Tumor-derived Extracellular Vesicles-mediated Oxidative Stress Transfer Activates Glycometabolic Reprogramming of CAFs

Tumor-derived Extracellular Vesicles-mediated Oxidative Stress Transfer Activates Glycometabolic Reprogramming of CAFs | 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 Tumor-derived Extracellular Vesicles-mediated Oxidative Stress Transfer Activates Glycometabolic Reprogramming of CAFs Erhui Jiang, Xiang Li, Xinyu Dou, Yuming Xu, Zhe Shao, Zhengjun Shang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5432071/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 Background Stressed by the adverse conditions of the tumor microenvironment (TME), hyperproliferation of tumor cells is accompanied by high production of reactive oxygen species (ROS). Tumor-derived extracellular vesicles (TEVs) exert a variety of biological functions in the interaction between the tumor cells and cancer-associated fibroblasts (CAFs). However, the intercellular transmission of ROS and its role in tumor-stroma communication remain unclear. Method The human gingival fibroblasts (HGFs), paracancerous normal fibroblasts (PNFs) and CAFs were isolated from the healthy gingival tissues of volunteers and six patients with OSCC. HGFs were treated with CAL27/SCC25 TEVs. In vitro , we assessed the level of autophagy and glycometabolism in PNFs/CAFs and HGFs/TEVs-treated HGFs by immunofluorescence and Western blot. Blockage or activation of autophagy was employed to investigate its effects on glycometabolism. Flow cytometry was used to detect whether TEVs play a crucial role in inducing fibroblast autophagy and glycolysis through ROS transfer. In vivo , xenograft models were established to validated the effect of TEVs. Results CAFs exhibited higher level in autophagy compared with PNFs. Treatment with autophagy inhibitors diminished autophagy-dependent glycometabolic reprogramming induced by TEVs, whereas activation of autophagy enhanced glycolysis in CAFs. Furthermore, ROS transferred by TEVs was confirmed to drive glycometabolic reprogramming through both autophagy-dependent mechanisms and the HIF-1α/PFKFB3 axis. In vivo , TEVs consistently promoted autophagy and glycometabolic reprogramming. Conclusion TEVs-induced intercellular transmission of ROS and the regulatory role of ROS-mediated autophagy in the glycometabolic reprogramming of CAFs, providing a novel rationale for the oxidative stress transfer model in tumor-stroma crosstalk. Tumor-derived extracellular vesicles Autophagy Glycometabolic reprogramming Oxidative stress CAFs OSCC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Despite significant improvements in diagnosis and therapy, the intractable TME is still a determinant of cancer cell behavior and disease progression[ 1 ]. As one of the most abundant stromal components in the TME, CAFs, an activated form of stromal fibroblasts, interplay with tumor cells to establish multiple and complex crosstalk mechanisms[ 2 , 3 ]. However, the TME for cells within the nascent tumor is harsh and imposes different types of stress on cells[ 4 ]. Malignant cells have developed adaptive mechanisms to survive under the extreme conditions of the TME such as restricted oxygen supply and nutrient deprivation[ 5 ]. Confronted with adverse conditions, hyperproliferation of tumor cells is accompanied by high ROS production[ 6 ]. ROS acting as a double-edged sword, influencing cancer evolution in apparently contradictory ways, either initiating/stimulating tumorigenesis[ 7 ] and supporting transformation/proliferation[ 8 ] of cancer cells or causing cell death[ 9 ]. Nonetheless, uncontrolled ROS generation is still recognized to damage fundamental cellular components, including nucleic acids, proteins, and lipids, which can lead to cell senescence, degeneration, or fatal lesions[ 10 ]. To maintain aberrant redox homeostasis, tumor cells thrive to adapt oxidative stress in various ways and transfer this oxidative burden away. Tumor-derived EVs (generally referred as TEVs) mediate the interaction and communication between tumor and stroma[ 11 ]. They exert various functions in intercellular communication by transferring biological cargo to recipient cells locally or at specific distant sites via circulation[ 12 ]. Recent studies showed that TEVs not only play a crucial role in CAFs’ activation [ 13 ] and glycometabolic reprogramming[ 14 ], but also help tumor cells transfer harmful byproducts and unnecessary organelles[ 15 ], thereby alleviating tumor’s burden. This transfer mechanism may be a strategy for tumor cells to maintain survival in a harsh microenvironment. However, it is unclear whether TEVs, as mediators of intercellular delivery[ 16 ], can transfer ROS to surrounding CAFs to mitigate tumor oxidative stress. Autophagy, an intracellular lysosome-dependent degradation system ubiquitous in eukaryotic cells, degrades and engulfs superfluous or damaged organelles and proteins in response to adverse conditions[ 17 , 18 ]. Recently, autophagy in stromal cells has been brought to the forefront. Jacob New et al. highlighted the important role for secretory autophagy in the supporting stromal CAFs, which enhances HNSCC progression[ 19 ]. In the non-contact co-culture system in vitro , human colorectal fibroblasts positively influenced the metabolism of colorectal cancer cells through its autophagy enhancement[ 20 ]. However, the molecular insight, by which the autophagy of CAFs is regulated, needs further study. Metabolic alterations are the classic theme of concern after autophagy occurs. The rapid progression of malignancy tumor often necessitates metabolic rewiring to meet the demands to forefront nutrient and oxygen deprivation, as well as increased anabolic requests to match high proliferation rate[ 21 ]. CAFs activated by epithelial tumor cells transform into a metabolic factory that produces energy-rich metabolites. The glycometabolic reprogramming in CAFs, driven by the host-parasite interaction, is referred as the “reverse Warburg effect”[ 22 ]. Our previous research has illustrated that primary CAFs derived from OSCC patients undergo PKFKFB3-driven glycometabolic reprogramming, characterized by an enhancement in aerobic glycolysis with a decrease in OXPHOS[ 23 ]. Additionally, TEVs-activated CAFs supply lactate to tumor cells by establishing glycometabolic crosstalk loop MCT1/MCT4[ 14 ]. During autophagy, the contents encapsulated by autophagosomes are degraded by specific hydrolases to produce lipids, amino acids, sugars, and other degradation products, which are catabolized through glycolysis, the citric acid cycle, β-oxidation, and oxidative phosphorylation to generate ATP and macromolecular precursors[ 24 ]. However, it remains unclear whether the glycometabolic reprogramming in CAFs is induced by TEVs-mediated enhancement of autophagy. As the sixth most common cancer worldwide[ 25 ], OSCC is an aggressive disease characterized by the intense fibrotic stromal response, with the tumor-stroma ratio determining the prognosis[ 26 ]. In this study, we originally propose the TEVs-mediated oxidative stress transfer model. To elucidate the role and mechanism of TEVs in tumor and stroma communication, TEVs were obtained from two OSCC cell lines (SCC25 and CAL27), and fibroblasts were isolated from healthy gingival tissues of volunteers and OSCC patients, respectively. Our results demonstrated that CAFs of OSCC exhibited high level of LC3B and TEVs enhanced autophagy in normal fibroblasts. Blockade of autophagy inhibited the glycometabolic reprogramming, whereas activation of autophagy promoted the glycometabolic reprogramming in CAFs. Furthermore, the transmission of ROS in TEVs played a crucial role in triggering autophagy and glycometabolic reprogramming in HGFs. In vivo , we validated that TEVs promoted autophagy and glycometabolic reprogramming in tumor stroma. In conclusion, our experiments focus on TEVs as mediators in autophagy-dependent glycometabolic reprogramming and propose a novel rationale for the “stress transfer model”. Materials and Methods Cell culture OSCC cell lines CAL27 and SCC25 were purchased from China Center for Type Culture Collection (Shanghai, China). Short tandem repeat was performed routinely on the cell line to confirm its authenticity, and Mycoplasma was routinely tested. SCC25 cells were cultivated in DMEM/F-12, and CAL27 cells were cultivated in DEME/HIGH GLUCOSE (Hyclone, UT, USA), both containing 10% FBS (Gibco, Australia). Isolation of TEVs The TEVs were isolation from OSCC cell (CAL27 and SCC25) supernatant, as previously reported by Jiang et al[14]. Isolation and culture of fibroblasts The human gingival fibroblasts (HGFs), PNFs and CAFs were isolated from the healthy gingival tissues of volunteers and 6 patients with OSCC, respectively, according to our previous study[14]. All procedures were in accordance with the Ethics Committee of School and Hospital of Stomatology at Wuhan University (2022LUNSHENZIA05). Transmission electron microscopy Transmission electron microscopy (TEM) was conducted as our previous study showed to observe TEVs[14]. TEM analysis was also conducted to observe the morphology and number of the autophagosome in the TEVs treated HGFs. Western blot Cells were lysed with mammalian protein extraction reagent (MPER) (Thermo Fisher Scientific) with protease inhibitor and phosphatase inhibitor (MilliporeSigma). Subsequently, a bicinchoninic acid protein assay was used to test the quantity of each sample. Afterward, the protein solutions were added with loading buffer (5x) and heated for 10 min at 95°C. Next, equal amounts of the protein samples (15 mg) were subjected to 8–12% SDS-PAGE (60 V, 30 min; 110 V, 60–70 min), and electrophoretically transferred to polyvinylidene fluoride membranes (MilliporeSigma) in the sodium dodecyl sulfate–electroblot buffer (25mMTris-Cl, 192mMglycine, 20%methanol, pH8.3) (200mA, 100 min). After blocking with 5% nonfat milk in Tris-buffered saline at room temperature for 90 min, the membranes were incubated with primary antibodies overnight at 4°C. Subsequently, the bound antibodies were tested using horseradish peroxidase–conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine). The antibodies were as follows: LC3B (CST, 83506), P62(Proteintech, 18420-1-AP), MCT4 (Proteintech, 22787-1-AP), GLUT1 (Proteintech, 21829-1-AP), PFKFB3 (Abcam, ab181861), PDK1 (CST, C47H1), PKM2 (Proteintech, 15822-1-AP), β-actin (CST, 4970), HIF-1α (Proteintech, 20960-1-AP), ATP1A1 (Proteintech, 55187-1-AP). Cell transfection The mRFP-GFP-LC3B adenovirus was purchased from GeneChem (Shanghai, China). Cells were transfected according to the manufacturer’s instruction. MDC staining The HGFs were seeded into Forty-eight-well plates at a density of 2x10 4 cell/well and treated with TEVs for 48 h. Then, HGFs were incubated with 0.05 mM monodansylcadaverine (MDC) (Promega, USA) at 37°C for 15 min, and then washed three times in PBS. Analyses were performed with a fluorescent microscope (Biozero BZ-8000, Keyence, Osaka, Japan). Flow cytometry Single-cell suspensions of HGFs were prepared at a density of 1x10 6 cell/ml. Next, the cells were incubated with dichloro-dihydro-fluorescein diacetate (DCFH-DA) using Reactive Oxygen Species Assay Kit (Promega, USA) in accordance with the manufacturer’s instructions. Then, flow cytometry (BD Biotechnology) was used to detect the relative level of ROS in HGFs. Apoptosis assay Briefly, the cells with treatments were stained using Annexin V/ propidium iodide (PI) (Beyotime, Shanghai, China). Labeled cells were washed and suspended in PBS. Flow cytometry and CytExpert software were used to analyze. CCK-8 assay For the assessment of the proliferation ability of cells, cells were plated and cultured in 96-well plates (5000 cells per well). On the 0, 12, 24, 48, 72, and 96 hours after cell adherence, the proliferation ability of cells was examined with the CCK-8 kit (Biosharp, China) following the manufacturer’s protocol. Immunohistochemistry For the immunohistochemical staining, surgical specimens from patients with OSCC or adjacent normal mucosa and xenografts from mice were fixed with 10% formalin and embedded in paraffin. Sections from the above specimens were incubated with corresponding antibodies at 4°C overnight and detected by peroxidase-conjugated secondary antibody. For the statistical analyses, the stained slides were scored according to the intensity of staining (-: 0; +: 1; ++:2; and+++: 3) (x200 magnification) and the percentage of the cells of interest staining positive for each antigen (0%: 0; 1–25%: 1; 26–50%: 2; 51–75%: 3; and 76–100%: 4) (x40 magnification). The intensity score was added to the percentage score to obtain a final score. Immunofluorescence For tissues, surgical specimens from patients with OSCC or adjacent normal mucosa were fixed with 10% formalin and embedded in paraffin. Sections from the above specimens were incubated with corresponding antibodies at 4°C overnight and then with Cy3-or FITC-conjugated secondary IgG (Servicebio, Wuhan). Analyses were performed with a fluorescent microscope (Biozero BZ-8000, Keyence, Osaka, Japan). For cells, PNFs and CAFs were fixed with 4% paraformaldehyde for 30 min and then washed with PBS for 3 times. After blocking with 5% BSA at room temperature for 30 min, cells were incubated with corresponding antibodies at 4°C overnight and then with Cy3-or FITC-conjugated secondary IgG (Servicebio, Wuhan). Analyses were performed with a fluorescent microscope (Biozero BZ-8000, Keyence, Osaka, Japan). Luciferase reporter assay The pGL3-Basic plasmid inserted with the promoter sequence of HIF-1α and Renilla luciferase plasmid (MiaoLing, Wuhan) were co-transfected into HGFs using Lipo3000, and then treated with TEVs. After 48h, luciferase activity was detected by Dual-Luciferase Reporter Assay Kit (Promega, USA) in accordance with the manufacturer’s instructions. Tumor xenografts in nude mice The in vivo studies were conducted following the guidance of the Ethical Committee on Animal Experiments of Animal Care Committee of Wuhan University (S07914060B). Briefly, female BALB/c nude mice (18-20 g, 4-6 weeks old) were used to establish tumor-bearing mouse models. The process and analysis were conducted as described in our previous study[14]. Statistical analysis All experiments were repeated a minimum of three times, and the results were presented as mean ± SD. Sample size determination did not involve any statistical method. Statistical analysis was performed using SPSS and GraphPad Prism 9 software, employing either one-way ANOVA or a non-parametric test. Each group exhibited similar variance. Statistical significance was defined as a P-value < 0.05. Results CAFs of OSCC exhibit high level of LC3B First, we analyzed the expression of LC3B in the TCGA-HNSCC dataset. The result showed that LC3B expression was significantly higher in tumor tissues compared to the normal group (Fig. 1Aⅰ). Further correlation analysis suggested that the upregulation of LC3B seemed closely associated with stroma, specifically CAFs (Fig. 1Aii). To verify this hypothesis, we detected the autophagic puncta by immunofluorescence staining of LC3B in OSCC tissues, and Vimentin staining was used to distinguish fibroblasts from tumor cells. The result also showed enhanced staining of LC3B in stroma (Fig. 1B). To further analyze the autophagy level in stroma cells, PNFs and CAFs were isolated from OSCC patients as our previous study[23]. Cell immunofluorescence and MDC staining were used to label the LC3B and acidic vesicular organelles in the autophagy process, respectively. The results showed that higher level of LC3B puncta and MDC-positive granular structures were present in CAFs than PNFs (Fig. 1C and D). Finally, we assessed the autophagy marker LC3B and the selective cargo receptor for autophagy, p62, by western blot. The results showed that LC3B expression in CAFs significantly elevated compared with PNFs. However, p62, the autophagic flux marker, which would decrease when autophagy level increased, was also higher in CAFs than PNFs (Fig. 1E). As cell autophagy is a dynamic process affected by the extracellular microenvironment and signals, we suspect that due to the lack of signals from tumor cells, the autophagy flux of primary fibroblasts that have been separated from the TME is blocked. TEVs enhance the level of autophagy in fibroblasts TEVs are important mediators of tumor-stroma interaction and paly critical roles during tumor progression. And TEVs from OSCC cells have been reported to induce the activation and reprogramming of fibroblasts in our previous study[14]. So, we want to figure out whether the tumor derived signals, TEVs, enhance the autophagy level of fibroblasts. First, we treated the PNFs and CAFs with TEVs (20 μg/ml) derived from CAL27 and SCC25, and assessed the expression of LC3B and p62. The results showed that both PNFs and CAFs exhibited significantly higher expression of comparative LC3B after treated with TEVs. But the expression of p62 exhibited the same increasing trend with LC3B in PNFs and CAFs (Fig. 2A and B). The results indicate that the overexpression of p62 in CAFs is not due to the lack of stimulation from tumor derived signals. Then TEVs were used to treat HGFs to generate TEVs-activated CAFs and to study their roles in autophagy of stromal fibroblasts. First, the influence of TEVs on proliferation and apoptosis of HGFs were evaluated. CCK-8 assay showed that proliferation of HGFs began to decrease after treated with TEVs for 48h (Fig. 2C). Western blot analysis of apoptotic marker Cl-caspase 3 showed no difference between TEVs-treated HGFs and negative control group (Fig. 2D). Besides, apoptotic assay was performed on TEVs-treated HGFs, the result showed that apoptosis of HGFs decreased after treated with TEVs for 48h (Fig. 2E ⅰ and ⅱ). The data above indicated that both proliferation and apoptosis of HGFs decrease after TEVs-treatment. Then we treated HGFs with TEVs for 1, 3, 6, 12 and 24h, respectively and evaluated the autophagy level of TEVs-treated HGFs. The results showed that as treatment time extension, the overall trend of comparative LC3B expression showed significant increase, although there was a little variation, while the expression of p62 maintained a steady enhancement (Fig. 2F and G). These results were in accordance with that in PNFs and CAFs. To better assess the autophagy level in TEVs-treated HGFs, we observed the autophagosomes with TEM and stained autophagic puncta by immunofluorescence of LC3B in TEVs-treated HGFs. Then we identified more abundant autophagosomes (Fig. 2H ⅰ and ⅱ) and augmented staining of LC3B (Fig. 2I) in TEVs-treated HGFs compared with control HGFs. However, the question that whether the increase in p62 means that the autophagy flux is blocked arises again. Then we utilized the tandem mCherry-GFP-LC3B adenovirus to construct HGFs expressing mCherry-GFP-LC3B fusion protein to confirm whether autophagy flux is unobstructed. The result showed that HGFs expressing mCherry-GFP-LC3B fusion protein was established successfully. When treating the constructed HGFs with TEVs for 24h, we observed accumulation of LC3B (green puncta) and increased red puncta that represents fusion of autophagosomes and lysosomes (Fig. 2G), indicating that autophagy flux is unobstructed in TEVs-treated HGFs. TEVs induce autophagy-dependent glycometabolic reprogramming in fibroblasts Autophagy was reported to be closely associated with cellular metabolism and we have validated that TEVs mediated glycometabolic reprogramming in CAFs[14]. We wonder whether the glycometabolic reprogramming in CAFs was associated with autophagy. First, we evaluated the level of glycolysis related proteins, GLUT-1, PFKFB3, PKM2, PDK1 and MCT4 by western blot. Although there was a little variation, the expression of GLUT-1, PFKFB3, PKM2, PDK1 and MCT4 increased when HGFs were treated with TEVs. Besides, PFKFB3 increased stably with the time expansion of TEVs-treatment (Fig. 3A and B). Furthermore, the elevated expression of GLUT-1, PFKFB3, PKM2, PDK1 and MCT4 was also detected in the primary CAFs compared with PNFs (Fig. 3C). Then we investigated the relationship between autophagy and metabolic reprogramming in CAFs and TEVs-treated HGFs. First, autophagy inhibitor chloroquine (CQ) and 3-Methyladenine(3-MA) were used to inhibit autophagy and Earle's balanced salt solution-induced starvation (STV) and rapamycin (RM) were used to enhance autophagy in CAFs. Western Blot results showed autophagy was inhibited or enhanced successfully, and the expression of GLUT-1, PFKFB3 and MCT4 were in accordance with the expression of LC3B, regardless of the expression of p62 (Fig. 3D). However, the expression of PDK1 and PKM2 had no significance (Fig. S1A). Interestingly, in the STV group, LC3B, GLUT-1, PFKFB3 and MCT4 all showed lower expression compared with other groups. Then we inhibited TEVs-induced autophagy in TEVs-treated HGFs with CQ and 3-MA. The results showed that the expression of GLUT-1, PFKFB3 and MCT4, especially the expression of PFKFB3, were strictly consistent with expression of LC3B (Fig. 3E). Similarly, the expression of PDK1 and PKM2 had no significance (Fig. S1B). These results indicated that TEVs drive glycometabolic reprogramming in fibroblasts through LC3B-dependent non-classical autophagy. Direct transfer of ROS via TEVs mediates the autophagy and glycometabolic reprogramming Tumor hyperproliferation is often accompanied with increased oxidative stress, characterized by elevated ROS production. Growing studies have shown that TEVs can transmit biological signaling molecules to regulate the receptor cells. Then, we speculated whether TEVs can directly transmit ROS to mediate the stress response of receptor fibroblasts. First, we detected the level of ROS in tumor cells and TEVs, and the results showed that ROS abound in tumor cells and TEVs, and ROS level in TEVs decreased with the prolongation of the time that TEVs detach from tumor cells (Fig. S2A, B and Fig. 4A, B). Therefore, in subsequent experiments, TEVs are used for experiments within 24 hours after acquisition to ensure that ROS level in TEVs is physiologically. Next, the ROS in TEVs was labeled with ROS probe DCF-DA and incubated with fibroblasts, and then observed under confocal microscopy. 1d-TEVs exhibited more intense fluorescence, while 7d-TEVs fluorescence was significantly reduced (Fig.4C and D). Furthermore, we detected the level of ROS uptake by fibroblasts within 24 hours via flow cytometry, the flow cytometry indicated plentiful ROS in HGFs at 12 hours (Fig.4E and F). NAC can clear ROS in TEVs effectively (Fig.4G and H). After HGFs incubated with NAC treated TEVs, flow cytometry showed that ROS internalized by HGFs were significantly inhibited (Fig.4I and J). Western Blot results showed that the expression of key glycolysis enzymes (PDK1, PKM2 and PFKFB3), GLUT1 and MCT4 were significantly upregulated under the stimulation of ROS derived from TEVs (Fig.4K and L). Meanwhile, the up-regulation of LC3B expression, regardless of p62, indicates that the transferred ROS enhanced the level of autophagy in HGFs (Fig.4K and L). And elimination of ROS in TEVs obviously reversed the enhancement of autophagy and glycolysis (Fig.4K and L). Interestingly, PFKFB3 expression closely mirrored that of LC3B. The aforementioned data revealed that transmission of ROS in TEVs plays a key role in HGFs autophagy and glycometabolic reprogramming. HIF-1α/PFKFB3 signal pathway is involved in the ROS-mediated glycometabolic reprogramming As an important binding site in the promoter sequence of the PFKFB3 gene, HIF-1α is an oxidative stress-inducible gene. Under the stimulation of TEVs, the expression of HIF-1α in fibroblasts is exhibited as the tendency to decrease after rising (Fig.5A and B), which was considered to be the stress response of fibroblasts before the elimination of extrinsic ROS. Furthermore, dual luciferase report assay revealed that TEVs promoted the transcription of HIF-1α genes in HGFs (Fig.5C and D). Western Blot indicated that CAFs have elevated HIF-1α level relative to PNFs (Fig.5E). Notably, under normoxic conditions, the level of HIF-1α in TEVs are minimal, allowing us to rule out the possibility that the increase in HIF-1α in CAFs after TEVs stimulation is due to the direct transfer of HIF-1α (Fig.5F). Consistently, after ROS eliminated in TEVs by NAC, the ability of TEVs to induce HIF-1α overexpression in HGFs was significantly reduced, which indicated that ROS delivered in TEVs is the key molecule in tumor oxidative stress transfer (Fig.5G and H). After selectively inhibiting the translation of HIF-1α protein through KC7F2, only the up-regulated expression of PFKFB3, a key enzyme of HGF glycolysis induced by TEVs, was significantly blocked (Fig.5I, J and S3A, B). Considered together, tumor cells transfer their ROS to CAFs through TEVs, subsequently inducing glycometabolic reprogramming via the HIF-1α/PFKFB3 axis. TEVs promote stroma autophagy and glycometabolic reprogramming in vivo To further verify in vitro experiments conclusions, the xenografts were established in vivo . CAL27 cells and HGFs were mixed at a ratio of 1:3 and co-injected into nude mouse subcutaneously. Exactly 7 days after the injection, TEVs (50 mg) or PBS was injected around the tumors every 3 days. Immunohistochemistry was conducted on the tumor dissection. Compared with the control group, the stromal cells of the TEVs-injected xenografts exhibited significantly higher LC3B, HIF-1α and PFKFB3 expression (Fig6A, B). Moreover, similar results were confirmed in OSCC tissues and healthy mucosa tissues (Fig.6C, D). Overall, in vivo results confirmed that TEVs elevated the autophagy levels and promoted glycolysis in the stroma. Discussion TME is a multicellular system with complex tumor-stromal interactions, playing critical roles in all stages of the neoplastic process[ 1 ]. CAFs constitute the most abundant stromal cell type of the TME. Confronting with harsh environment caused by the rapid process of cancer, tumor and stromal cells continuously engage in signal crosstalk and energy exchanges, thereby creating an environment conducive to tumor growth[ 21 ]. As an important medium of communication between tumor and stromal cells, TEVs were confirmed to exerts variety of biological functions[ 13 ]. However, under adverse conditions of high oxidative stress caused by rapid tumor proliferation, it remains unclear what role TEVs play in the crosstalk between tumor cells and surrounding stromal cells. Here, we revealed that surrounding stromal cells could shoulder tumor oxidative stress via accepting ROS-rich TEVs. The ROS delivered by TEVs increase the autophagy levels in CAFs, thereby activating glycometabolic reprogramming to produce energy-rich metabolites for tumor cells. These findings shed light on the crucial role of TEVs in the stress transfer model and offer a theoretical rationale for ROS-mediated autophagy in the glycometabolic reprogramming of CAFs (Fig. 7 ). Autophagy, an intracellular lysosome-dependent degradation system, serves as a self-regulatory mechanism that enables cells to adapt to adverse conditions. By degrading and recycling dysfunctional or unnecessary cellular components, autophagy helps maintain cellular homeostasis and provides essential nutrients and energy during periods of stress or nutrient deprivation[ 18 ]. Our previous research indicated that the activation of autophagy is essential for the acquisition of cancer stem cells properties to resist chemotherapy, starvation, or hypoxic conditions[ 17 ]. Besides, accumulating findings highlight that autophagy in stroma influences the TME[ 27 ]. Pavlides et al.[ 28 ] metabolomics analysis of the mammary fat pads of WT and Cav-1–deficient mice, combined with human breast cancer transcriptomic data[ 29 ], demonstrated that extensive stromal catabolism directly promoted tumor anabolic growth via a parasitic mechanism, and that blocking stromal autophagy would inhibit energy transfer to epithelial cancer cells. In this study, we confirmed that autophagy levels are significantly elevated in the OSCC stroma, as evidenced by higher LC3B expression and an increase in MDC-positive granular structures of CAFs. More importantly, although our previous research had confirmed that primary CAFs in OSCC undergo PFKFB3-driven metabolic reprogramming of glycolysis[ 23 ], the upstream switch that initiates this process requires further investigation. In this work, we discovered that when autophagy was blocked by CQ and 3-MA, the downregulation of the glycolysis key enzyme PFKFB3, and the reduction in the import and export of glucose fluxes, GLUT1 and MCT4, were detected together. Conversely, activation of autophagy enhanced the expression of PFKFB3, GLUT1 and MCT4. Therefore, we propose that autophagy induces PFKFB3-driven glycolysis, providing a reasonable explanation for the glycometabolic reprogramming in CAFs. To further determine the factors affecting autophagy and glycometabolic reprogramming in tumor stromal cells, TEVs were purified as reported in our previous study[ 13 ]. TEVs, a class of extracellular vesicles which exhibit higher correlations with the original cells in terms of protein level, can transfer bioinformation of cancer cells to the surrounding stromal cells and alter the phenotype of the recipient cells. In our previous research, TEVs were capable of mediating the FAP-dependent transition of normal fibroblasts to CAFs and activating the glycometabolic reprogramming through the transfer of p-ERK/2 protein[ 14 ]. Interestingly, in this study we surprising found that the proliferation of TEVs-treated normal fibroblasts was not active, and apoptosis also remained at a low level, indicating that the normal fibroblasts stimulated by TEVs were in a quiescent protective state[ 30 ]. By evaluating autophagy levels, we validated that TEVs-mediated enhancement of fibroblast autophagy explained this phenomenon. Furthermore, inhibition of TEVs-mediated autophagy, partially blocks the enhancement of glycolysis. Unexpectedly, we observed a steady increase in p62 expression during the process of TEVs-treated fibroblasts. This seem suggest that the mechanism of TEVs-mediated autophagy appeared to bypass the need for p62, hinting at a potentially distinct regulatory pathway that governs this form of metabolic adaptation. For instance, recent studies have shown that some forms of autophagy can proceed via alternate pathways involving Beclin-1-independent[ 31 ] or LC3B-associated phagocytosis[ 32 , 33 ], which do not require the conventional autophagy machinery. Therefore, exploring whether TEVs-mediated autophagy in fibroblasts also follows such unconventional routes could provide deeper insights into the complexity of tumor-stroma interactions and the versatile nature of cellular metabolism under the influence of tumor-derived factors. Hyperproliferation of tumor cells is accompanied by high ROS production. As a by-product of aerobic respiration, endogenous ROS are generated from mitochondrial metabolism, peroxisomes and the activity of the family of transmembrane NADPH oxidases (NOXs)[ 34 ]. Therefore, the conventional view is that oxidative burden pushes redox balance away from a reduced state by increasing their antioxidant status to resist hyperproliferation-driven ROS, while at the same time avoiding ROS thresholds that would trigger senescence or apoptosis[ 35 ]. Interestingly, we found that ROS release from tumor via TEVs is another way to balance the elevated levels of intracellular oxidative stress. After labeling TEVs-derived ROS with DCF-DA probe, further detection revealed that HGFs took in ROS transmitted by tumors and elevated autophagy, suggesting that stromal cells niche around the tumor play an important role in sharing tumor oxidative stress. Meanwhile, When ROS concentrations are elevated, the SQSTM1 gene encoding p62 can be transcriptional activated by the transcription factor Nrf2 and plays an important role in overall antioxidant and detoxification responses, which explains the stable increase in p62 in fibroblasts following TEVs stimulation[ 36 ]. Moreover, ROS transmitted by TEVs could activate glycolysis through both LC3B-dependent and HIF-1α/PFKFB3 pathways, and eliminating ROS effectively blocked glycometabolic reprogramming in CAFs. These findings highlight an intriguing mechanism where tumor cells, upon encountering stress stimuli, generate ROS not just as by-products of oxidative metabolism but as active signaling molecules. By releasing ROS via TEVs, tumor cells effectively transfer the oxidative burden to nearby CAFs, thereby outsourcing their oxidative stress. This transfer of stress induces autophagy and metabolic reprogramming within CAFs, which appear to undergo a self-sacrificial process to support the survival and growth of tumor cells. In conclusion, our discovery establishes a novel model of tumor oxidative stress via TEVs. The transferred TEVs, rich in ROS, drive glycometabolic reprogramming in CAFs through dual pathways by enhancing autophagy and the HIF-1α/PFKFB3 axis. Declarations Funding This study was supported by National Natural Science Foundation of China (Grant No. 82103382), National Key R&D Programme of China (Grant No. 2022YFC2504200), National Natural Science Foundation of China (Grant No. 82273306) and Key Research Project of Hubei Province (Grant No. 2023BCB135). Competing Interests The authors declare that they have no conflict of interest. Ethics Approval and Consent to Participate Registry and the Registration No. of the study: Ethics Committee of School and Hospital of Stomatology at Wuhan University (2022LUNSHENZIA05). Written informed consents were obtained from all patients participated. Animal works were approved by the Ethical Committee on Animal Experiments of the Animal Care Committee of Wuhan University (S07914060B). Author Contributions Research design: EJ; Manuscripts writing: XL, EJ; Experiments performed: EJ, XL and XD; Data analysis: YX; Supervision: ZS and ZS. Data Availability The datasets generated during the current study are available from the corresponding author upon reasonable request. Consent for Publication Not applicable. References Chen X, Song E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev Drug Discov. 2019;18:99–115. Mezawa Y, Orimo A. The roles of tumor- and metastasis-promoting carcinoma-associated fibroblasts in human carcinomas. Cell Tissue Res. 2016;365:675–89. Zhan H, Zhou B, Cheng Y, Xu J, Wang L, Zhang G, et al. Crosstalk between stromal cells and cancer cells in pancreatic cancer: New insights into stromal biology. Cancer Lett. 2017;392:83–93. Vitale I, Manic G, Galassi C, Galluzzi L. Stress responses in stromal cells and tumor homeostasis. Pharmacol Ther. 2019;200:55–68. Policastro LL, Ibañez IL, Notcovich C, Duran HA, Podhajcer OL. The Tumor Microenvironment: Characterization, Redox Considerations, and Novel Approaches for Reactive Oxygen Species-Targeted Gene Therapy. Antioxid Redox Signal. 2013;19:854–95. Hayes JD, Dinkova-Kostova AT, Tew KD. Oxidative Stress in Cancer. Cancer Cell. 2020;38:167–97. Weinberg F, Hamanaka R, Wheaton WW, Weinberg S, Joseph J, Lopez M et al. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proceedings of the National Academy of Sciences. 2010;107:8788–93. Kong H, Reczek CR, McElroy GS, Steinert EM, Wang T, Sabatini DM et al. Metabolic determinants of cellular fitness dependent on mitochondrial reactive oxygen species. Sci Adv 6:eabb7272. Reczek CR, Birsoy K, Kong H, Martínez-Reyes I, Wang T, Gao P, et al. A CRISPR screen identifies a pathway required for paraquat-induced cell death. Nat Chem Biol. 2017;13:1274–9. Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39:44–84. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367:eaau6977. Thakur A, Ke X, Chen Y-W, Motallebnejad P, Zhang K, Lian Q, et al. The mini player with diverse functions: extracellular vesicles in cell biology, disease, and therapeutics. Protein Cell. 2022;9:631–54. Zhao X-P, Wang M, Song Y, Song K, Yan T, Wang L, et al. Membrane microvesicles as mediators for melanoma-fibroblasts communication: Roles of the VCAM-1/VLA-4 axis and the ERK1/2 signal pathway. Cancer Lett. 2015;360:125–33. Jiang E, Xu Z, Wang M, Yan T, Huang C, Zhou X, et al. Tumoral microvesicle–activated glycometabolic reprogramming in fibroblasts promotes the progression of oral squamous cell carcinoma. FASEB j. 2019;33:5690–703. Harmati M, Bukva M, Böröczky T, Buzás K, Gyukity-Sebestyén E. The role of the metabolite cargo of extracellular vesicles in tumor progression. Cancer Metastasis Rev. 2021;40:1203–21. Yang X, Li Y, Zou L, Zhu Z. Role of Exosomes in Crosstalk Between Cancer-Associated Fibroblasts and Cancer Cells. Front Oncol. 2019;9. Chen Y, Zhao H, Liang W, Jiang E, Zhou X, Shao Z, et al. Autophagy regulates the cancer stem cell phenotype of head and neck squamous cell carcinoma through the noncanonical FOXO3/SOX2 axis. Oncogene. 2022;41:634–46. Levy JMM, Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer. 2017;17:528–42. New J, Arnold L, Ananth M, Alvi S, Thornton M, Werner L, et al. Secretory Autophagy in Cancer-Associated Fibroblasts Promotes Head and Neck Cancer Progression and Offers a Novel Therapeutic Target. Cancer Res. 2017;77:6679–91. Zhou W, Xu G, Wang Y, Xu Z, Liu X, Xu X, et al. Oxidative stress induced autophagy in cancer associated fibroblast enhances proliferation and metabolism of colorectal cancer cells. Cell Cycle. 2017;16:73–81. Chiarugi P, Cirri P. Metabolic exchanges within tumor microenvironment. Cancer Lett. 2016;380:272–80. Pavlides S, Whitaker-Menezes D, Castello-Cros R, Flomenberg N, Witkiewicz AK, Frank PG, et al. The reverse Warburg effect: Aerobic glycolysis in cancer associated fibroblasts and the tumor stroma. Cell Cycle. 2009;8:3984–4001. Li X, Jiang E, Zhao H, Chen Y, Xu Y, Feng C, et al. Glycometabolic reprogramming-mediated proangiogenic phenotype enhancement of cancer-associated fibroblasts in oral squamous cell carcinoma: role of PGC-1α/PFKFB3 axis. Br J Cancer. 2022;3:449–61. Feng Y, Chen Y, Wu X, Chen J, Zhou Q, Liu B, et al. Interplay of energy metabolism and autophagy. Autophagy. 2024;1:4–14. Chow LQM. Head and Neck Cancer. N Engl J Med. 2020;382:60–72. Qiu J, Jiang E, Shang Z. Prognostic value of tumor–stroma ratio in oral carcinoma: Role of cancer-associated fibroblasts. Oral Dis. 2023;29:1967–78. Rudnick JA, Debnath J. Autophagy in host stromal fibroblasts supports tumor desmoplasia. Autophagy. 2021;17:4497–8. Pavlides S, Tsirigos A, Migneco G, Whitaker-Menezes D, Chiavarina B, Flomenberg N, et al. The autophagic tumor stroma model of cancer. Cell Cycle. 2010;9:3485–505. Pavlides S, Tsirigos A, Vera I, Flomenberg N, Frank PG, Casimiro MC, et al. Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the reverse Warburg effect: A transcriptional informatics analysis with validation. Cell Cycle. 2010;9:2201–19. Wang L, Shang Z, Zhou Y, Hu X, Chen Y, Fan Y, et al. Autophagy mediates glucose starvation-induced glioblastoma cell quiescence and chemoresistance through coordinating cell metabolism, cell cycle, and survival. Cell Death Dis. 2018;9:1–17. Trocoli A, Mathieu J, Priault M, Reiffers J, Souquère S, Pierron G, et al. ATRA-induced upregulation of Beclin 1 prolongs the life span of differentiated acute promyelocytic leukemia cells. Autophagy. 2011;7:1108–14. Sanjuan MA, Dillon CP, Tait SWG, Moshiach S, Dorsey F, Connell S, et al. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature. 2007;450:1253–7. Deretic V, Jiang S, Dupont N. Autophagy intersections with conventional and unconventional secretion in tissue development, remodeling and inflammation. Trends Cell Biol. 2012;22:397–406. Sies H, Jones DP. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol. 2020;21:363–83. Forman HJ, Zhang H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat Rev Drug Discov. 2021;20:689–709. Simon H-U, Friis R, Tait SWG, Ryan KM. Retrograde signaling from autophagy modulates stress responses. Sci Signal. 2017;10:eaag2791. Additional Declarations No competing interests reported. Supplementary Files originalwb.zip supplementfig.tif supplementaryfigurelegends.docx 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5432071","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":378440812,"identity":"278c261e-ce26-4ae9-9346-6108c38b1c23","order_by":0,"name":"Erhui Jiang","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Erhui","middleName":"","lastName":"Jiang","suffix":""},{"id":378440813,"identity":"e9c8dbd3-8511-43b0-8cd8-e4b1cfa782c3","order_by":1,"name":"Xiang Li","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Li","suffix":""},{"id":378440814,"identity":"a486ccb4-8d1a-4763-8c4b-5da18901f480","order_by":2,"name":"Xinyu Dou","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Dou","suffix":""},{"id":378440815,"identity":"3a29830e-dd8f-4220-a028-bb05bc2d20ab","order_by":3,"name":"Yuming Xu","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuming","middleName":"","lastName":"Xu","suffix":""},{"id":378440816,"identity":"185d0a84-f502-443e-aaca-38fb01ccdee0","order_by":4,"name":"Zhe Shao","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Shao","suffix":""},{"id":378440817,"identity":"c401234d-afc1-4275-a624-1c6bd5193375","order_by":5,"name":"Zhengjun Shang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYDACCTB5AEQkMDBUgAiYIHFazpCohYGBsY0ILfKzm589/PLnjpw5/4Jn0oXzDucZHGA+eJuHwS4PlxaDO8fMjWV4nhlbzniQJj1z2+FigwNsydY8DMnFOLVIJJhJS0gcTtxw40CaNO+224kbDvCYSfMwHEhswOWwGenfpCUMDtdDtMwBaeH/hlcLw40cM8kPCYcTDM43ALU0gG1hw6vF4EZOmTTDgcOGG24wAL1w7H/izMNsxpZzDJLxOWyb5I8/h+UNzp9JvM1Tk5bYd7z54Y03FXa4HQYEzDwgUiInAcoF245HPRAw/gCR/McP4Fc2CkbBKBgFIxYAAINoX5+5zmYXAAAAAElFTkSuQmCC","orcid":"","institution":"Wuhan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zhengjun","middleName":"","lastName":"Shang","suffix":""}],"badges":[],"createdAt":"2024-11-11 12:38:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5432071/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5432071/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71728154,"identity":"09916a57-89d9-4a23-a3fd-827dbe362eea","added_by":"auto","created_at":"2024-12-18 06:28:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22771342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAFs of OSCC exhibit high level of LC3B\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Ai) \u003c/strong\u003eLC3B mRNA expression in tumor and normal groups based on the TCGA-HNSCC dataset. \u003cstrong\u003e(ii) \u003c/strong\u003eThe pair-wise gene expression correlation analysis between LC3B and CAFs markers was evaluated from the TCGA-HNSCC dataset. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative tissue immunofluorescence staining in OSCC and adjacent normal mucosa tissues. \u003cstrong\u003e(C) \u003c/strong\u003eImmunofluorescence staining of phalloidin and LC3B in CAFs and PNFs. \u003cstrong\u003e(D) \u003c/strong\u003eAcidic vesicular organelles were detected by MDC staining in CAFs and PNFs. \u003cstrong\u003e(E) \u003c/strong\u003eWestern blot analysis of LC3B and p62 expression in six pairs of CAFs and PNFs. Full-length blots are presented in Supplementary files. Data are presented as means±SD. Results are representative of at least three independent experiments. *P \u0026lt;0.05, **P\u0026lt; 0.01, ***P \u0026lt; 0.001, and ns represents no significance.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/22c460ac802dba3808d84943.png"},{"id":71728158,"identity":"3b83a94a-dfc3-4e9b-b312-774798b9f94a","added_by":"auto","created_at":"2024-12-18 06:28:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19891503,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEVs enhance the level of autophagy in fibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003e Western blot analysis of p62 and LC3B expression in PNFs and CAFs treated with CAL27/SCC25 TEVs for 0 to 24 hours. \u003cstrong\u003e(C) \u003c/strong\u003eAssessment of HGFs proliferation ability after 48 hours of TEVs treatment. \u003cstrong\u003e(D) \u003c/strong\u003eWestern blot analysis of Cl-caspase 3 expression in HGFs treated with TEVs for 0 to 48 hours. \u003cstrong\u003e(Ei)\u003c/strong\u003e Flow cytometry analysis of apoptosis in HGFs after TEVs treatment. \u003cstrong\u003e(ii) \u003c/strong\u003eQuantitative analysis of the percentage of apoptotic cells in HGFs. \u003cstrong\u003e(F, G) \u003c/strong\u003eWestern blot analysis of p62 and LC3B expression in HGFs treated with CAL27/SCC25 TEVs for 0 to 24 hours. \u003cstrong\u003e(Hi) \u003c/strong\u003eTransmission electron microscope (TEM) was conducted to observe autophagosomes in TEVs-treated HGFs. \u003cstrong\u003e(ii)\u003c/strong\u003e Quantitative analysis of the number of autophagosomes. \u003cstrong\u003e(I) \u003c/strong\u003eImmunofluorescence staining of phalloidin and LC3B in NC-HGFs and TEVs-treated HGFs. \u003cstrong\u003e(J) \u003c/strong\u003eImmunofluorescence was used to observe the autophagy flux in NC-HGFs and TEVs-treated HGFs. Full-length blots are presented in Supplementary file. Data are presented as means±SD. Results are representative of at least three independent experiments. *P \u0026lt;0.05, **P\u0026lt; 0.01, ***P \u0026lt; 0.001, and ns represents no significance.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/499fb82ff3548ed7a266ac7b.png"},{"id":71728755,"identity":"28a89b9e-e66c-4186-9812-772334aaf4a1","added_by":"auto","created_at":"2024-12-18 06:36:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6999888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEVs induce autophagy-dependent glycometabolic reprogramming in fibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B) \u003c/strong\u003eWestern blot analysis of glycolytic transporter proteins (MCT4, GLUT1) and glycolytic rate-limiting enzymes (PDK1, PKM2 and PFKFB3) expression in HGFs treated with CAL27/SCC25 TEVs for 0 to 24 hours. \u003cstrong\u003e(C) \u003c/strong\u003eWestern blot analysis of protein expression in three pairs of CAFs and PNFs. \u003cstrong\u003e(D) \u003c/strong\u003eWestern blot analysis of glycolysis-related and autophagy-related proteins expression in CAFs when autophagy was inhibited (CQ and 3-MA) or enhanced (starvation and RM). \u003cstrong\u003e(E)\u003c/strong\u003eWestern blot analysis of glycolysis-related and autophagy-related proteins expression in TEVs-treated HGFswhen autophagy was inhibited. Full-length blots are presented in Supplementary file. Data are presented as means±SD. Results are representative of at least three independent experiments. *P \u0026lt;0.05, **P\u0026lt; 0.01, ***P \u0026lt; 0.001, and ns represents no significance.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/625fbd11c28569c4de0a0831.png"},{"id":71728754,"identity":"af3a607b-72cd-4431-b04e-7089e2a42c2c","added_by":"auto","created_at":"2024-12-18 06:36:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18505900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDirect transfer of ROS via TEVs mediates autophagy and glycometabolic reprogramming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Ai, Bi) \u003c/strong\u003eFlow cytometry analysis of ROS levels and their duration in CAL27/SCC25 TEVs. \u003cstrong\u003e(Aii, Bii) \u003c/strong\u003eQuantitative analysis of the relative ROS levels from 1 to 7 day.\u003cstrong\u003e (C, D)\u003c/strong\u003e Representative bright field and fluorescence images of fibroblasts after incubation with DCF-DA labeled CAL27/SCC25 TEVs.\u003cstrong\u003e (Ei, Fi) \u003c/strong\u003eFlow cytometry analysis of ROS levels in fibroblasts after uptake of CAL27/SCC25 TEVs over 24 hours. \u003cstrong\u003e(Eii, Fii) \u003c/strong\u003eQuantitative analysis of the relative ROS levels in CAFs.\u003cstrong\u003e (Gi, Hi) \u003c/strong\u003eFlow cytometry showed the effect of NAC on the clearance of ROS in CAL27/SCC25 TEVs. \u003cstrong\u003e(Gii, Hii) \u003c/strong\u003eQuantitative analysis of the relative ROS levels in CAL27/SCC25 TEVs after NAC treatment.\u003cstrong\u003e (Ii, Ji) \u003c/strong\u003eFlow cytometry analysis of ROS levels in HGFs after incubation with treated CAL27/SCC25 TEVs. \u003cstrong\u003e(Iii, Jii)\u003c/strong\u003e Quantitative analysis of the relative ROS levels in HGFs.\u003cstrong\u003e (K, L)\u003c/strong\u003eWestern blot analysis of proteins expression in HGFs after CAL27/SCC25 TEVs treatment. Full-length blots are presented in Supplementary file.\u003cstrong\u003e \u003c/strong\u003eData are presented as means±SD. Results are representative of at least three independent experiments. *P \u0026lt;0.05, **P\u0026lt; 0.01, ***P \u0026lt; 0.001, and ns represents no significance.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/6a7e5ca2e2658a4826023b3c.png"},{"id":71728156,"identity":"b243b184-0116-484a-9037-c3db62ccf832","added_by":"auto","created_at":"2024-12-18 06:28:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7990435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIF-1α/PFKFB3 signal pathway is involved in the ROS-mediated glycometabolic reprogramming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B) \u003c/strong\u003eWestern blot analysis of HIF-1α expression in HGFs treated with CAL27/SCC25 TEVs for 0 to 24 hours. \u003cstrong\u003e(C, D) \u003c/strong\u003eHIF-1α luciferase reporter assay was performed in HGFs after incubation with CAL27/SCC25 TEVs. \u003cstrong\u003e(E) \u003c/strong\u003eWestern blot analysis of HIF-1α expression in three pairs of PNFs and CAFs. \u003cstrong\u003e(F) \u003c/strong\u003eWestern blot analysis of HIF-1α expression in CAL27/SCC25 TEVs under normoxic and hypoxic conditions. \u003cstrong\u003e(G, H) \u003c/strong\u003eWestern blot analysis of HIF-1α expression in HGFs afterCAL27/SCC25 TEVs treatment. \u003cstrong\u003e(I, J) \u003c/strong\u003eWestern blot analysis of PFKFB3 and HIF-1α expression in HGFs after CAL27/SCC25 TEVs and KC7F2 treatment. Full-length blots are presented in Supplementary file. Data are presented as means±SD. Results are representative of at least three independent experiments. *P \u0026lt;0.05, **P\u0026lt; 0.01, ***P \u0026lt; 0.001, and ns represents no significance.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/b591a64e3620b30bd1343b11.png"},{"id":71728163,"identity":"c7ab9828-8e77-4979-8210-c2537ac1da93","added_by":"auto","created_at":"2024-12-18 06:28:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":65180732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEVs promote stroma autophagy and glycometabolic reprogramming \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRepresentative IHC images of LC3B, HIF-1α and PFKFB3 in tumor sections from the xenografts injected with TEVs or PBS. Tumor epithelium as T, and the stroma as S. \u003cstrong\u003e(B) \u003c/strong\u003eQuantification of LC3B, HIF-1α and PFKFB3expression levels in stroma of the xenografts injected with TEVs or PBS. N=6. \u003cstrong\u003e(C) \u003c/strong\u003eRepresentative IHC images of LC3B, HIF-1α and PFKFB3 in OSCC and adjacent normal mucosa tissues. Normal epithelium is denoted as E, tumor epithelium as T, and the stroma as S. Tumor epithelium as T, mucosa epithelium as E and the stroma as S. \u003cstrong\u003e(D)\u003c/strong\u003eQuantification of LC3B, HIF-1α and PFKFB3 expression levels in stroma of OSCC and adjacent normal mucosa tissues. OSCC=67 Mucosa =9.Data are presented as means±SD. Results are representative of at least three independent experiments. *P \u0026lt;0.05, **P\u0026lt; 0.01, ***P \u0026lt; 0.001, and ns represents no significance.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/66527efd6b7e88981c052ec5.png"},{"id":71728752,"identity":"c5530619-5fa9-4ef5-8bf7-470815bfe350","added_by":"auto","created_at":"2024-12-18 06:36:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2999222,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the tumor oxidative stress transfer model mediated by TEVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor cells transfer their oxidative stress by secreting TEVs enriched with ROS. CAFs uptake these ROS-rich TEVs, alleviating the tumor stress and driving glycometabolic reprogramming to provide energy for the tumor through dual pathways: enhancing autophagy and the HIF-1α/PFKFB3 axis.\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/29f8cae7c252474542e6f957.png"},{"id":89062820,"identity":"91c0afd5-5cdf-4c4d-97b4-3720a7ba61a7","added_by":"auto","created_at":"2025-08-14 09:45:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":92777907,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/c88f716f-94ed-4fc2-bd58-4586ae4398e7.pdf"},{"id":71728162,"identity":"23375cd6-9efa-4d77-8e27-c9ab216e9e8f","added_by":"auto","created_at":"2024-12-18 06:28:33","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":38359348,"visible":true,"origin":"","legend":"","description":"","filename":"originalwb.zip","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/9711a5cd3914a169c7f1a872.zip"},{"id":71728751,"identity":"6da311d7-766a-4d5f-9298-f2082f7eae44","added_by":"auto","created_at":"2024-12-18 06:36:32","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5118236,"visible":true,"origin":"","legend":"","description":"","filename":"supplementfig.tif","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/679583e7d302aa919a8bbd41.tif"},{"id":71730294,"identity":"639798eb-83f5-474d-ba7b-3a4ed802ebff","added_by":"auto","created_at":"2024-12-18 06:44:33","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18212,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-5432071/v1/08232aa7061f5d6150de64da.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tumor-derived Extracellular Vesicles-mediated Oxidative Stress Transfer Activates Glycometabolic Reprogramming of CAFs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDespite significant improvements in diagnosis and therapy, the intractable TME is still a determinant of cancer cell behavior and disease progression[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As one of the most abundant stromal components in the TME, CAFs, an activated form of stromal fibroblasts, interplay with tumor cells to establish multiple and complex crosstalk mechanisms[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the TME for cells within the nascent tumor is harsh and imposes different types of stress on cells[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Malignant cells have developed adaptive mechanisms to survive under the extreme conditions of the TME such as restricted oxygen supply and nutrient deprivation[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Confronted with adverse conditions, hyperproliferation of tumor cells is accompanied by high ROS production[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. ROS acting as a double-edged sword, influencing cancer evolution in apparently contradictory ways, either initiating/stimulating tumorigenesis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and supporting transformation/proliferation[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] of cancer cells or causing cell death[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Nonetheless, uncontrolled ROS generation is still recognized to damage fundamental cellular components, including nucleic acids, proteins, and lipids, which can lead to cell senescence, degeneration, or fatal lesions[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To maintain aberrant redox homeostasis, tumor cells thrive to adapt oxidative stress in various ways and transfer this oxidative burden away.\u003c/p\u003e \u003cp\u003eTumor-derived EVs (generally referred as TEVs) mediate the interaction and communication between tumor and stroma[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. They exert various functions in intercellular communication by transferring biological cargo to recipient cells locally or at specific distant sites via circulation[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recent studies showed that TEVs not only play a crucial role in CAFs\u0026rsquo; activation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and glycometabolic reprogramming[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], but also help tumor cells transfer harmful byproducts and unnecessary organelles[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], thereby alleviating tumor\u0026rsquo;s burden. This transfer mechanism may be a strategy for tumor cells to maintain survival in a harsh microenvironment. However, it is unclear whether TEVs, as mediators of intercellular delivery[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], can transfer ROS to surrounding CAFs to mitigate tumor oxidative stress.\u003c/p\u003e \u003cp\u003eAutophagy, an intracellular lysosome-dependent degradation system ubiquitous in eukaryotic cells, degrades and engulfs superfluous or damaged organelles and proteins in response to adverse conditions[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recently, autophagy in stromal cells has been brought to the forefront. Jacob New et al. highlighted the important role for secretory autophagy in the supporting stromal CAFs, which enhances HNSCC progression[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the non-contact co-culture system \u003cem\u003ein vitro\u003c/em\u003e, human colorectal fibroblasts positively influenced the metabolism of colorectal cancer cells through its autophagy enhancement[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the molecular insight, by which the autophagy of CAFs is regulated, needs further study.\u003c/p\u003e \u003cp\u003eMetabolic alterations are the classic theme of concern after autophagy occurs. The rapid progression of malignancy tumor often necessitates metabolic rewiring to meet the demands to forefront nutrient and oxygen deprivation, as well as increased anabolic requests to match high proliferation rate[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. CAFs activated by epithelial tumor cells transform into a metabolic factory that produces energy-rich metabolites. The glycometabolic reprogramming in CAFs, driven by the host-parasite interaction, is referred as the \u0026ldquo;reverse Warburg effect\u0026rdquo;[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our previous research has illustrated that primary CAFs derived from OSCC patients undergo PKFKFB3-driven glycometabolic reprogramming, characterized by an enhancement in aerobic glycolysis with a decrease in OXPHOS[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, TEVs-activated CAFs supply lactate to tumor cells by establishing glycometabolic crosstalk loop MCT1/MCT4[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. During autophagy, the contents encapsulated by autophagosomes are degraded by specific hydrolases to produce lipids, amino acids, sugars, and other degradation products, which are catabolized through glycolysis, the citric acid cycle, β-oxidation, and oxidative phosphorylation to generate ATP and macromolecular precursors[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, it remains unclear whether the glycometabolic reprogramming in CAFs is induced by TEVs-mediated enhancement of autophagy.\u003c/p\u003e \u003cp\u003eAs the sixth most common cancer worldwide[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], OSCC is an aggressive disease characterized by the intense fibrotic stromal response, with the tumor-stroma ratio determining the prognosis[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, we originally propose the TEVs-mediated oxidative stress transfer model. To elucidate the role and mechanism of TEVs in tumor and stroma communication, TEVs were obtained from two OSCC cell lines (SCC25 and CAL27), and fibroblasts were isolated from healthy gingival tissues of volunteers and OSCC patients, respectively. Our results demonstrated that CAFs of OSCC exhibited high level of LC3B and TEVs enhanced autophagy in normal fibroblasts. Blockade of autophagy inhibited the glycometabolic reprogramming, whereas activation of autophagy promoted the glycometabolic reprogramming in CAFs. Furthermore, the transmission of ROS in TEVs played a crucial role in triggering autophagy and glycometabolic reprogramming in HGFs. \u003cem\u003eIn vivo\u003c/em\u003e, we validated that TEVs promoted autophagy and glycometabolic reprogramming in tumor stroma. In conclusion, our experiments focus on TEVs as mediators in autophagy-dependent glycometabolic reprogramming and propose a novel rationale for the \u0026ldquo;stress transfer model\u0026rdquo;.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOSCC cell lines CAL27 and SCC25 were purchased from China Center for Type Culture Collection (Shanghai, China). Short tandem repeat was performed routinely on the cell line to confirm its authenticity, and Mycoplasma was routinely tested. SCC25 cells were cultivated in DMEM/F-12, and CAL27 cells were cultivated in DEME/HIGH GLUCOSE (Hyclone, UT, USA), both containing 10% FBS (Gibco, Australia).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIsolation of TEVs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe TEVs were isolation from OSCC cell (CAL27 and SCC25) supernatant, as previously reported by Jiang et al[14].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIsolation and culture of fibroblasts\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe human gingival fibroblasts (HGFs), PNFs and CAFs were isolated from the healthy gingival tissues of volunteers and 6 patients with OSCC, respectively, according to our previous study[14]. All procedures were in accordance with the Ethics Committee of School and Hospital of Stomatology at Wuhan University (2022LUNSHENZIA05).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTransmission electron microscopy\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy (TEM) was conducted as our previous study showed to observe TEVs[14]. TEM analysis was also conducted to observe the morphology and number of the autophagosome in the TEVs treated HGFs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWestern blot\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed with mammalian protein extraction reagent (MPER) (Thermo Fisher Scientific) with protease inhibitor and phosphatase inhibitor (MilliporeSigma). Subsequently, a bicinchoninic acid protein assay was used to test the quantity of each sample. Afterward, the protein solutions were added with loading buffer (5x) and heated for 10 min at 95\u0026deg;C. Next, equal amounts of the protein samples (15 mg) were subjected to 8\u0026ndash;12% SDS-PAGE (60 V, 30 min; 110 V, 60\u0026ndash;70 min), and electrophoretically transferred to polyvinylidene fluoride membranes (MilliporeSigma) in the sodium dodecyl sulfate\u0026ndash;electroblot buffer (25mMTris-Cl, 192mMglycine, 20%methanol, pH8.3) (200mA, 100 min). After blocking with 5% nonfat milk in Tris-buffered saline at room temperature for 90 min, the membranes were incubated with primary antibodies overnight at 4\u0026deg;C. Subsequently, the bound antibodies were tested using horseradish peroxidase\u0026ndash;conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine). The antibodies were as follows: LC3B (CST, 83506), P62(Proteintech, 18420-1-AP), MCT4 (Proteintech, 22787-1-AP), GLUT1 (Proteintech, 21829-1-AP), PFKFB3 (Abcam, ab181861), PDK1 (CST, C47H1), PKM2 (Proteintech, 15822-1-AP), \u0026beta;-actin (CST, 4970), HIF-1\u0026alpha; (Proteintech, 20960-1-AP), ATP1A1 (Proteintech, 55187-1-AP).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell transfection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe mRFP-GFP-LC3B adenovirus was purchased from GeneChem (Shanghai, China). Cells were transfected according to the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMDC staining\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe HGFs were seeded into Forty-eight-well plates at a density of 2x10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003ecell/well and treated with TEVs for 48 h. Then, HGFs were incubated with 0.05 mM monodansylcadaverine (MDC) (Promega, USA) at 37\u0026deg;C for 15 min, and then washed three times in PBS. Analyses were performed with a fluorescent microscope (Biozero BZ-8000, Keyence, Osaka, Japan).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFlow cytometry\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell suspensions of HGFs were prepared at a density of 1x10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ecell/ml. Next, the cells were incubated with dichloro-dihydro-fluorescein diacetate (DCFH-DA) using Reactive Oxygen Species Assay Kit (Promega, USA) in accordance with the manufacturer\u0026rsquo;s instructions. Then, flow cytometry (BD Biotechnology) was used to detect the relative level of ROS in HGFs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eApoptosis assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBriefly, the cells with treatments were stained using Annexin V/ propidium iodide (PI) (Beyotime, Shanghai, China). Labeled cells were washed and suspended in PBS. Flow cytometry and CytExpert software were used to analyze.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCCK-8 assay\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor the assessment of the proliferation ability of cells, cells were plated and cultured in 96-well plates (5000 cells per well). On the 0, 12, 24, 48, 72, and 96 hours after cell adherence, the proliferation ability of cells was examined with the CCK-8 kit (Biosharp, China) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunohistochemistry\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor the immunohistochemical staining, surgical specimens from patients with OSCC or adjacent normal mucosa and xenografts from mice were fixed with 10% formalin and embedded in paraffin. Sections from the above specimens were incubated with corresponding antibodies at 4\u0026deg;C overnight and detected by peroxidase-conjugated secondary antibody. For the statistical analyses, the stained slides were scored according to the intensity of staining (-: 0; +: 1; ++:2; and+++: 3) (x200 magnification) and the percentage of the cells of interest staining positive for each antigen (0%: 0; 1\u0026ndash;25%: 1; 26\u0026ndash;50%: 2; 51\u0026ndash;75%: 3; and 76\u0026ndash;100%: 4) (x40 magnification). The intensity score was added to the percentage score to obtain a final score.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunofluorescence\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor tissues, surgical specimens from patients with OSCC or adjacent normal mucosa were fixed with 10% formalin and embedded in paraffin. Sections from the above specimens were incubated with corresponding antibodies at 4\u0026deg;C overnight and then with Cy3-or FITC-conjugated secondary IgG (Servicebio, Wuhan). Analyses were performed with a fluorescent microscope (Biozero BZ-8000, Keyence, Osaka, Japan).\u003c/p\u003e\n\u003cp\u003eFor cells, PNFs and CAFs were fixed with 4% paraformaldehyde for 30 min and then washed with PBS for 3 times. After blocking with 5% BSA at room temperature for 30 min, cells were incubated with corresponding antibodies at 4\u0026deg;C overnight and then with Cy3-or FITC-conjugated secondary IgG (Servicebio, Wuhan). Analyses were performed with a fluorescent microscope (Biozero BZ-8000, Keyence, Osaka, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLuciferase reporter assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe pGL3-Basic plasmid inserted with the promoter sequence of HIF-1\u0026alpha; and Renilla luciferase plasmid (MiaoLing, Wuhan) were co-transfected into HGFs using Lipo3000, and then treated with TEVs. After 48h, luciferase activity was detected by Dual-Luciferase Reporter Assay Kit (Promega, USA) in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTumor xenografts in nude mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe in vivo studies were conducted following the guidance of the Ethical Committee on Animal Experiments of Animal Care Committee of Wuhan University (S07914060B). Briefly, female BALB/c nude mice (18-20 g, 4-6 weeks old) were used to establish tumor-bearing mouse models. The process and analysis were conducted as described in our previous study[14].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were repeated a minimum of three times, and the results were presented as mean \u0026plusmn; SD. Sample size determination did not involve any statistical method. Statistical analysis was performed using SPSS and GraphPad Prism 9 software, employing either one-way ANOVA or a non-parametric test. Each group exhibited similar variance. Statistical significance was defined as a P-value \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCAFs of OSCC exhibit high level of LC3B\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, we analyzed the expression of LC3B in the TCGA-HNSCC dataset. The result showed that LC3B expression was significantly higher in tumor tissues compared to the normal group (Fig. 1Aⅰ). Further correlation analysis suggested that the upregulation of LC3B seemed closely associated with stroma, specifically CAFs (Fig. 1Aii).\u0026nbsp;To verify this hypothesis, we detected the autophagic puncta by immunofluorescence staining of LC3B in OSCC tissues, and Vimentin staining was used to distinguish fibroblasts from tumor cells. The result also showed enhanced staining of LC3B in stroma (Fig. 1B). To further analyze the autophagy level in stroma cells, PNFs and CAFs were isolated from OSCC patients as our previous study[23]. Cell immunofluorescence and MDC staining were used to label the LC3B and acidic vesicular organelles in the autophagy process, respectively. The results showed that higher level of LC3B puncta and MDC-positive granular structures were present in CAFs than PNFs (Fig. 1C and D). Finally, we assessed the autophagy marker LC3B and the selective cargo receptor for autophagy, p62, by western blot. The results showed that LC3B expression in CAFs significantly elevated compared with PNFs. However, p62, the autophagic flux marker, which would decrease when autophagy level increased, was also higher in CAFs than PNFs (Fig. 1E). As cell autophagy is a dynamic process affected by the extracellular microenvironment and signals, we suspect that due to the lack of signals from tumor cells, the autophagy flux of primary fibroblasts that have been separated from the TME is blocked.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTEVs enhance the level of autophagy in fibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEVs are important mediators of tumor-stroma interaction and paly critical roles during tumor progression. And TEVs from OSCC cells have been reported to induce the activation and reprogramming of fibroblasts in our previous study[14]. So, we want to figure out whether the tumor derived signals, TEVs, enhance the autophagy level of fibroblasts. First, we treated the PNFs and CAFs with TEVs (20 \u0026mu;g/ml) derived from CAL27 and SCC25, and assessed the expression of LC3B and p62. The results showed that both PNFs and CAFs exhibited significantly higher expression of comparative LC3B after treated with TEVs. But the expression of p62 exhibited the same increasing trend with LC3B in PNFs and CAFs (Fig. 2A and B). The results indicate that the overexpression of p62 in CAFs is not due to the lack of stimulation from tumor derived signals. Then TEVs were used to treat HGFs to generate TEVs-activated CAFs and to study their roles in autophagy of stromal fibroblasts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFirst, the influence of TEVs on proliferation and apoptosis of HGFs were evaluated. CCK-8 assay showed that proliferation of HGFs began to decrease after treated with TEVs for 48h (Fig. 2C). Western blot analysis of apoptotic marker Cl-caspase 3 showed no difference between TEVs-treated HGFs and negative control group (Fig. 2D). Besides, apoptotic assay was performed on TEVs-treated HGFs, the result showed that apoptosis of HGFs decreased after treated with TEVs for 48h (Fig. 2E ⅰ and ⅱ). The data above indicated that both proliferation and apoptosis of HGFs decrease after TEVs-treatment. Then we treated HGFs with TEVs for 1, 3, 6, 12 and 24h, respectively and evaluated the autophagy level of TEVs-treated HGFs. The results showed that as treatment time extension, the overall trend of comparative LC3B expression showed significant increase, although there was a little variation, while the expression of p62 maintained a steady enhancement (Fig. 2F and G). These results were in accordance with that in PNFs and CAFs. To better assess the autophagy level in TEVs-treated HGFs, we observed the autophagosomes with TEM and stained autophagic puncta by immunofluorescence of LC3B in TEVs-treated HGFs. Then we identified more abundant autophagosomes (Fig. 2H ⅰ and ⅱ) and augmented staining of LC3B (Fig. 2I) in TEVs-treated HGFs compared with control HGFs. However, the question that whether the increase in p62 means that the autophagy flux is blocked arises again. Then we utilized the tandem mCherry-GFP-LC3B adenovirus to construct HGFs expressing mCherry-GFP-LC3B fusion protein to confirm whether autophagy flux is unobstructed. The result showed that HGFs expressing mCherry-GFP-LC3B fusion protein was established successfully. When treating the constructed HGFs with TEVs for 24h, we observed accumulation of LC3B (green puncta) and increased red puncta that represents fusion of autophagosomes and lysosomes (Fig. 2G), indicating that autophagy flux is unobstructed in TEVs-treated HGFs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTEVs induce autophagy-dependent glycometabolic reprogramming in fibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAutophagy was reported to be closely associated with cellular metabolism and we have validated that TEVs mediated glycometabolic reprogramming in CAFs[14]. We wonder whether the glycometabolic reprogramming in CAFs was associated with autophagy. First, we evaluated the level of glycolysis related proteins, GLUT-1, PFKFB3, PKM2, PDK1 and MCT4 by western blot. Although there was a little variation, the expression of GLUT-1, PFKFB3, PKM2, PDK1 and MCT4 increased when HGFs were treated with TEVs. Besides, PFKFB3 increased stably with the time expansion of TEVs-treatment (Fig. 3A and B). Furthermore, the elevated expression of GLUT-1, PFKFB3, PKM2, PDK1 and MCT4 was also detected in the primary CAFs compared with PNFs (Fig. 3C). Then we investigated the relationship between autophagy and metabolic reprogramming in CAFs and TEVs-treated HGFs. First, autophagy inhibitor chloroquine (CQ) and 3-Methyladenine(3-MA) were used to inhibit autophagy and Earle\u0026apos;s balanced salt solution-induced starvation (STV) and rapamycin (RM) were used to enhance autophagy in CAFs. Western Blot results showed autophagy was inhibited or enhanced successfully, and the expression of GLUT-1, PFKFB3 and MCT4 were in accordance with the expression of LC3B, regardless of the expression of p62 (Fig. 3D). However, the expression of PDK1 and PKM2 had no significance (Fig. S1A).\u0026nbsp;Interestingly, in the STV group, LC3B, GLUT-1, PFKFB3 and MCT4 all showed lower expression compared with other groups. Then we inhibited TEVs-induced autophagy in TEVs-treated HGFs with CQ and 3-MA. The results showed that the expression of GLUT-1, PFKFB3 and MCT4, especially the expression of PFKFB3, were strictly consistent with expression of LC3B (Fig. 3E). Similarly, the expression of PDK1 and PKM2 had no significance (Fig. S1B). These results indicated that TEVs drive glycometabolic reprogramming in fibroblasts through LC3B-dependent non-classical autophagy.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDirect transfer of ROS via TEVs mediates the autophagy and glycometabolic reprogramming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor hyperproliferation is often accompanied with increased oxidative stress, characterized by elevated ROS production. Growing studies have shown that TEVs can transmit biological signaling molecules to regulate the receptor cells. Then, we speculated whether TEVs can directly transmit ROS to mediate the stress response of receptor fibroblasts. First, we detected the level of ROS in tumor cells and TEVs, and the results showed that ROS abound in tumor cells and TEVs, and ROS level in TEVs decreased with the prolongation of the time that TEVs detach from tumor cells (Fig. S2A, B and Fig. 4A, B). Therefore, in subsequent experiments, TEVs are used for experiments within 24 hours after acquisition to ensure that ROS level in TEVs is physiologically. Next, the ROS in TEVs was labeled with ROS probe DCF-DA and incubated with fibroblasts, and then observed under confocal microscopy. 1d-TEVs exhibited more intense fluorescence, while 7d-TEVs fluorescence was significantly reduced (Fig.4C and D). Furthermore, we detected the level of ROS uptake by fibroblasts within 24 hours via flow cytometry, the flow cytometry indicated plentiful ROS in HGFs at 12 hours (Fig.4E and F). NAC can clear ROS in TEVs effectively (Fig.4G and H). After HGFs incubated with NAC treated TEVs, flow cytometry showed that ROS internalized by HGFs were significantly inhibited (Fig.4I and J). Western Blot results showed that the expression of key glycolysis enzymes (PDK1, PKM2 and PFKFB3), GLUT1 and MCT4 were significantly upregulated under the stimulation of ROS derived from TEVs (Fig.4K and L). Meanwhile, the up-regulation of LC3B expression, regardless of p62, indicates that the transferred ROS enhanced the level of autophagy in HGFs (Fig.4K and L). And elimination of ROS in TEVs obviously reversed the enhancement of autophagy and glycolysis (Fig.4K and L). Interestingly, PFKFB3 expression closely mirrored that of LC3B. The aforementioned data revealed that transmission of ROS in TEVs plays a key role in HGFs autophagy and glycometabolic reprogramming.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHIF-1\u0026alpha;/PFKFB3 signal pathway\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;is involved in the ROS-mediated glycometabolic reprogramming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs an important binding site in the promoter sequence of the PFKFB3 gene, HIF-1\u0026alpha; is an oxidative stress-inducible gene. Under the stimulation of TEVs, the expression of HIF-1\u0026alpha; in fibroblasts is exhibited as the tendency to decrease after rising (Fig.5A and B), which was considered to be the stress response of fibroblasts before the elimination of extrinsic ROS. Furthermore, dual luciferase report assay revealed that TEVs promoted the transcription of HIF-1\u0026alpha; genes in HGFs (Fig.5C and D). Western Blot indicated that CAFs have elevated HIF-1\u0026alpha; level relative to PNFs (Fig.5E). Notably, under normoxic conditions, the level of HIF-1\u0026alpha; in TEVs are minimal, allowing us to rule out the possibility that the increase in HIF-1\u0026alpha; in CAFs after TEVs stimulation is due to the direct transfer of HIF-1\u0026alpha; (Fig.5F).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eConsistently, after ROS eliminated in TEVs by NAC, the ability of TEVs to induce HIF-1\u0026alpha; overexpression in HGFs was significantly reduced, which indicated that ROS delivered in TEVs is the key molecule in tumor oxidative stress transfer (Fig.5G and H). After selectively inhibiting the translation of HIF-1\u0026alpha; protein through KC7F2, only the up-regulated expression of PFKFB3, a key enzyme of HGF glycolysis induced by TEVs, was significantly blocked (Fig.5I, J and S3A, B). Considered together, tumor cells transfer their ROS to CAFs through TEVs, subsequently inducing glycometabolic reprogramming via the HIF-1\u0026alpha;/PFKFB3 axis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTEVs promote stroma autophagy and glycometabolic reprogramming \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further verify \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eexperiments conclusions, the xenografts were established \u003cem\u003ein vivo\u003c/em\u003e. CAL27 cells and HGFs were mixed at a ratio of 1:3 and co-injected into nude mouse subcutaneously. Exactly 7 days after the injection, TEVs (50 mg) or PBS was injected around the tumors every 3 days. Immunohistochemistry was conducted on the tumor dissection. Compared with the control group, the stromal cells of the TEVs-injected xenografts exhibited significantly higher LC3B, HIF-1\u0026alpha; and PFKFB3 expression (Fig6A, B). Moreover, similar results were confirmed in OSCC tissues and healthy mucosa tissues (Fig.6C, D). Overall,\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e results confirmed that TEVs elevated the autophagy levels and promoted glycolysis in the stroma.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTME is a multicellular system with complex tumor-stromal interactions, playing critical roles in all stages of the neoplastic process[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. CAFs constitute the most abundant stromal cell type of the TME. Confronting with harsh environment caused by the rapid process of cancer, tumor and stromal cells continuously engage in signal crosstalk and energy exchanges, thereby creating an environment conducive to tumor growth[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As an important medium of communication between tumor and stromal cells, TEVs were confirmed to exerts variety of biological functions[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, under adverse conditions of high oxidative stress caused by rapid tumor proliferation, it remains unclear what role TEVs play in the crosstalk between tumor cells and surrounding stromal cells. Here, we revealed that surrounding stromal cells could shoulder tumor oxidative stress via accepting ROS-rich TEVs. The ROS delivered by TEVs increase the autophagy levels in CAFs, thereby activating glycometabolic reprogramming to produce energy-rich metabolites for tumor cells. These findings shed light on the crucial role of TEVs in the stress transfer model and offer a theoretical rationale for ROS-mediated autophagy in the glycometabolic reprogramming of CAFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAutophagy, an intracellular lysosome-dependent degradation system, serves as a self-regulatory mechanism that enables cells to adapt to adverse conditions. By degrading and recycling dysfunctional or unnecessary cellular components, autophagy helps maintain cellular homeostasis and provides essential nutrients and energy during periods of stress or nutrient deprivation[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our previous research indicated that the activation of autophagy is essential for the acquisition of cancer stem cells properties to resist chemotherapy, starvation, or hypoxic conditions[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Besides, accumulating findings highlight that autophagy in stroma influences the TME[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Pavlides et al.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] metabolomics analysis of the mammary fat pads of WT and Cav-1\u0026ndash;deficient mice, combined with human breast cancer transcriptomic data[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], demonstrated that extensive stromal catabolism directly promoted tumor anabolic growth via a parasitic mechanism, and that blocking stromal autophagy would inhibit energy transfer to epithelial cancer cells. In this study, we confirmed that autophagy levels are significantly elevated in the OSCC stroma, as evidenced by higher LC3B expression and an increase in MDC-positive granular structures of CAFs. More importantly, although our previous research had confirmed that primary CAFs in OSCC undergo PFKFB3-driven metabolic reprogramming of glycolysis[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the upstream switch that initiates this process requires further investigation. In this work, we discovered that when autophagy was blocked by CQ and 3-MA, the downregulation of the glycolysis key enzyme PFKFB3, and the reduction in the import and export of glucose fluxes, GLUT1 and MCT4, were detected together. Conversely, activation of autophagy enhanced the expression of PFKFB3, GLUT1 and MCT4. Therefore, we propose that autophagy induces PFKFB3-driven glycolysis, providing a reasonable explanation for the glycometabolic reprogramming in CAFs.\u003c/p\u003e \u003cp\u003eTo further determine the factors affecting autophagy and glycometabolic reprogramming in tumor stromal cells, TEVs were purified as reported in our previous study[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. TEVs, a class of extracellular vesicles which exhibit higher correlations with the original cells in terms of protein level, can transfer bioinformation of cancer cells to the surrounding stromal cells and alter the phenotype of the recipient cells. In our previous research, TEVs were capable of mediating the FAP-dependent transition of normal fibroblasts to CAFs and activating the glycometabolic reprogramming through the transfer of p-ERK/2 protein[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Interestingly, in this study we surprising found that the proliferation of TEVs-treated normal fibroblasts was not active, and apoptosis also remained at a low level, indicating that the normal fibroblasts stimulated by TEVs were in a quiescent protective state[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. By evaluating autophagy levels, we validated that TEVs-mediated enhancement of fibroblast autophagy explained this phenomenon. Furthermore, inhibition of TEVs-mediated autophagy, partially blocks the enhancement of glycolysis. Unexpectedly, we observed a steady increase in p62 expression during the process of TEVs-treated fibroblasts. This seem suggest that the mechanism of TEVs-mediated autophagy appeared to bypass the need for p62, hinting at a potentially distinct regulatory pathway that governs this form of metabolic adaptation. For instance, recent studies have shown that some forms of autophagy can proceed via alternate pathways involving Beclin-1-independent[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] or LC3B-associated phagocytosis[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which do not require the conventional autophagy machinery. Therefore, exploring whether TEVs-mediated autophagy in fibroblasts also follows such unconventional routes could provide deeper insights into the complexity of tumor-stroma interactions and the versatile nature of cellular metabolism under the influence of tumor-derived factors.\u003c/p\u003e \u003cp\u003eHyperproliferation of tumor cells is accompanied by high ROS production. As a by-product of aerobic respiration, endogenous ROS are generated from mitochondrial metabolism, peroxisomes and the activity of the family of transmembrane NADPH oxidases (NOXs)[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, the conventional view is that oxidative burden pushes redox balance away from a reduced state by increasing their antioxidant status to resist hyperproliferation-driven ROS, while at the same time avoiding ROS thresholds that would trigger senescence or apoptosis[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Interestingly, we found that ROS release from tumor via TEVs is another way to balance the elevated levels of intracellular oxidative stress. After labeling TEVs-derived ROS with DCF-DA probe, further detection revealed that HGFs took in ROS transmitted by tumors and elevated autophagy, suggesting that stromal cells niche around the tumor play an important role in sharing tumor oxidative stress. Meanwhile, When ROS concentrations are elevated, the SQSTM1 gene encoding p62 can be transcriptional activated by the transcription factor Nrf2 and plays an important role in overall antioxidant and detoxification responses, which explains the stable increase in p62 in fibroblasts following TEVs stimulation[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, ROS transmitted by TEVs could activate glycolysis through both LC3B-dependent and HIF-1α/PFKFB3 pathways, and eliminating ROS effectively blocked glycometabolic reprogramming in CAFs. These findings highlight an intriguing mechanism where tumor cells, upon encountering stress stimuli, generate ROS not just as by-products of oxidative metabolism but as active signaling molecules. By releasing ROS via TEVs, tumor cells effectively transfer the oxidative burden to nearby CAFs, thereby outsourcing their oxidative stress. This transfer of stress induces autophagy and metabolic reprogramming within CAFs, which appear to undergo a self-sacrificial process to support the survival and growth of tumor cells.\u003c/p\u003e \u003cp\u003eIn conclusion, our discovery establishes a novel model of tumor oxidative stress via TEVs. The transferred TEVs, rich in ROS, drive glycometabolic reprogramming in CAFs through dual pathways by enhancing autophagy and the HIF-1α/PFKFB3 axis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by\u0026nbsp;National Natural Science Foundation of China (Grant No. 82103382), National Key R\u0026amp;D Programme of China (Grant No. 2022YFC2504200), National Natural Science Foundation of China (Grant No. 82273306) and Key Research Project of Hubei Province (Grant No. 2023BCB135).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRegistry and the Registration No. of the study: Ethics Committee of School and Hospital of Stomatology at Wuhan University (2022LUNSHENZIA05). Written informed consents were obtained from all patients participated. Animal works were approved by the Ethical Committee on Animal Experiments of the Animal Care Committee of Wuhan University (S07914060B).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch design: EJ;\u0026nbsp;Manuscripts writing: XL, EJ; Experiments performed: EJ, XL and XD; Data analysis: YX; Supervision: ZS and ZS.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available from the corresponding author upon reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen X, Song E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev Drug Discov. 2019;18:99\u0026ndash;115.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMezawa Y, Orimo A. The roles of tumor- and metastasis-promoting carcinoma-associated fibroblasts in human carcinomas. Cell Tissue Res. 2016;365:675\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhan H, Zhou B, Cheng Y, Xu J, Wang L, Zhang G, et al. Crosstalk between stromal cells and cancer cells in pancreatic cancer: New insights into stromal biology. Cancer Lett. 2017;392:83\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVitale I, Manic G, Galassi C, Galluzzi L. Stress responses in stromal cells and tumor homeostasis. Pharmacol Ther. 2019;200:55\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePolicastro LL, Iba\u0026ntilde;ez IL, Notcovich C, Duran HA, Podhajcer OL. The Tumor Microenvironment: Characterization, Redox Considerations, and Novel Approaches for Reactive Oxygen Species-Targeted Gene Therapy. Antioxid Redox Signal. 2013;19:854\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayes JD, Dinkova-Kostova AT, Tew KD. Oxidative Stress in Cancer. Cancer Cell. 2020;38:167\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeinberg F, Hamanaka R, Wheaton WW, Weinberg S, Joseph J, Lopez M et al. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proceedings of the National Academy of Sciences. 2010;107:8788\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong H, Reczek CR, McElroy GS, Steinert EM, Wang T, Sabatini DM et al. Metabolic determinants of cellular fitness dependent on mitochondrial reactive oxygen species. Sci Adv 6:eabb7272.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReczek CR, Birsoy K, Kong H, Mart\u0026iacute;nez-Reyes I, Wang T, Gao P, et al. A CRISPR screen identifies a pathway required for paraquat-induced cell death. Nat Chem Biol. 2017;13:1274\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39:44\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367:eaau6977.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThakur A, Ke X, Chen Y-W, Motallebnejad P, Zhang K, Lian Q, et al. The mini player with diverse functions: extracellular vesicles in cell biology, disease, and therapeutics. Protein Cell. 2022;9:631\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X-P, Wang M, Song Y, Song K, Yan T, Wang L, et al. Membrane microvesicles as mediators for melanoma-fibroblasts communication: Roles of the VCAM-1/VLA-4 axis and the ERK1/2 signal pathway. Cancer Lett. 2015;360:125\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang E, Xu Z, Wang M, Yan T, Huang C, Zhou X, et al. Tumoral microvesicle\u0026ndash;activated glycometabolic reprogramming in fibroblasts promotes the progression of oral squamous cell carcinoma. FASEB j. 2019;33:5690\u0026ndash;703.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarmati M, Bukva M, B\u0026ouml;r\u0026ouml;czky T, Buz\u0026aacute;s K, Gyukity-Sebesty\u0026eacute;n E. The role of the metabolite cargo of extracellular vesicles in tumor progression. Cancer Metastasis Rev. 2021;40:1203\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Li Y, Zou L, Zhu Z. Role of Exosomes in Crosstalk Between Cancer-Associated Fibroblasts and Cancer Cells. Front Oncol. 2019;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Zhao H, Liang W, Jiang E, Zhou X, Shao Z, et al. Autophagy regulates the cancer stem cell phenotype of head and neck squamous cell carcinoma through the noncanonical FOXO3/SOX2 axis. Oncogene. 2022;41:634\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevy JMM, Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer. 2017;17:528\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNew J, Arnold L, Ananth M, Alvi S, Thornton M, Werner L, et al. Secretory Autophagy in Cancer-Associated Fibroblasts Promotes Head and Neck Cancer Progression and Offers a Novel Therapeutic Target. Cancer Res. 2017;77:6679\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou W, Xu G, Wang Y, Xu Z, Liu X, Xu X, et al. Oxidative stress induced autophagy in cancer associated fibroblast enhances proliferation and metabolism of colorectal cancer cells. Cell Cycle. 2017;16:73\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiarugi P, Cirri P. Metabolic exchanges within tumor microenvironment. Cancer Lett. 2016;380:272\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavlides S, Whitaker-Menezes D, Castello-Cros R, Flomenberg N, Witkiewicz AK, Frank PG, et al. The reverse Warburg effect: Aerobic glycolysis in cancer associated fibroblasts and the tumor stroma. Cell Cycle. 2009;8:3984\u0026ndash;4001.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Jiang E, Zhao H, Chen Y, Xu Y, Feng C, et al. Glycometabolic reprogramming-mediated proangiogenic phenotype enhancement of cancer-associated fibroblasts in oral squamous cell carcinoma: role of PGC-1α/PFKFB3 axis. Br J Cancer. 2022;3:449\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng Y, Chen Y, Wu X, Chen J, Zhou Q, Liu B, et al. Interplay of energy metabolism and autophagy. Autophagy. 2024;1:4\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChow LQM. Head and Neck Cancer. N Engl J Med. 2020;382:60\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu J, Jiang E, Shang Z. Prognostic value of tumor\u0026ndash;stroma ratio in oral carcinoma: Role of cancer-associated fibroblasts. Oral Dis. 2023;29:1967\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRudnick JA, Debnath J. Autophagy in host stromal fibroblasts supports tumor desmoplasia. Autophagy. 2021;17:4497\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavlides S, Tsirigos A, Migneco G, Whitaker-Menezes D, Chiavarina B, Flomenberg N, et al. The autophagic tumor stroma model of cancer. Cell Cycle. 2010;9:3485\u0026ndash;505.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavlides S, Tsirigos A, Vera I, Flomenberg N, Frank PG, Casimiro MC, et al. Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the reverse Warburg effect: A transcriptional informatics analysis with validation. Cell Cycle. 2010;9:2201\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Shang Z, Zhou Y, Hu X, Chen Y, Fan Y, et al. Autophagy mediates glucose starvation-induced glioblastoma cell quiescence and chemoresistance through coordinating cell metabolism, cell cycle, and survival. Cell Death Dis. 2018;9:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrocoli A, Mathieu J, Priault M, Reiffers J, Souqu\u0026egrave;re S, Pierron G, et al. ATRA-induced upregulation of Beclin 1 prolongs the life span of differentiated acute promyelocytic leukemia cells. Autophagy. 2011;7:1108\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanjuan MA, Dillon CP, Tait SWG, Moshiach S, Dorsey F, Connell S, et al. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature. 2007;450:1253\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeretic V, Jiang S, Dupont N. Autophagy intersections with conventional and unconventional secretion in tissue development, remodeling and inflammation. Trends Cell Biol. 2012;22:397\u0026ndash;406.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSies H, Jones DP. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol. 2020;21:363\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForman HJ, Zhang H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat Rev Drug Discov. 2021;20:689\u0026ndash;709.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimon H-U, Friis R, Tait SWG, Ryan KM. Retrograde signaling from autophagy modulates stress responses. Sci Signal. 2017;10:eaag2791.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Tumor-derived extracellular vesicles, Autophagy, Glycometabolic reprogramming, Oxidative stress, CAFs, OSCC","lastPublishedDoi":"10.21203/rs.3.rs-5432071/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5432071/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eStressed by the adverse conditions of the tumor microenvironment (TME), hyperproliferation of tumor cells is accompanied by high production of reactive oxygen species (ROS). Tumor-derived extracellular vesicles (TEVs) exert a variety of biological functions in the interaction between the tumor cells and cancer-associated fibroblasts (CAFs). However, the intercellular transmission of ROS and its role in tumor-stroma communication remain unclear.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e \u003cp\u003eThe human gingival fibroblasts (HGFs), paracancerous normal fibroblasts (PNFs) and CAFs were isolated from the healthy gingival tissues of volunteers and six patients with OSCC. HGFs were treated with CAL27/SCC25 TEVs. \u003cem\u003eIn vitro\u003c/em\u003e, we assessed the level of autophagy and glycometabolism in PNFs/CAFs and HGFs/TEVs-treated HGFs by immunofluorescence and Western blot. Blockage or activation of autophagy was employed to investigate its effects on glycometabolism. Flow cytometry was used to detect whether TEVs play a crucial role in inducing fibroblast autophagy and glycolysis through ROS transfer. \u003cem\u003eIn vivo\u003c/em\u003e, xenograft models were established to validated the effect of TEVs.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCAFs exhibited higher level in autophagy compared with PNFs. Treatment with autophagy inhibitors diminished autophagy-dependent glycometabolic reprogramming induced by TEVs, whereas activation of autophagy enhanced glycolysis in CAFs. Furthermore, ROS transferred by TEVs was confirmed to drive glycometabolic reprogramming through both autophagy-dependent mechanisms and the HIF-1α/PFKFB3 axis. \u003cem\u003eIn vivo\u003c/em\u003e, TEVs consistently promoted autophagy and glycometabolic reprogramming.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eTEVs-induced intercellular transmission of ROS and the regulatory role of ROS-mediated autophagy in the glycometabolic reprogramming of CAFs, providing a novel rationale for the oxidative stress transfer model in tumor-stroma crosstalk.\u003c/p\u003e","manuscriptTitle":"Tumor-derived Extracellular Vesicles-mediated Oxidative Stress Transfer Activates Glycometabolic Reprogramming of CAFs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-18 06:28:27","doi":"10.21203/rs.3.rs-5432071/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":"ec00b26b-36cf-4334-aa4b-21f05478175a","owner":[],"postedDate":"December 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-30T10:24:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-18 06:28:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5432071","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5432071","identity":"rs-5432071","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}