secretion in fibroblasts, thereby advancing the progression of oral pre-cancer. The conditioned medium from ANE-treated human gingival fibroblasts, along with recombinant GM-CSF, elevated EGFR phosphorylation and facilitated oncogenic transformation in DOK dysplastic oral keratinocytes. Moreover, in a hamster model, daily application of ANE on buccal pouches for 37 weeks replicated the in vitro findings for ANE-induced GM-CSF expression in fibroblasts and EGFR phosphorylation in mucosa epithelial cells. In conclusion, this investigation provides in vitro and in vivo evidence suggesting that ANE promotes EMT and GM-CSF secretion in fibroblasts, which activates EGFR and the malignant transformation of oral pre-cancer cells.\u003c/p\u003e","manuscriptTitle":"Metabolic Reprogramming and GM-CSF Secretion in Areca Nut-Activated Fibroblast Drives Oral Precancer Progression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 18:54:32","doi":"10.21203/rs.3.rs-7567949/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9bae6e3-f0fe-4184-91f0-91c90bf29b8a","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-15T10:53:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-03 18:54:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7567949","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7567949","identity":"rs-7567949","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}