M1 macrophage-derived exosomal miR-20b promotes radiosensitization in HPV + HNSC

M1 macrophage-derived exosomal miR-20b promotes radiosensitization in HPV + HNSC | 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 M1 macrophage-derived exosomal miR-20b promotes radiosensitization in HPV + HNSC Huan Liu, Siwei Zhang, Wanlin Li, Zengchen Liu, Tingdan Gong, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5372230/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 Human papillomavirus (HPV) is a significant risk factor for head and neck squamous cell carcinoma (HNSC). M1 macrophages enhance the radiosensitivity of HPV + HNSC. Research has demonstrated that M1 macrophage-derived exosomes (M1 exos) possess a more potent anti-tumor function, and these exosomes serve as crucial mediators of communication between tumor cells and the tumor microenvironment. However, the role of M1 exos in the radiation sensitivity of HNSC remains unclear. Materials and Methods HPV status and macrophage infiltration levels in the tissues of 25 HNSC were evaluated using IHC. M1 macrophages were induced and cultured in vitro, and exosomes were extracted through differential ultracentrifugation. The effect of M1 macrophage exosomes on the radiotherapy sensitivity of HPV + HNSC was investigated using an in vitro co-culture system. The expression level of γ-H2AX was assessed by immunofluorescence. Data from TCGA and GEO databases were utilized to evaluate the levels of miR-20b in HNSC and its relationship with radiotherapy sensitivity and prognosis. Additionally, the radiosensitivity of SCC090 cells overexpressing miR-20b was assessed through cell experiments to determine the functional role of miR-20b. Finally, bioinformatics methods were employed to elucidate the mechanism by which miR-20b enhances radiotherapy sensitivity. Results In HPV + HNSC, M1 macrophages were highly infiltrated and played a crucial role in enhancing the sensitivity of HPV + HNSC to radiotherapy. M1 exos infiltrated HPV + HNSCC, increasing their sensitivity to radiation. Meanwhile, M1 macrophages were abundant in miR-20b than M2 macrophages, and the radiation sensitivity of HPV + HNSC was significantly increased by transfecting them with a miR-20b mimic. The target genes of miR-20b were involved in DNA damage repair and cell cycle regulation. By analyzing the function of the target genes, CCND1 was identified as a key gene through which miR-20b enhanced radiotherapy sensitivity in HPV + HNSC. Conclusion In this study, our data suggest that M1 exos, enriched with miR-20b, regulate the DNA damage repair pathway in tumor cells by targeting CCND1, thereby enhancing the sensitivity of tumors to radiotherapy. Consequently, miR-20b may represent a potential therapeutic strategy for HNSC. HPV HNSC Exosome miR-20b Radiotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Head and neck cancer, the sixth most prevalent cancer worldwide, presents in various anatomical locations within the head and neck region. In 2022, more than 890,000 new cases were confirmed, and approximately 450,000 deaths [ 1 , 2 ]. Approximately 90% of head and neck malignancies are classified as head and neck squamous cell carcinomas (HNSC), primarily due to factors such as smoking, alcohol abuse, and human papillomavirus (HPV) infections. With the control of tobacco and alcohol, the incidence of HNSC caused by HPV infection is increasing annually. According to the status of HPV infection, HNSC is divided into HPV + HNSC and HPV - HNSC. Compared to HPV - HNSC, HPV + HNSC is a special heterogeneous tumor with unique molecular and clinical features. HPV + HNSC patients are generally younger than HPV - HNSC, have smaller tumors, and show a greater responsiveness to radiation treatment [ 3 ]. As one of the primary methods of HNSC therapy, the enhancement of radiotherapy sensitivity by HPV may result from modifications to the cell cycle, delayed DNA damage repair, and changes in immune infiltration within the tumor microenvironment. These factors collectively contribute to increased radiotherapy sensitivity. However, the precise mechanisms underlying these effects require further investigation. Tumor-associated macrophages (TAMs) represent the predominant cell type within the tumor microenvironment, constituting roughly 50% of its cellular makeup [ 4 – 6 ]. They are essential to the processes of tumor initiation and progression. TAMs originate from circulating monocytes. Following stimulation, they polarize to M1 macrophages, which are characterized by anti-tumor functions, and M2 macrophages, which are associated with pro-tumor activities [ 7 ]. M1 macrophages inhibit tumor growth by secreting inflammatory factors, chemokines and exosomes. Studies have demonstrated that M1 macrophages significantly influence the sensitivity of tumor radiotherapy. HPV + HNSC enhances the infiltration of M1 macrophages through the release of interleukin-6 and miR-9, improving radiotherapy sensitivity [ 8 , 9 ]. During tumor radiotherapy, a significant number of M1 macrophage-derived exosomes (M1 exos) accumulate in the tumor microenvironment. This accumulation reduces the infiltration of immunosuppressive tumor cells and enhances the sensitivity of the tumor to radiotherapy [ 10 , 11 ]. Engineered M1 exos modify the tumor microenvironment, promote the repolarization of M2 macrophages, enhance phagocytosis, and serve as sensitizers for radiotherapy [ 12 ]. However, the impact of M1 exos on the radiation sensitivity of HNSC remains unclear. Exosomes are lipid bilayer vesicles characterized by a diameter between 30 and 200 nanometers [ 13 ]. These vesicles can be secreted by stromal cells, immune cells, and tumor cells in various pathological and physiological conditions. Exosomes consist of various bioactive molecules, including miRNAs, proteins, and mRNAs, which facilitate their distribution within the organism and play a crucial role in intercellular communication [ 14 , 15 ]. miRNAs, contained within exosomes, consist of small single-stranded RNA molecules that rang in length from 19 to 25 nucleotides and are significant contributors to tumor progression [ 16 ]. miRNAs target mRNA and inhibits gene expression to regulate cell growth, development, and metabolism. miR-122-3p and miR-340-5p suppress tumor growth and metastasis by regulating the expression levels of GRK4 and GTF2E2. miR-20b-5p inhibits the progression of thyroid and bladder cancer by modulating the MAPK-Erk signaling pathways and proteins associated with the cell cycle [ 17 , 18 ]. In oropharyngeal cancer, miR-20b is upregulated in HPV + HNSC and is the miRNA most significantly associated with HPV p16. Elevated levels of miR-20b are linked to a favorable prognosis in HNSCC [ 19 , 20 ]. As a member of the miR-17 family, miR-20b inhibits tumor progression by suppressing cell proliferation and inducing G1 phase cell cycle arrest. miR-20b can enhance tumor radiosensitivity and improve prognosis when combined with immune checkpoint inhibitors [ 21 ]. The levels of miR-20b in M1 macrophages are significantly higher than those in M2 macrophages [ 22 ]. However, the impact of elevated miR-20b levels in M1 macrophages on the radiotherapy sensitivity of HPV + HNSC is still unclear. In this study, we demonstrated that M1 exos enhanced the sensitivity of HPV + HNSC to radiotherapy by targeting the expression of CCND1 which associated with DNA damage repair pathway and cell cycle regulation. Our findings elucidate the mechanism by which M1 macrophages increase radiotherapy sensitivity through the exosomal pathway, thereby addressing the gap in understanding the functional role of M1 exos in HNSC. This research provides a novel direction for anti-tumor strategies in HNSC and establishes a theoretical foundation for the development of engineered exosomes from M1 macrophages. Material and Methods Patients samples The samples were collected from 25 patients diagnosed with HNSC at Shenzhen Third People’s Hospital from 2021 to 2023. The research ethics committee of Shenzhen Third People’s Hospital approved this study in accordance with the Declaration of Helsinki (2021-056). Clinical information and written informed consent were obtained from all participants involved in the study. Cell lines The HPV + HNSC cell line SCC090 was purchased from the American Type Culture Collection and maintained in high-glucose DMEM (ThermoFisher, C11995500BT) supplemented with 10% fetal bovine serum (FBS) (GIBCO, 10099141C) and 1% penicillin-streptomycin solution (PS) (P1400). The cells were tested for mycoplasma contamination, and no mycoplasma was detected. The cell lines were cultured in a humidified incubator at 37°C with 5% CO 2 . Macrophage differentiation and polarization The human monocyte cell line THP-1 was obtained from the American Type Culture Collection and cultured in RPMI 1640 medium (ThermoFisher, C11875500BT) supplemented with 10% FBS and 1% PS. All cells were maintained at 37 ℃ in a 5% CO 2 atmosphere. Macrophage polarization was performed as previously described [ 8 ]. Briefly, THP-1 cells were seeded in 12-well plates at a density of 5 × 10 5 cells per well and treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) (MCE, HY18739) to induce macrophage differentiation. After 24 hours, 100 ng/mL lipopolysaccharide (LPS) (Beyotime, S1732) and 20 ng/mL interferon-gamma (IFN-γ) (Beyotime, P5664) were added for an additional 48 hours to induce M1 macrophages. To obtain M1 exos without the influence of FBS-derived exosomes, the FBS was ultracentrifuged at 120,000 g for 20 hours prior to use. Exosome isolation and characterization The M1 exos were isolated from the cell supernatant using differential ultracentrifugation [ 9 ]. Briefly, the culture supernatant of M1 macrophages was collected and centrifuged at 200 g for 15 min, followed by centrifugation at 2,000 g for 20 min. This was followed by ultracentrifugation at 10,000 g for 30 min, and finally, centrifugation at 100,000 g for 70 min. The exosomes were then re-suspended in 50–100 µL of pre-cooled PBS and stored at -80°C. All ultracentrifugation procedures were conducted using a Beckman Coulter centrifuge. The temperature during all centrifugation steps was maintained at 4°C, and the operations were performed on ice. Particle size and concentration were analyzed using nanoparticle tracking analysis (NTA) with a Zetaview instrument from Particle Metrix (Germany). The isolated exosomes (10 µL) were placed on a copper mesh for 5 to 10 min, and the excess liquid was absorbed with filter paper in preparation for transmission electron microscopy (TEM) analysis. The samples were then visualized using a Hitachi HT7700 transmission electron microscope. M1 exos tracking In order to verify whether the exosomes were taken up by SCC090 cells, the M1 exos were labeled with a green fluorescent dye (PKH67, Sigma-Aldrich) according to the manufacturer’s instructions. These labeled exosomes were then incubated with SCC090 cells at 37°C for 2 h in the dark. The results were observed using a Zeiss LSM 980 confocal microscope. Immunohistochemistry Immunohistochemical staining of the tissue was performed using the method described previously [ 8 ]. Briefly, tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned to a thickness of 4 µm. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min, and goat serum (Bioss, C01-03001) was applied for 15 min. Sections were stained overnight at 4°C with antibodies against iNOS (1:100; Proteintech, 80517-1-RR), CD163 (1:200; Proteintech, 16646-1-AP), and p16 (1:150; ZSGB-Bio, ZM-0205), followed by incubation with a two-step IHC reagent (ZSGB-Bio, PV9000). The results were evaluated by two experienced pathologists in a double-blinded manner and graded according to previous research. Scores were assigned based on the proportion of positively stained cells and the intensity of staining: 0 (no positive cells), 1 ( 50% positive cells). The intensity of staining was evaluated using a defined scale: 0 indicates no staining, 1 represents weak staining (light yellow), 2 denotes medium staining (tan), and 3 signifies strong staining (brown). The staining index was calculated by multiplying the staining intensity score by the proportional score. Staining indices of 0, 1, 2, 3, 4, 6, and 9 were utilized for the assessment of the IHC. RNA scope Paraffin tissue sections were processed using the RNAscope® 2.5 HD Detection Kit-BROWN (ACD, 322310) in accordance with the manufacturer's instructions. Cell radiation assay The SCC090 cells, cultured in a 12-well plate, were co-cultured with various treatments for 24 h and then irradiated with 2 Gy X-ray using a RadSource RS2000 (US). After 24 h, the cells were fixed with 10% neutral formaldehyde for immunofluorescence staining of γ-H2AX. Immunofluorescence staining The SCC090 cells were fixed in 10% neutral formaldehyde for 15 min, permeabilized with 0.5% Triton X-100, and blocked with 5% goat serum for 30 min. Rabbit anti-human γ-H2AX (1:200; Abcam, ab81299) was incubated overnight at 4°C. After washing with PBS three times, the cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1:200; Proteintech Group, RGAR002) for 1 h at 37°C in the dark, followed by three washes with PBS. Finally, the samples were stained with DAPI (Beyotime, P0131) to visualize the nuclei. Sections were imaged using a Zeiss LSM 980 confocal microscope. Cell transfection SCC090 cells in the logarithmic growth phase were seeded in 6-well plates at 5 × 10 5 cells per well. The hsa-miR-20b-5p mimic, along with corresponding negative and positive controls (50 nM, Abm, MIH01532), were transfected into SCC090 cells using Lipofectamine 2000 (Invitrogen, 11668030) according to the manufacturer's instructions. Opti-MEM™ I medium (GIBCO, 31985070) was used as the dilution reagent. After 6 h, the transfection reagent was replaced with complete medium, and the cells were cultured for an additional 48 h for subsequent functional experiments. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from cells using Trizol® (Invitrogen, 15596018CN). The RNA was reverse transcribed using the PrimeScript RT Reagent Kit (Takara, RR047A) along with specific miR-20b stem-loop primers. TB Green Premix Ex Taq (Takara, RR820A) was utilized for qRT-PCR. U6 was served as an endogenous control. All experiments were conducted in triplicate, and the data were analyzed using the 2 −ΔΔCt method. The miR-20b primer sequences were designed using miRNA Design V1.01. The forward and reverse primers are detailed in the supplementary materials: Table S1 . Bioinformatics analysis HNSC data were downloaded from The Cancer Genome Atlas (TCGA) ( https://portal.gdc.cancer.gov/ ), which included 421 HPV − HNSC samples and 97 HPV + HNSC samples. Macrophage infiltration and survival analyses were conducted using TIMER 2.0 ( http://timer.cistrome.org/ ) [ 23 ]. The macrophage miRNA-seq expression profile data were obtained from the Gene Expression Omnibus (GEO) ( https://www.ncbi.nlm.nih.gov/geo/ ) [ 24 ]. MiRDB ( https://mirdb.org/ ) was utilized to analyze the target genes of miR-20b, with a target score greater than 50 set as the cutoff value. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) ( https://david.ncifcrf.gov ) was employed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. STRING ( https://string-db.org ) was used to analyze gene interaction relationships, which were visualized using Cytoscape software (v3.10.1). Gene enrichment results were plotted using an online platform for data analysis and visualization ( https://www.bioinformatics.com.cn ). Statistical analysis GraphPad Prism 10 was utilized for data analysis. The results are presented as mean ± SD. The Student's t-test or one-way ANOVA was employed to compare two or more independent groups. The Kaplan-Meier method was used for survival analysis. Pearson chi-squared tests were conducted to evaluate the correlation between two variables. Data were collected from a minimum of three independent experiments. Statistical significance was defined as p < 0.05. Results The infiltration of M1 macrophages increased in HPV + HNSC The dataset comprising 518 patients with HNSC was obtained from the TCGA database, which included 421 patients with HPV - HNSC and 97 patients with HPV + HNSC. By integrating data on macrophage infiltration in HPV - HNSC and HPV + HNSC from the CIBSORT-ABS database, we analyzed the influence of HPV HNSC on the infiltration of various macrophage subtypes. The results indicated that M0 macrophages and M2 macrophages did not differ significantly between HPV - HNSC and HPV + HNSC. However, M1 macrophages were significantly elevated in HPV + HNSC compared to HPV - HNSC ( p < 0.05) (Fig. 1 A). Twenty-five samples were collected from patients with HNSCC to assess HPV status and macrophage infiltration using IHC and RNA scope (Fig. 1 B). Then, the expression of iNOS (M1 macrophages) and CD163 (M2 macrophages) was analyzed using IHC. The results indicated that iNOS levels were significantly elevated in HPV + HNSC ( p < 0.05) (Fig. 1 C, D). In contrast, CD163 levels were significantly increased in HPV − HNSC ( p < 0.001) (Fig. 1 E, F). These results suggested that M1 macrophages were elevated in HPV + HNSC. Our histological findings were consistent with those from TCGA. Using the TIMER 2.0 database indicated that increased infiltration of M1 macrophages in HPV + HNSC was correlated with improved prognosis (Fig. S1 B). However, M0 macrophages and M2 macrophages did not impact the prognosis of either HPV + HNSC or HPV − HNSC (Fig. S1 A, C). M1 macrophages enhanced the radiosensitivity of HPV + HNSC This study aims to determine the effect of macrophages infiltration on the sensitivity of HNSC to radiotherapy. By analyzing the levels of macrophage infiltration in 518 patients with varying sensitivities to radiotherapy, we found that M0 and M2 macrophages did not influence the radiotherapy response (Fig. 2 A, C). In contrast, M1 macrophages were associated with increased radiosensitivity in HNSC (Fig. 2 B) ( p < 0.001). The flow chart was followed when conducting in vitro cell tests (Fig. 2 D). The amount of γ-H2AX indicated the radiotherapy sensitivity following 2Gy irradiation of HNSC. Compared to SCC090 or SCC090 and M0 macrophages, the addition of M1 macrophages to SCC090 cells was observed to improve radiosensitivity ( p < 0.01) (Fig. 2 E, F). This suggested that M1 macrophages were essential for raising the radiosensitivity of HPV + HSNC. MiR-20b of M1 exos elevated the radiosensitivity of HPV + HNSC Exosomes play a crucial role in intercellular communication, and M1 exos are significant in anti-tumor responses. This study aims to explore the role of M1 exos in enhancing the sensitivity of HNSC to radiotherapy. First, M1 macrophages were induced according to the diagram, and M1 exos were isolated from the cell culture medium using differential ultracentrifugation (Fig. 3 A). TEM analysis revealed a characteristic two-disc structure (Fig. 3 C). NTA analysis revealed high exosome concentrations in the range of 50–150 nm (Fig. 3 D). Next, M1 exos were labeled with fluorescent PKH67 and incubated with SCC090 cells, which demonstrated that M1 exos were internalized by SCC090 cells (Fig. 3 B). The radiosensitivity of SCC090 cells, both with and without M1 exos, was evaluated using IF to detect γ-H2AX foci (Fig. 3 E, F). The results indicated that M1 exos enhanced sensitivity to radiation therapy in HPV + HSNC. Exosomal miRNAs serve as a crucial medium for information exchange between cells and play a significant role in the pathogenesis of various tumors. miR-20b is the miRNA most closely associated with HPV 16 and linked to an improved prognosis in HPV + HSNC. Therefore, we analyzed the miRNA-seq expression profile data of macrophages from GSE53107 and found that the expression of miR-20b in M1 macrophages was significantly higher than that in M2 macrophages ( p < 0.01) (Fig. 4 A). Additionally, miRNA data from TCGA indicated that miR-20b was significantly elevated in radio-sensitive patients with HNSC ( p < 0.01) (Fig. 4 B). Furthermore, compared to HPV − HNSC, miR-20b levels were significantly higher and positively correlated with a favorable prognosis in HPV + HNSC compared to HPV − HNSC ( p < 0.001) (Fig. 4 C, D). To clarify the role of miR-20b in the radiosensitivity of HPV + HNSC, the miR-20b mimic was utilized to upregulate the expression of miR-20b in SCC090 cells, as illustrated in the schematic diagram (Fig. 4 E). qRT-PCR was employed to assess the expression level of miR-20b, which the miR-20b mimic was transfected after 24 h (Fig. S2). SCC090 cells, both with and without miR-20b mimics, were subsequently exposed to 2 Gy X-ray radiation and analyzed for gamma-H2AX foci using immunofluorescence (Fig. 4 F). Quantitative immunofluorescence analysis demonstrated that, in comparison to the control group and the simulated negative control group, the γ-H2AX foci in SCC090 cells treated with the miR-20b mimic were significantly elevated (Fig. 4 G). Those results indicated that M1 macrophages played a crucial role in the sensitization of HPV + HNSC through exosomal miR-20b. CCND1 is a hub gene in the enhancement of radiosensitivity in HPV + HNSC by miR-20b. To elucidate the mechanism by which miR-20b enhanced radiosensitivity, we analyzed and identified 1,974 genes that are negatively correlated with miR-20b expression in HNSC from TCGA. The top 50 genes that exhibit a negative correlation with miR-20b were showed (Fig. 5 A). Target genes of miR-20b were retrieved from the miRDB database, yielding 1,315 genes with a target score > 50. Among the 1,974 genes identified in TCGA, we found 265 overlapping target genes in the miRDB dataset (Fig. 5 B). The 265 genes were visualized using Cytoscape and organized according to their degree scores, revealing seven genes with the highest levels of interaction (Fig. 5 C). To clarify the function of miR-20b target genes, a functional enrichment analysis was conducted on 265 genes. The results of the GO enrichment analysis indicated that miR-20b was significantly associated with cytokine response, DNA damage repair response, and the G1/S transition of the mitotic cell cycle (Fig. 5 D). Additionally, the KEGG enrichment analysis revealed that the target genes were enriched in cancer pathways, as well as the JAK-STAT and PI3K-Akt signaling pathways (Fig. 5 E). These findings suggest that miR-20b target genes are linked to tumorigenesis, cell proliferation, and DNA damage response pathways. Notably, MAPK1, PDGFRB, CCND1, MMP2, HIF1A, and PXDN are all implicated in DNA damage repair and cell cycle-related pathways. To identify the primary target genes of miR-20b that influence the DNA damage repair pathway and cell cycle transition, we analyzed the expression levels of these genes across different HPV statuses and radiotherapy sensitivities. Using Pearson correlation analysis, we assessed the relationship between the target genes, M1 macrophage infiltration, and the expression levels of miR-20b. The results indicated that the expression of CCND1, PDGFRB, HIF1A, PDXN and MMP2 was significantly reduced in the HPV + HNSC, and the expression of MAPK1 was no difference in HPV + HNSC (Fig. 6 A, Fig. S3). In completely response of radiotherapy cohorts, the expression of CCND1, MAPK1, PDGFRB and PDXN was decreased, and the expression of HIF1A and MMP2 was no no effect on radiotherapy sensitivity(Fig. 6 B, Fig. S4). In order to investigate the influence of target genes on M1 macrophage infiltration, correlation analysis revealed that only CCND1 exhibited a negative correlation with M1 macrophage infiltration in HPV + HNSC (Fig. 6 C). However, other target genes did not show a significant association with M1 macrophage infiltration in either HPV + HNSC (Fig. S5). Using the same method, a correlation analysis of target genes and miR-20b expression was conducted. The results indicated that all target genes were negatively correlated with miR-20b, consistent with the original findings regarding these target genes (Fig. 6 D, Fig. S6). To further elucidate the relationship between target genes and the outcome of HPV + HNSC, an analysis using Timer 2.0 revealed that only low expression levels of CCND1 and PXDN were associated with a favorable prognosis for HPV + HNSC. In contrast, the expression levels of the other target genes did not show a significant correlation with prognosis (Fig. 6 E, Fig. S7). However, regardless of HPV status, the expression level of CCND1 did not influence the prognosis of HNSC (Fig. 6 F). Therefore, based on this analysis, we reasonably speculate that miR-20b primarily alters DNA damage repair and cell cycle-related pathways through the targeted regulation of CCND1 expression. Discussion HPV is a significant risk factor for the development of HNSC and is associated with various clinical characteristics and the biological behavior of the tumor, including response to radiation therapy [ 25 ]. Notably, HPV + HNSC exhibits a better prognosis and greater sensitivity to radiotherapy compared to HPV − HNSC [ 26 ]. As the primary modality for tumor treatment, the effectiveness of radiotherapy on cancer cells is influenced by DNA damage repair mechanisms and cell cycle distribution [ 27 , 28 ]. Currently, HPV enhances the sensitivity of HNSC to radiotherapy primarily through its oncogenes, which lead to delayed DNA damage repair, cell cycle arrest, and immune modulation, thereby increasing the susceptibility of tumor cells to radiotherapy [ 29 , 30 ]. Research has demonstrated that HPV improves tumor prognosis by promoting the polarization of TAMs towards the M1 macrophages, which subsequently alters the tumor's response to radiotherapy [ 8 ]. As the primary immune cells in the tumor microenvironment, macrophages can be polarized into M1 and M2 macrophages following stimulation, which play a “good” or “bad” role in tumor progression [ 31 ]. M1 macrophages suppress tumor cells either directly or indirectly by releasing inflammatory factors and activating other immune cells. In contrast, M2 macrophages promote tumor cell growth by secreting growth factors and participating in the remodeling of the tumor extracellular matrix. With the advancement of nanotechnology, the exchange of information between tumor cells and surrounding cells has been identified through the exosome pathway, including the regulation of macrophage polarization. In laryngeal squamous cell carcinoma, tumor cells induce mononuclear macrophages to polarize into M2 macrophages via long non-coding RNA HOX transcript antisense RNA contained in exosomes, which diminishes the anti-tumor effect [ 32 ]. In HNSC, the status of HPV influences the release of exosomes by tumor cells. HPV − HNSC tumor cells release more exosomes than HPV + HNSCC tumor cells, and the substances contained within these exosomes also differ significantly [ 33 ]. Our previous results demonstrated that exosomes released by HPV + HNSC promoted the polarization of mononuclear macrophages to M1 macrophages, improving tumor prognosis [ 9 ]. M1 macrophages may also regulate tumor cell function through the exosomal pathway. Numerous studies have shown that M1 macrophages, as natural nanovesicles, exhibit excellent biocompatibility and stable drug-carrying capacity, making them suitable for targeted tumor therapy [ 12 ]. Engineered M1 exos act as sensitizing agents for tumor radiotherapy. However, the increased infiltration of M1 macrophages enhances sensitivity to radiotherapy in HPV + HNSC, the underlying molecular mechanisms remain unclear. In this study, we found that M1 exos increased the sensitivity of HPV + HNSC to radiotherapy by releasing exosomes. The results indicated that M1 exos were a significant component of the tumor microenvironment and facilitated information exchange between tumor cells and immune cells. Exosomes are extracellular vesicles composed of a lipid bilayer that are released by various cells, including tumor cells. They contain varieties of active substances, such as miRNAs. Exosomes serve as important mediators of intercellular communication and play a role in anti-tumor activity through the delivery of miRNAs [ 34 ]. Dysregulation of miRNA expression is closely associated with cancer and can either promote or inhibit tumor progression. Studies have demonstrated that miRNAs are transferred to target cells via exosomes, thereby regulating the functions of those target cells [ 35 , 36 ]. MiR-20b, a member of the miR-17 family, is significantly elevated and associated with a favorable prognosis in HPV + HNSC [ 37 ]. Research indicates that miR-20b, recognized as a biomarker for cancer, plays a crucial role in regulating the cell cycle, cell proliferation, and apoptosis. It exhibits both anti-tumor and pro-tumor effects in various tumors [ 38 ]. The increased levels of miR-20b in breast and prostate cancers promote tumor cell proliferation by inhibiting the expression of phosphatase and tensin homolog [ 39 , 40 ]. Conversely, miR-20b can also suppress tumor cell proliferation by inhibiting cyclin-dependent kinases and can impede the proliferation of aggressive bladder cancer cells by targeting cyclins to block the G1 phase of the cell cycle. Additionally, it inhibits the expression of matrix metalloproteinases, thereby reducing tumor invasion [ 41 , 42 ]. In this study, Analysis of data from TCGA revealed that miR-20b levels are elevated in M1 macrophages, and this increase is significantly correlated with sensitivity to radiotherapy. In an in vitro co-culture system, it was demonstrated that M1 exos increased the sensitivity of HPV + HNSC to radiotherapy. Through the analysis of target genes and the functional enrichment of miR-20b, as well as the correlation of target genes with M1 macrophage infiltration, radiotherapy sensitivity, and HPV status, CCND1 emerged as a prominent gene, which had been identified as the key gene through which miR-20b regulates radiotherapy sensitivity in HPV + HNSC. CCND1 is a member of the cyclin family that plays a crucial role in regulating cell cycle progression and transcription. It is involved in the transition of cells from the G1 phase to the S phase by forming complexes with cell cycle-related kinases, thereby promoting cell proliferation [ 43 , 44 ]. Research has demonstrated that CCND1 serves as a biomarker for tumor phenotype and progression [ 7 ]. In various cancers, including liver cancer, thyroid cancer, and HNSC, the overexpression of CCND1 has been shown to enhance the proliferation and invasion of tumor cells [ 45 ]. In HNSC, CCND1 is recognized as a marker of poor prognosis. HPV infection appears to influence the expression of CCND1 in HNSC. The downregulation of CCND1 in HPV + HNSC can inhibit the activation of the DNA damage repair pathway, enhance sensitivity to radiation, and improve tumor prognosis [ 46 , 47 ]. Additionally, miR-20b has been shown to inhibit tumor growth in colon and bladder cancers by downregulating CCND1 [ 48 ]. Our study demonstrated that miR-20b delayed DNA damage repair and increased the sensitivity of tumor cells to radiotherapy by negatively regulating the expression of CCND1. In summary, based on the observation that increased infiltration of M1 macrophages improves the prognosis of HPV + HNSC, we demonstrated that high expression levels of exosomal miR-20b enhanced the radiotherapy sensitivity of HPV + HNSC. M1 macrophages derived exosomal miR-20b by downregulating the expression of CCND1, which inhibits the activation of the DNA damage repair pathway and arrest the cell cycle, thereby increasing the radiotherapy sensitivity of HNSC (Fig. 7 ). We elucidated the role of miR-20b in the radiotherapy sensitivity of HNSC, which not only clarifies the mechanism by which M1 macrophages confer radiotherapy sensitivity to HPV + HNSC but may also offer a potential therapeutic strategy for treating HNSC. Abbreviations CCND1 Cyclin D1 DMEM Dulbecco’s modified Eagle’s medium FBS Fetal bovine serum GO Gene Ontology HIF1A hypoxia-inducible factor 1 subunit alpha HNSC Head and neck squamous cell cancer HPV Human papillomavirus IHC Immunohistochemistry iNOS Inducible nitric oxide sythase RPMI Roswell Park Memorial Institute KEGG Kyoto Encyclopedia of Genes and Genomes M1 exos M1 macrophage-derived exosomes MAPK1 mitogen-activated protein kinase 1 MMP2 matrix metalloproteinase-2 NTA Nanoparticle tracking analysis PDGFRB platelet-derived growth factor receptor beta PMA Phorbol 12-myristate 13-acetat PS Penicillin-streptomycin solution PXDN peroxidasin qRT-PCR quantitative reverse transcription polymerase chain reaction SD Standard deviation TAMs Tumor-associated macrophages TCGA The Cancer Genome Atlas TEM Transmission electron microscopy TIMER Tumor Immune Estimation Resource. Declarations Data availability statement The data that support the findings of our study are available from the corresponding author, upon reasonable request. Author contribution statement Huan Liu, Siwei Zhang, Shuang Pan and Lanlan Wei designed the study strategy. Huan Liu, Siwei Zhang, Wanlin Li and Zengchen Liu worked with cell function experiments, such as Immunohistochemistry, immunofluorescence, exosome characterization. Siyu Duan and Tingdan Gong cultured cells. Tianyang Liu and Fangjia Tong analyzed the data. Tianyang Liu, Fangjia Tong, Shuang Pan and Lanlan Wei revised the paper. Lanlan Wei, Shuang Pan and Huan Liu supported the funding. All the authors read and approved the final manuscript. Funding This work was supported by the Shenzhen Science and Technology Innovation Program [grant number KCXFZ20211020163544002]; the Natural Science Foundation of Guangdong [grant number 2023A1515220104]; the National Natural Science Foundation of China Young Scientist Fund [grant number 8210100763]; the Shenzhen Science and Technology R&D Fund [grant number JCYJ20210324131607019]; the Postgraduate research and practice innovation project of Harbin Medical University [grant number YJSCX2024-29HYD]. Ethical approval Declarations The research ethics committee of Shenzhen Third People’s Hospital approved this study in accordance with the Declaration of Helsinki (approval no.2021-056). Consent for publication Declarations All authors have read and agreed to all the contents for publication. Competing interests All authors declare no competing interests. References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. 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CCND1 amplification contributes to immunosuppression and is associated with a poor prognosis to immune checkpoint inhibitors in solid tumors. Front Immunol. 2020;11:1620. Xu J, Lin DI. Oncogenic c-terminal cyclin D1 (CCND1) mutations are enriched in endometrioid endometrial adenocarcinomas. PLoS ONE. 2018;13(7):e0199688. Feng Z, Guo W, Zhang C, Xu Q, Zhang P, Sun J, Zhu H, Wang Z, Li J, Wang L, et al. CCND1 as a predictive biomarker of neoadjuvant chemotherapy in patients with locally advanced head and neck squamous cell carcinoma. PLoS ONE. 2011;6(10):e26399. Novotný J, Bandúrová V, Strnad H, Chovanec M, Hradilová M, Šáchová J, Šteffl M, Grušanović J, Kodet R, Pačes V, et al. Analysis of HPV-positive and HPV-negative head and neck squamous cell carcinomas and paired normal mucosae reveals cyclin D1 deregulation and compensatory effect of cyclin D2. Cancers (Basel). 2020;12(4):792. Dubot C, Bernard V, Sablin MP, Vacher S, Chemlali W, Schnitzler A, Pierron G, Ait Rais K, Bessoltane N, Jeannot E, et al. Comprehensive genomic profiling of head and neck squamous cell carcinoma reveals FGFR1 amplifications and tumour genomic alterations burden as prognostic biomarkers of survival. Eur J Cancer. 2018;91:47–55. Park SL, Cho TM, Won SY, Song JH, Noh DH, Kim WJ, Moon SK. MicroRNA-20b inhibits the proliferation, migration and invasion of bladder cancer EJ cells via the targeting of cell cycle regulation and Sp-1-mediated MMP-2 expression. Oncol Rep. 2015;34(3):1605–12. Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.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. 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09:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5372230/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5372230/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69077842,"identity":"8759da85-49ce-494f-bf7a-a16aae439111","added_by":"auto","created_at":"2024-11-15 11:24:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3842102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated M1 macrophage infiltration in HPV\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e HNSC.\u003c/strong\u003e (A) CIBERSORT-ABS analysis of M0, M1, M2 in HPV\u003csup\u003e+\u003c/sup\u003e and HPV\u003csup\u003e- \u003c/sup\u003eHNSC tissues. (B) p16 was analyzed using IHC and RNA\u003cem\u003e scope \u003c/em\u003ein HNSC. Scal bar = 50 μm. (C) IHC analysis iNOS expression in HNSC. Scal bar = 20 μm. (D) Quantification of iNOS for IHC. (E) IHC analysis CD163 expression in HNSC. Scal bar = 20 μm. (F) Quantification of CD163 for IHC. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5372230/v1/506adbe29f8370ceae46e695.jpg"},{"id":69077879,"identity":"1d07e061-71af-4206-ab5d-42a0532c21ec","added_by":"auto","created_at":"2024-11-15 11:24:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1273458,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eM1 macrophages infiltration promoted the radiosensitivity of HPV\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e HNSC. \u003c/strong\u003e(A-C) The relationship between M0, M1 and M2 macrophages infiltration contribute to radiosensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC and HPV\u003csup\u003e- \u003c/sup\u003eHNSC from TCGA. (D) The model showed the steps of functional test. (E) Immunofluorescence staining for γ-H2AX foci in SCC090 cells after irradiation 24 h (scale bar = 20 μm). (F) Quantitation of γ-H2AX foci after irradiation 24 h. ns, not significance. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5372230/v1/8b2327720a8e3a5232a87125.jpg"},{"id":69077876,"identity":"f700d558-f9d4-4d7c-8d3e-aca72e3fe404","added_by":"auto","created_at":"2024-11-15 11:24:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":692865,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eM1-exo promoted radiosensitization of HPV\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e HNSC.\u003c/strong\u003e (A) Shematic of M1-exo extraction and M1 macrophages polarization. The detailed description was shown in the Materials and Methods. (B) Representative immunofluorescence images of PKH67-labeled M1-exo (green) internalized into SCC090 cells (red). (C) TEM image of M1-exo. (D) NTA of M1-exo. (E) Immunofluorescence staining for γ-H2AX foci in SCC090 cells treated or not with M1-exo 24h after irradiation (scale bar = 20 μm). (F) Quantitation of γ-H2AX foci 24 h after irradiation. M1-exo, M1 macrophage derived exosome; NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy. ns, not significance; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5372230/v1/903533bd27f1c4ccbe6969c3.jpg"},{"id":69077883,"identity":"7cbf96ca-db5f-442e-9aac-f36d28c69ad6","added_by":"auto","created_at":"2024-11-15 11:24:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1320904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased radiosensitivity of HPV\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e HNSC by miR-20b.\u003c/strong\u003e (A) The expression of miR-20b in M1 and M2 macrophages by miRNA-seq from GEO. (B) MiR-20b expression between complete response group and no response group from TCGA. Data serve as mean ± SD. Student’s t-test. (C) MiR-20b expression between HPV\u003csup\u003e-\u003c/sup\u003e and HPV\u003csup\u003e+\u003c/sup\u003e HNSC from TCGA. (D) miR-20b expression and the outcome in HNSC from TCGA. (E) miR-20b mimic transfection flowchart. (F) Immunofluorescence staining for γ-H2AX foci in SCC090 cells treated or not with miR-20b mimic 24h after irradiation (scale bar = 20 μm). (G) Quantitation of γ-H2AX foci after irradiation 24 h. ns, not significant; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5372230/v1/75ab5542ed7b2b98988df65b.jpg"},{"id":69077881,"identity":"2f38dfe7-8a44-4823-9008-792bfaf089f0","added_by":"auto","created_at":"2024-11-15 11:24:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3600534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-20b regulates the expression of genes associated with DNA damage repair pathway.\u003c/strong\u003e (A) The genes which negative correlated with miR-20b were analysed in HNSC (Top 50); (B) The intersection of the genes negatively correlated with miR-20b and the genes predicted to be targeted by miR-20b; (C) Cytoscape was used to visualized 265 crossover genes, and the genes with the most crossovers were screened. (D-E) The results of mRNA-seq were analyzed by GO network (D) and KEGG network (E) analyses. ns, no significance; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5372230/v1/c2da74f594e3bd996b6836b4.jpg"},{"id":69077846,"identity":"ddfe8af3-0d92-481f-9a3b-b340a9f8d44c","added_by":"auto","created_at":"2024-11-15 11:24:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":790619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCND1 is a hub gene for promoting the radiosensitivity of HPV\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e HNSC.\u003c/strong\u003e\u0026nbsp; (A) Expression of CCND1 in HPV\u003csup\u003e+\u003c/sup\u003e HNSC and HPV\u003csup\u003e- \u003c/sup\u003eHNSC from TCGA. (B) The expression of CCND1 in radiotherapy-sensitive HNSC. (C) Correlation analysis of CCND1 expression and M1 macrophages infiltration in HPV\u003csup\u003e+\u003c/sup\u003e HNSC. (D) Correlation analysis of CCND1 and miR-20b expression. (E-F) HPV\u003csup\u003e+\u003c/sup\u003e HNSC (E) and HNSCC (F) survival respectively relative to the expression of CCND1. CCND1, cyclin D1. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e ***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5372230/v1/a964de93258e64e148997fbe.jpg"},{"id":69077875,"identity":"783c6177-552e-4bd5-bf18-c4b1dddb7ce0","added_by":"auto","created_at":"2024-11-15 11:24:35","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":475696,"visible":true,"origin":"","legend":"\u003cp\u003eThe schematic diagram of miR-20b promoting the radiosensitization of HPV\u003csup\u003e+\u003c/sup\u003e HNSC by inhibiting the expression of CCND1.\u003c/p\u003e","description":"","filename":"Fig7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5372230/v1/bec371dd736044f2f2bd6955.jpg"},{"id":89654381,"identity":"48999384-c5f2-4583-9b94-ba1e6d402553","added_by":"auto","created_at":"2025-08-22 10:17:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13003212,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5372230/v1/1c03d94e-b093-444e-a6f5-59abc91e4c3e.pdf"},{"id":69077839,"identity":"95867d66-c344-4118-809a-dadd5fe113a4","added_by":"auto","created_at":"2024-11-15 11:24:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3977238,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-5372230/v1/1f9a413218f6cd3660b941b2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"M1 macrophage-derived exosomal miR-20b promotes radiosensitization in HPV + HNSC","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHead and neck cancer, the sixth most prevalent cancer worldwide, presents in various anatomical locations within the head and neck region. In 2022, more than 890,000 new cases were confirmed, and approximately 450,000 deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Approximately 90% of head and neck malignancies are classified as head and neck squamous cell carcinomas (HNSC), primarily due to factors such as smoking, alcohol abuse, and human papillomavirus (HPV) infections. With the control of tobacco and alcohol, the incidence of HNSC caused by HPV infection is increasing annually. According to the status of HPV infection, HNSC is divided into HPV\u003csup\u003e+\u003c/sup\u003e HNSC and HPV\u003csup\u003e-\u003c/sup\u003e HNSC. Compared to HPV\u003csup\u003e-\u003c/sup\u003e HNSC, HPV\u003csup\u003e+\u003c/sup\u003e HNSC is a special heterogeneous tumor with unique molecular and clinical features. HPV\u003csup\u003e+\u003c/sup\u003e HNSC patients are generally younger than HPV\u003csup\u003e-\u003c/sup\u003e HNSC, have smaller tumors, and show a greater responsiveness to radiation treatment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As one of the primary methods of HNSC therapy, the enhancement of radiotherapy sensitivity by HPV may result from modifications to the cell cycle, delayed DNA damage repair, and changes in immune infiltration within the tumor microenvironment. These factors collectively contribute to increased radiotherapy sensitivity. However, the precise mechanisms underlying these effects require further investigation.\u003c/p\u003e \u003cp\u003eTumor-associated macrophages (TAMs) represent the predominant cell type within the tumor microenvironment, constituting roughly 50% of its cellular makeup [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. They are essential to the processes of tumor initiation and progression. TAMs originate from circulating monocytes. Following stimulation, they polarize to M1 macrophages, which are characterized by anti-tumor functions, and M2 macrophages, which are associated with pro-tumor activities [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. M1 macrophages inhibit tumor growth by secreting inflammatory factors, chemokines and exosomes. Studies have demonstrated that M1 macrophages significantly influence the sensitivity of tumor radiotherapy. HPV\u003csup\u003e+\u003c/sup\u003e HNSC enhances the infiltration of M1 macrophages through the release of interleukin-6 and miR-9, improving radiotherapy sensitivity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. During tumor radiotherapy, a significant number of M1 macrophage-derived exosomes (M1 exos) accumulate in the tumor microenvironment. This accumulation reduces the infiltration of immunosuppressive tumor cells and enhances the sensitivity of the tumor to radiotherapy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Engineered M1 exos modify the tumor microenvironment, promote the repolarization of M2 macrophages, enhance phagocytosis, and serve as sensitizers for radiotherapy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the impact of M1 exos on the radiation sensitivity of HNSC remains unclear.\u003c/p\u003e \u003cp\u003eExosomes are lipid bilayer vesicles characterized by a diameter between 30 and 200 nanometers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These vesicles can be secreted by stromal cells, immune cells, and tumor cells in various pathological and physiological conditions. Exosomes consist of various bioactive molecules, including miRNAs, proteins, and mRNAs, which facilitate their distribution within the organism and play a crucial role in intercellular communication [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. miRNAs, contained within exosomes, consist of small single-stranded RNA molecules that rang in length from 19 to 25 nucleotides and are significant contributors to tumor progression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. miRNAs target mRNA and inhibits gene expression to regulate cell growth, development, and metabolism. miR-122-3p and miR-340-5p suppress tumor growth and metastasis by regulating the expression levels of GRK4 and GTF2E2. miR-20b-5p inhibits the progression of thyroid and bladder cancer by modulating the MAPK-Erk signaling pathways and proteins associated with the cell cycle [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In oropharyngeal cancer, miR-20b is upregulated in HPV\u003csup\u003e+\u003c/sup\u003e HNSC and is the miRNA most significantly associated with HPV p16. Elevated levels of miR-20b are linked to a favorable prognosis in HNSCC [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As a member of the miR-17 family, miR-20b inhibits tumor progression by suppressing cell proliferation and inducing G1 phase cell cycle arrest. miR-20b can enhance tumor radiosensitivity and improve prognosis when combined with immune checkpoint inhibitors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The levels of miR-20b in M1 macrophages are significantly higher than those in M2 macrophages [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the impact of elevated miR-20b levels in M1 macrophages on the radiotherapy sensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC is still unclear.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that M1 exos enhanced the sensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC to radiotherapy by targeting the expression of CCND1 which associated with DNA damage repair pathway and cell cycle regulation. Our findings elucidate the mechanism by which M1 macrophages increase radiotherapy sensitivity through the exosomal pathway, thereby addressing the gap in understanding the functional role of M1 exos in HNSC. This research provides a novel direction for anti-tumor strategies in HNSC and establishes a theoretical foundation for the development of engineered exosomes from M1 macrophages.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients samples\u003c/h2\u003e \u003cp\u003eThe samples were collected from 25 patients diagnosed with HNSC at Shenzhen Third People\u0026rsquo;s Hospital from 2021 to 2023. The research ethics committee of Shenzhen Third People\u0026rsquo;s Hospital approved this study in accordance with the Declaration of Helsinki (2021-056). Clinical information and written informed consent were obtained from all participants involved in the study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines\u003c/h3\u003e\n\u003cp\u003eThe HPV\u003csup\u003e+\u003c/sup\u003e HNSC cell line SCC090 was purchased from the American Type Culture Collection and maintained in high-glucose DMEM (ThermoFisher, C11995500BT) supplemented with 10% fetal bovine serum (FBS) (GIBCO, 10099141C) and 1% penicillin-streptomycin solution (PS) (P1400). The cells were tested for mycoplasma contamination, and no mycoplasma was detected. The cell lines were cultured in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eMacrophage differentiation and polarization\u003c/h3\u003e\n\u003cp\u003eThe human monocyte cell line THP-1 was obtained from the American Type Culture Collection and cultured in RPMI 1640 medium (ThermoFisher, C11875500BT) supplemented with 10% FBS and 1% PS. All cells were maintained at 37 ℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Macrophage polarization was performed as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Briefly, THP-1 cells were seeded in 12-well plates at a density of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) (MCE, HY18739) to induce macrophage differentiation. After 24 hours, 100 ng/mL lipopolysaccharide (LPS) (Beyotime, S1732) and 20 ng/mL interferon-gamma (IFN-γ) (Beyotime, P5664) were added for an additional 48 hours to induce M1 macrophages. To obtain M1 exos without the influence of FBS-derived exosomes, the FBS was ultracentrifuged at 120,000 g for 20 hours prior to use.\u003c/p\u003e\n\u003ch3\u003eExosome isolation and characterization\u003c/h3\u003e\n\u003cp\u003eThe M1 exos were isolated from the cell supernatant using differential ultracentrifugation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Briefly, the culture supernatant of M1 macrophages was collected and centrifuged at 200 g for 15 min, followed by centrifugation at 2,000 g for 20 min. This was followed by ultracentrifugation at 10,000 g for 30 min, and finally, centrifugation at 100,000 g for 70 min. The exosomes were then re-suspended in 50\u0026ndash;100 \u0026micro;L of pre-cooled PBS and stored at -80\u0026deg;C. All ultracentrifugation procedures were conducted using a Beckman Coulter centrifuge. The temperature during all centrifugation steps was maintained at 4\u0026deg;C, and the operations were performed on ice.\u003c/p\u003e \u003cp\u003eParticle size and concentration were analyzed using nanoparticle tracking analysis (NTA) with a Zetaview instrument from Particle Metrix (Germany). The isolated exosomes (10 \u0026micro;L) were placed on a copper mesh for 5 to 10 min, and the excess liquid was absorbed with filter paper in preparation for transmission electron microscopy (TEM) analysis. The samples were then visualized using a Hitachi HT7700 transmission electron microscope.\u003c/p\u003e\n\u003ch3\u003eM1 exos tracking\u003c/h3\u003e\n\u003cp\u003eIn order to verify whether the exosomes were taken up by SCC090 cells, the M1 exos were labeled with a green fluorescent dye (PKH67, Sigma-Aldrich) according to the manufacturer\u0026rsquo;s instructions. These labeled exosomes were then incubated with SCC090 cells at 37\u0026deg;C for 2 h in the dark. The results were observed using a Zeiss LSM 980 confocal microscope.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining of the tissue was performed using the method described previously [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Briefly, tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned to a thickness of 4 \u0026micro;m. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min, and goat serum (Bioss, C01-03001) was applied for 15 min. Sections were stained overnight at 4\u0026deg;C with antibodies against iNOS (1:100; Proteintech, 80517-1-RR), CD163 (1:200; Proteintech, 16646-1-AP), and p16 (1:150; ZSGB-Bio, ZM-0205), followed by incubation with a two-step IHC reagent (ZSGB-Bio, PV9000). The results were evaluated by two experienced pathologists in a double-blinded manner and graded according to previous research. Scores were assigned based on the proportion of positively stained cells and the intensity of staining: 0 (no positive cells), 1 (\u0026lt;\u0026thinsp;10% positive cells), 2 (10\u0026ndash;50% positive cells), and 3 (\u0026gt;\u0026thinsp;50% positive cells). The intensity of staining was evaluated using a defined scale: 0 indicates no staining, 1 represents weak staining (light yellow), 2 denotes medium staining (tan), and 3 signifies strong staining (brown). The staining index was calculated by multiplying the staining intensity score by the proportional score. Staining indices of 0, 1, 2, 3, 4, 6, and 9 were utilized for the assessment of the IHC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA\u003c/b\u003e \u003cb\u003escope\u003c/b\u003e\u003c/p\u003e \u003cp\u003eParaffin tissue sections were processed using the RNAscope\u0026reg; 2.5 HD Detection Kit-BROWN (ACD, 322310) in accordance with the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell radiation assay\u003c/h3\u003e\n\u003cp\u003eThe SCC090 cells, cultured in a 12-well plate, were co-cultured with various treatments for 24 h and then irradiated with 2 Gy X-ray using a RadSource RS2000 (US). After 24 h, the cells were fixed with 10% neutral formaldehyde for immunofluorescence staining of γ-H2AX.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eThe SCC090 cells were fixed in 10% neutral formaldehyde for 15 min, permeabilized with 0.5% Triton X-100, and blocked with 5% goat serum for 30 min. Rabbit anti-human γ-H2AX (1:200; Abcam, ab81299) was incubated overnight at 4\u0026deg;C. After washing with PBS three times, the cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1:200; Proteintech Group, RGAR002) for 1 h at 37\u0026deg;C in the dark, followed by three washes with PBS. Finally, the samples were stained with DAPI (Beyotime, P0131) to visualize the nuclei. Sections were imaged using a Zeiss LSM 980 confocal microscope.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003eSCC090 cells in the logarithmic growth phase were seeded in 6-well plates at 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. The hsa-miR-20b-5p mimic, along with corresponding negative and positive controls (50 nM, Abm, MIH01532), were transfected into SCC090 cells using Lipofectamine 2000 (Invitrogen, 11668030) according to the manufacturer's instructions. Opti-MEM\u0026trade; I medium (GIBCO, 31985070) was used as the dilution reagent. After 6 h, the transfection reagent was replaced with complete medium, and the cells were cultured for an additional 48 h for subsequent functional experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells using Trizol\u0026reg; (Invitrogen, 15596018CN). The RNA was reverse transcribed using the PrimeScript RT Reagent Kit (Takara, RR047A) along with specific miR-20b stem-loop primers. TB Green Premix Ex Taq (Takara, RR820A) was utilized for qRT-PCR. U6 was served as an endogenous control. All experiments were conducted in triplicate, and the data were analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. The miR-20b primer sequences were designed using miRNA Design V1.01. The forward and reverse primers are detailed in the supplementary materials: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eHNSC data were downloaded from The Cancer Genome Atlas (TCGA) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which included 421 HPV\u0026thinsp;\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026thinsp;HNSC samples and 97 HPV\u003csup\u003e+\u003c/sup\u003e HNSC samples. Macrophage infiltration and survival analyses were conducted using TIMER 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://timer.cistrome.org/\u003c/span\u003e\u003cspan address=\"http://timer.cistrome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The macrophage miRNA-seq expression profile data were obtained from the Gene Expression Omnibus (GEO) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. MiRDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mirdb.org/\u003c/span\u003e\u003cspan address=\"https://mirdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to analyze the target genes of miR-20b, with a target score greater than 50 set as the cutoff value. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org\u003c/span\u003e\u003cspan address=\"https://string-db.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze gene interaction relationships, which were visualized using Cytoscape software (v3.10.1). Gene enrichment results were plotted using an online platform for data analysis and visualization (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 10 was utilized for data analysis. The results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The Student's t-test or one-way ANOVA was employed to compare two or more independent groups. The Kaplan-Meier method was used for survival analysis. Pearson chi-squared tests were conducted to evaluate the correlation between two variables. Data were collected from a minimum of three independent experiments. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThe infiltration of M1 macrophages increased in HPV\u003csup\u003e+\u003c/sup\u003e HNSC\u003c/h2\u003e \u003cp\u003eThe dataset comprising 518 patients with HNSC was obtained from the TCGA database, which included 421 patients with HPV\u003csup\u003e-\u003c/sup\u003e HNSC and 97 patients with HPV\u003csup\u003e+\u003c/sup\u003e HNSC. By integrating data on macrophage infiltration in HPV\u003csup\u003e-\u003c/sup\u003e HNSC and HPV\u003csup\u003e+\u003c/sup\u003e HNSC from the CIBSORT-ABS database, we analyzed the influence of HPV HNSC on the infiltration of various macrophage subtypes. The results indicated that M0 macrophages and M2 macrophages did not differ significantly between HPV\u003csup\u003e-\u003c/sup\u003e HNSC and HPV\u003csup\u003e+\u003c/sup\u003e HNSC. However, M1 macrophages were significantly elevated in HPV\u003csup\u003e+\u003c/sup\u003e HNSC compared to HPV\u003csup\u003e-\u003c/sup\u003e HNSC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eTwenty-five samples were collected from patients with HNSCC to assess HPV status and macrophage infiltration using IHC and RNA \u003cem\u003escope\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Then, the expression of iNOS (M1 macrophages) and CD163 (M2 macrophages) was analyzed using IHC. The results indicated that iNOS levels were significantly elevated in HPV\u003csup\u003e+\u003c/sup\u003e HNSC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). In contrast, CD163 levels were significantly increased in HPV\u0026thinsp;\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026thinsp;HNSC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F). These results suggested that M1 macrophages were elevated in HPV\u003csup\u003e+\u003c/sup\u003e HNSC. Our histological findings were consistent with those from TCGA. Using the TIMER 2.0 database indicated that increased infiltration of M1 macrophages in HPV\u003csup\u003e+\u003c/sup\u003e HNSC was correlated with improved prognosis (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). However, M0 macrophages and M2 macrophages did not impact the prognosis of either HPV\u003csup\u003e+\u003c/sup\u003e HNSC or HPV\u0026thinsp;\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026thinsp;HNSC (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eM1 macrophages enhanced the radiosensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC\u003c/h2\u003e \u003cp\u003eThis study aims to determine the effect of macrophages infiltration on the sensitivity of HNSC to radiotherapy. By analyzing the levels of macrophage infiltration in 518 patients with varying sensitivities to radiotherapy, we found that M0 and M2 macrophages did not influence the radiotherapy response (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C). In contrast, M1 macrophages were associated with increased radiosensitivity in HNSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The flow chart was followed when conducting in vitro cell tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The amount of γ-H2AX indicated the radiotherapy sensitivity following 2Gy irradiation of HNSC. Compared to SCC090 or SCC090 and M0 macrophages, the addition of M1 macrophages to SCC090 cells was observed to improve radiosensitivity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). This suggested that M1 macrophages were essential for raising the radiosensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HSNC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMiR-20b of M1 exos elevated the radiosensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC\u003c/h2\u003e \u003cp\u003eExosomes play a crucial role in intercellular communication, and M1 exos are significant in anti-tumor responses. This study aims to explore the role of M1 exos in enhancing the sensitivity of HNSC to radiotherapy. First, M1 macrophages were induced according to the diagram, and M1 exos were isolated from the cell culture medium using differential ultracentrifugation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). TEM analysis revealed a characteristic two-disc structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). NTA analysis revealed high exosome concentrations in the range of 50\u0026ndash;150 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Next, M1 exos were labeled with fluorescent PKH67 and incubated with SCC090 cells, which demonstrated that M1 exos were internalized by SCC090 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The radiosensitivity of SCC090 cells, both with and without M1 exos, was evaluated using IF to detect γ-H2AX foci (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). The results indicated that M1 exos enhanced sensitivity to radiation therapy in HPV\u003csup\u003e+\u003c/sup\u003e HSNC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExosomal miRNAs serve as a crucial medium for information exchange between cells and play a significant role in the pathogenesis of various tumors. miR-20b is the miRNA most closely associated with HPV 16 and linked to an improved prognosis in HPV\u003csup\u003e+\u003c/sup\u003e HSNC. Therefore, we analyzed the miRNA-seq expression profile data of macrophages from GSE53107 and found that the expression of miR-20b in M1 macrophages was significantly higher than that in M2 macrophages (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, miRNA data from TCGA indicated that miR-20b was significantly elevated in radio-sensitive patients with HNSC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Furthermore, compared to HPV\u0026thinsp;\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026thinsp;HNSC, miR-20b levels were significantly higher and positively correlated with a favorable prognosis in HPV\u003csup\u003e+\u003c/sup\u003e HNSC compared to HPV\u0026thinsp;\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026thinsp;HNSC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). To clarify the role of miR-20b in the radiosensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC, the miR-20b mimic was utilized to upregulate the expression of miR-20b in SCC090 cells, as illustrated in the schematic diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). qRT-PCR was employed to assess the expression level of miR-20b, which the miR-20b mimic was transfected after 24 h (Fig. S2). SCC090 cells, both with and without miR-20b mimics, were subsequently exposed to 2 Gy X-ray radiation and analyzed for gamma-H2AX foci using immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Quantitative immunofluorescence analysis demonstrated that, in comparison to the control group and the simulated negative control group, the γ-H2AX foci in SCC090 cells treated with the miR-20b mimic were significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Those results indicated that M1 macrophages played a crucial role in the sensitization of HPV\u003csup\u003e+\u003c/sup\u003e HNSC through exosomal miR-20b.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCCND1 is a hub gene in the enhancement of radiosensitivity in HPV\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eHNSC by miR-20b.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the mechanism by which miR-20b enhanced radiosensitivity, we analyzed and identified 1,974 genes that are negatively correlated with miR-20b expression in HNSC from TCGA. The top 50 genes that exhibit a negative correlation with miR-20b were showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Target genes of miR-20b were retrieved from the miRDB database, yielding 1,315 genes with a target score\u0026thinsp;\u0026gt;\u0026thinsp;50. Among the 1,974 genes identified in TCGA, we found 265 overlapping target genes in the miRDB dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The 265 genes were visualized using Cytoscape and organized according to their degree scores, revealing seven genes with the highest levels of interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To clarify the function of miR-20b target genes, a functional enrichment analysis was conducted on 265 genes. The results of the GO enrichment analysis indicated that miR-20b was significantly associated with cytokine response, DNA damage repair response, and the G1/S transition of the mitotic cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Additionally, the KEGG enrichment analysis revealed that the target genes were enriched in cancer pathways, as well as the JAK-STAT and PI3K-Akt signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These findings suggest that miR-20b target genes are linked to tumorigenesis, cell proliferation, and DNA damage response pathways. Notably, MAPK1, PDGFRB, CCND1, MMP2, HIF1A, and PXDN are all implicated in DNA damage repair and cell cycle-related pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the primary target genes of miR-20b that influence the DNA damage repair pathway and cell cycle transition, we analyzed the expression levels of these genes across different HPV statuses and radiotherapy sensitivities. Using Pearson correlation analysis, we assessed the relationship between the target genes, M1 macrophage infiltration, and the expression levels of miR-20b. The results indicated that the expression of CCND1, PDGFRB, HIF1A, PDXN and MMP2 was significantly reduced in the HPV\u003csup\u003e+\u003c/sup\u003e HNSC, and the expression of MAPK1 was no difference in HPV\u003csup\u003e+\u003c/sup\u003e HNSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, Fig. S3). In completely response of radiotherapy cohorts, the expression of CCND1, MAPK1, PDGFRB and PDXN was decreased, and the expression of HIF1A and MMP2 was no no effect on radiotherapy sensitivity(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Fig. S4). In order to investigate the influence of target genes on M1 macrophage infiltration, correlation analysis revealed that only CCND1 exhibited a negative correlation with M1 macrophage infiltration in HPV\u003csup\u003e+\u003c/sup\u003e HNSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). However, other target genes did not show a significant association with M1 macrophage infiltration in either HPV\u003csup\u003e+\u003c/sup\u003e HNSC (Fig. S5). Using the same method, a correlation analysis of target genes and miR-20b expression was conducted. The results indicated that all target genes were negatively correlated with miR-20b, consistent with the original findings regarding these target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, Fig. S6). To further elucidate the relationship between target genes and the outcome of HPV\u003csup\u003e+\u003c/sup\u003e HNSC, an analysis using Timer 2.0 revealed that only low expression levels of CCND1 and PXDN were associated with a favorable prognosis for HPV\u003csup\u003e+\u003c/sup\u003e HNSC. In contrast, the expression levels of the other target genes did not show a significant correlation with prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, Fig. S7). However, regardless of HPV status, the expression level of CCND1 did not influence the prognosis of HNSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Therefore, based on this analysis, we reasonably speculate that miR-20b primarily alters DNA damage repair and cell cycle-related pathways through the targeted regulation of CCND1 expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHPV is a significant risk factor for the development of HNSC and is associated with various clinical characteristics and the biological behavior of the tumor, including response to radiation therapy [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Notably, HPV\u003csup\u003e+\u003c/sup\u003e HNSC exhibits a better prognosis and greater sensitivity to radiotherapy compared to HPV\u0026thinsp;\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026thinsp;HNSC [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As the primary modality for tumor treatment, the effectiveness of radiotherapy on cancer cells is influenced by DNA damage repair mechanisms and cell cycle distribution [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Currently, HPV enhances the sensitivity of HNSC to radiotherapy primarily through its oncogenes, which lead to delayed DNA damage repair, cell cycle arrest, and immune modulation, thereby increasing the susceptibility of tumor cells to radiotherapy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Research has demonstrated that HPV improves tumor prognosis by promoting the polarization of TAMs towards the M1 macrophages, which subsequently alters the tumor's response to radiotherapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs the primary immune cells in the tumor microenvironment, macrophages can be polarized into M1 and M2 macrophages following stimulation, which play a \u0026ldquo;good\u0026rdquo; or \u0026ldquo;bad\u0026rdquo; role in tumor progression [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. M1 macrophages suppress tumor cells either directly or indirectly by releasing inflammatory factors and activating other immune cells. In contrast, M2 macrophages promote tumor cell growth by secreting growth factors and participating in the remodeling of the tumor extracellular matrix. With the advancement of nanotechnology, the exchange of information between tumor cells and surrounding cells has been identified through the exosome pathway, including the regulation of macrophage polarization. In laryngeal squamous cell carcinoma, tumor cells induce mononuclear macrophages to polarize into M2 macrophages via long non-coding RNA HOX transcript antisense RNA contained in exosomes, which diminishes the anti-tumor effect [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In HNSC, the status of HPV influences the release of exosomes by tumor cells. HPV\u003csup\u003e\u0026minus;\u003c/sup\u003e HNSC tumor cells release more exosomes than HPV\u003csup\u003e+\u003c/sup\u003e HNSCC tumor cells, and the substances contained within these exosomes also differ significantly [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our previous results demonstrated that exosomes released by HPV\u003csup\u003e+\u003c/sup\u003e HNSC promoted the polarization of mononuclear macrophages to M1 macrophages, improving tumor prognosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. M1 macrophages may also regulate tumor cell function through the exosomal pathway. Numerous studies have shown that M1 macrophages, as natural nanovesicles, exhibit excellent biocompatibility and stable drug-carrying capacity, making them suitable for targeted tumor therapy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Engineered M1 exos act as sensitizing agents for tumor radiotherapy. However, the increased infiltration of M1 macrophages enhances sensitivity to radiotherapy in HPV\u003csup\u003e+\u003c/sup\u003e HNSC, the underlying molecular mechanisms remain unclear. In this study, we found that M1 exos increased the sensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC to radiotherapy by releasing exosomes. The results indicated that M1 exos were a significant component of the tumor microenvironment and facilitated information exchange between tumor cells and immune cells.\u003c/p\u003e \u003cp\u003eExosomes are extracellular vesicles composed of a lipid bilayer that are released by various cells, including tumor cells. They contain varieties of active substances, such as miRNAs. Exosomes serve as important mediators of intercellular communication and play a role in anti-tumor activity through the delivery of miRNAs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Dysregulation of miRNA expression is closely associated with cancer and can either promote or inhibit tumor progression. Studies have demonstrated that miRNAs are transferred to target cells via exosomes, thereby regulating the functions of those target cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. MiR-20b, a member of the miR-17 family, is significantly elevated and associated with a favorable prognosis in HPV\u003csup\u003e+\u003c/sup\u003e HNSC [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Research indicates that miR-20b, recognized as a biomarker for cancer, plays a crucial role in regulating the cell cycle, cell proliferation, and apoptosis. It exhibits both anti-tumor and pro-tumor effects in various tumors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The increased levels of miR-20b in breast and prostate cancers promote tumor cell proliferation by inhibiting the expression of phosphatase and tensin homolog [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Conversely, miR-20b can also suppress tumor cell proliferation by inhibiting cyclin-dependent kinases and can impede the proliferation of aggressive bladder cancer cells by targeting cyclins to block the G1 phase of the cell cycle. Additionally, it inhibits the expression of matrix metalloproteinases, thereby reducing tumor invasion [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, Analysis of data from TCGA revealed that miR-20b levels are elevated in M1 macrophages, and this increase is significantly correlated with sensitivity to radiotherapy. In an in vitro co-culture system, it was demonstrated that M1 exos increased the sensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC to radiotherapy.\u003c/p\u003e \u003cp\u003eThrough the analysis of target genes and the functional enrichment of miR-20b, as well as the correlation of target genes with M1 macrophage infiltration, radiotherapy sensitivity, and HPV status, CCND1 emerged as a prominent gene, which had been identified as the key gene through which miR-20b regulates radiotherapy sensitivity in HPV\u003csup\u003e+\u003c/sup\u003e HNSC. CCND1 is a member of the cyclin family that plays a crucial role in regulating cell cycle progression and transcription. It is involved in the transition of cells from the G1 phase to the S phase by forming complexes with cell cycle-related kinases, thereby promoting cell proliferation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Research has demonstrated that CCND1 serves as a biomarker for tumor phenotype and progression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In various cancers, including liver cancer, thyroid cancer, and HNSC, the overexpression of CCND1 has been shown to enhance the proliferation and invasion of tumor cells [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In HNSC, CCND1 is recognized as a marker of poor prognosis. HPV infection appears to influence the expression of CCND1 in HNSC. The downregulation of CCND1 in HPV\u003csup\u003e+\u003c/sup\u003e HNSC can inhibit the activation of the DNA damage repair pathway, enhance sensitivity to radiation, and improve tumor prognosis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Additionally, miR-20b has been shown to inhibit tumor growth in colon and bladder cancers by downregulating CCND1 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Our study demonstrated that miR-20b delayed DNA damage repair and increased the sensitivity of tumor cells to radiotherapy by negatively regulating the expression of CCND1.\u003c/p\u003e \u003cp\u003eIn summary, based on the observation that increased infiltration of M1 macrophages improves the prognosis of HPV\u003csup\u003e+\u003c/sup\u003e HNSC, we demonstrated that high expression levels of exosomal miR-20b enhanced the radiotherapy sensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC. M1 macrophages derived exosomal miR-20b by downregulating the expression of CCND1, which inhibits the activation of the DNA damage repair pathway and arrest the cell cycle, thereby increasing the radiotherapy sensitivity of HNSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). We elucidated the role of miR-20b in the radiotherapy sensitivity of HNSC, which not only clarifies the mechanism by which M1 macrophages confer radiotherapy sensitivity to HPV\u003csup\u003e+\u003c/sup\u003e HNSC but may also offer a potential therapeutic strategy for treating HNSC.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCND1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCyclin D1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHIF1A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehypoxia-inducible factor 1 subunit alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHNSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHead and neck squamous cell cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman papillomavirus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eiNOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInducible nitric oxide sythase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRPMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRoswell Park Memorial Institute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eM1 exos\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eM1 macrophage-derived exosomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMAPK1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitogen-activated protein kinase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMP2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ematrix metalloproteinase-2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNanoparticle tracking analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDGFRB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eplatelet-derived growth factor receptor beta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhorbol 12-myristate 13-acetat\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePenicillin-streptomycin solution\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePXDN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eperoxidasin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative reverse transcription polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTAMs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor-associated macrophages\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Cancer Genome Atlas\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransmission electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTIMER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor Immune Estimation Resource.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eThe data that support the findings of our study are available from the corresponding author, upon reasonable request.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuan Liu, Siwei Zhang, Shuang Pan and Lanlan Wei designed the study strategy. Huan Liu, Siwei Zhang, Wanlin Li and Zengchen Liu worked with cell function experiments, such as Immunohistochemistry, immunofluorescence, exosome characterization. Siyu Duan and Tingdan Gong cultured cells. Tianyang Liu and Fangjia Tong analyzed the data. Tianyang Liu, Fangjia Tong, Shuang Pan and Lanlan Wei revised the paper. Lanlan Wei, Shuang Pan and Huan Liu supported the funding. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the\u0026nbsp;Shenzhen Science and Technology Innovation Program\u0026nbsp;[grant number KCXFZ20211020163544002]; the Natural Science Foundation of Guangdong [grant number 2023A1515220104]; the National Natural Science Foundation of China Young Scientist Fund [grant number 8210100763]; \u0026nbsp;the Shenzhen Science and Technology R\u0026amp;D Fund [grant number JCYJ20210324131607019]; the Postgraduate research and practice innovation project of Harbin Medical University [grant number YJSCX2024-29HYD].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research ethics committee of Shenzhen Third People\u0026rsquo;s Hospital approved this study in accordance with the Declaration of Helsinki (approval no.2021-056).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to all the contents for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. 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PLoS ONE. 2018;13(7):e0199688.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng Z, Guo W, Zhang C, Xu Q, Zhang P, Sun J, Zhu H, Wang Z, Li J, Wang L, et al. CCND1 as a predictive biomarker of neoadjuvant chemotherapy in patients with locally advanced head and neck squamous cell carcinoma. PLoS ONE. 2011;6(10):e26399.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNovotn\u0026yacute; J, Band\u0026uacute;rov\u0026aacute; V, Strnad H, Chovanec M, Hradilov\u0026aacute; M, Š\u0026aacute;chov\u0026aacute; J, Šteffl M, Grušanović J, Kodet R, Pačes V, et al. Analysis of HPV-positive and HPV-negative head and neck squamous cell carcinomas and paired normal mucosae reveals cyclin D1 deregulation and compensatory effect of cyclin D2. Cancers (Basel). 2020;12(4):792.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDubot C, Bernard V, Sablin MP, Vacher S, Chemlali W, Schnitzler A, Pierron G, Ait Rais K, Bessoltane N, Jeannot E, et al. Comprehensive genomic profiling of head and neck squamous cell carcinoma reveals FGFR1 amplifications and tumour genomic alterations burden as prognostic biomarkers of survival. Eur J Cancer. 2018;91:47\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark SL, Cho TM, Won SY, Song JH, Noh DH, Kim WJ, Moon SK. MicroRNA-20b inhibits the proliferation, migration and invasion of bladder cancer EJ cells via the targeting of cell cycle regulation and Sp-1-mediated MMP-2 expression. Oncol Rep. 2015;34(3):1605\u0026ndash;12.\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":"HPV, HNSC, Exosome, miR-20b, Radiotherapy","lastPublishedDoi":"10.21203/rs.3.rs-5372230/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5372230/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHuman papillomavirus (HPV) is a significant risk factor for head and neck squamous cell carcinoma (HNSC). M1 macrophages enhance the radiosensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC. Research has demonstrated that M1 macrophage-derived exosomes (M1 exos) possess a more potent anti-tumor function, and these exosomes serve as crucial mediators of communication between tumor cells and the tumor microenvironment. However, the role of M1 exos in the radiation sensitivity of HNSC remains unclear.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eHPV status and macrophage infiltration levels in the tissues of 25 HNSC were evaluated using IHC. M1 macrophages were induced and cultured in vitro, and exosomes were extracted through differential ultracentrifugation. The effect of M1 macrophage exosomes on the radiotherapy sensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC was investigated using an in vitro co-culture system. The expression level of γ-H2AX was assessed by immunofluorescence. Data from TCGA and GEO databases were utilized to evaluate the levels of miR-20b in HNSC and its relationship with radiotherapy sensitivity and prognosis. Additionally, the radiosensitivity of SCC090 cells overexpressing miR-20b was assessed through cell experiments to determine the functional role of miR-20b. Finally, bioinformatics methods were employed to elucidate the mechanism by which miR-20b enhances radiotherapy sensitivity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn HPV\u003csup\u003e+\u003c/sup\u003e HNSC, M1 macrophages were highly infiltrated and played a crucial role in enhancing the sensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC to radiotherapy. M1 exos infiltrated HPV\u003csup\u003e+\u003c/sup\u003e HNSCC, increasing their sensitivity to radiation. Meanwhile, M1 macrophages were abundant in miR-20b than M2 macrophages, and the radiation sensitivity of HPV\u003csup\u003e+\u003c/sup\u003e HNSC was significantly increased by transfecting them with a miR-20b mimic. The target genes of miR-20b were involved in DNA damage repair and cell cycle regulation. By analyzing the function of the target genes, CCND1 was identified as a key gene through which miR-20b enhanced radiotherapy sensitivity in HPV\u003csup\u003e+\u003c/sup\u003e HNSC.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn this study, our data suggest that M1 exos, enriched with miR-20b, regulate the DNA damage repair pathway in tumor cells by targeting CCND1, thereby enhancing the sensitivity of tumors to radiotherapy. Consequently, miR-20b may represent a potential therapeutic strategy for HNSC.\u003c/p\u003e","manuscriptTitle":"M1 macrophage-derived exosomal miR-20b promotes radiosensitization in HPV + HNSC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-15 11:23:50","doi":"10.21203/rs.3.rs-5372230/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":"913b0de1-7ef5-41fc-bfd4-af11a446e83c","owner":[],"postedDate":"November 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-22T10:09:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-15 11:23:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5372230","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5372230","identity":"rs-5372230","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}