B7-H3 regulates HK2 expression and promotes the PI3K/AKT/mTOR pathway

B7-H3 regulates HK2 expression and promotes the PI3K/AKT/mTOR pathway | 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 B7-H3 regulates HK2 expression and promotes the PI3K/AKT/mTOR pathway Shiyang Han, Yuantao Wu, Yan Zhang, Yan Zhou, Qiuyan Xia, Yi Wu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5738861/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 Lung cancer is the foremost cause of cancer-related mortality globally. B7-H3, an immune checkpoint, not only has immunomodulatory functions but also promotes tumor cell proliferation, drug resistance, metastasis, and aerobic glycolysis. The specific role of B7-H3 in aerobic glycolysis in lung cancer has not yet been elucidated. Methods B7-H3 expression was validated in lung cancer tissues and lung cancer cell lines. Overexpression and knockout experiments were conducted in lung cancer cell lines to assess cell proliferation, migration, lactate production, and glucose consumption. The expression of hexokinase 2 (HK2) and the PI3K/Akt/mTOR signaling pathway were analyzed. Additionally, soluble B7-H3 (sB7-H3) along with lactate levels in the plasma of lung cancer patients were measured, and the clinical characteristics were analysed. Results B7-H3 was highly expressed in lung cance tissue and lung cancer cell lines, and enhanced the proliferation and migration, along with increased lactate production and glucose consumption rates. Our study also demonstrated that B7-H3 regulates the expression of HK2, thereby promoting the proliferation and migration of lung cancer cells through the PI3K/Akt/mTOR signaling pathway. Furthermore, sB7-H3 levels was postively correlated with the lacte levels in the plasma of lung cancer patients, and high sB7-H3 levels were associated with a poorer prognosis. Conclusion The research suggests that B7-H3 affects lung cancer cell proliferation and migration by enhancing aerobic glycolysis via HK2, providing a new perspective for the application of B7-H3 in clinical prognosis and treatment. B7-H3 glycolysis HK2 lung cancer PI3K/AKT/mTOR signalling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Lung cancer is one of the most common malignant tumours in the world, and it is also the most common cancer type (20.8%) and the main cause of cancer related death in China( 1 ). Non-small cell lung cancer, accounting for approximately 85% of the pathological types of lung cancer, is divided into lung adenocarcinoma and lung squamous cell carcinoma( 2 ). Immune checkpoint inhibitors (PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies) have become the standard first-line therapy( 3 , 4 ). B7-H3, also known as CD276, is a membrane protein of the B7-CD28 family of immunomodulatory proteins, and is a type I membrane protein similar to the extracellular domain sequence of programmed death receptor-1(PD-L1)( 5 ). As a negative costimulatory molecule, B7-H3 inhibits immune cells through negative signals, which inactivates the functions of T cells and NK cells and then leads to tumour immune escape ( 6 , 7 ). On the other hand, many studies have shown that B7-H3 can directly act on tumour cells without depending on the immune system, including the NF-κB pathway, STAT-3 pathway and ROS-1 pathway, to promote the proliferation, invasion and metastasis of tumour cells( 8 ). At present, studies have shown that B7-H3 can promote glycolysis in colon cancer, oral squamous cell carcinoma and breast cancer cells( 9 – 12 ). A century ago, Otto Warburg reported that tumour tissue slices in vitro use glucose to produce a large amount of lactic acid, which is referred to as aerobic glycolysis. Some studies have shown that targeting key glycolytic enzymes can alleviate and treat tumours. At present, the targets under study are GLUT, SGLT, HK, GK, and PK ( 13 ). Among these key enzymes in glycolysis, HK is the first key enzyme in glycolysis, and HK2 is highly expressed in many tumour cells( 14 , 15 ). By studying the relationship between B7-H3 and HK2 in lung cancer and their influence on glycolysis, this study elucidates the mechanism of inhibiting the proliferation of lung cancer cells by targeting B7-H3. Materials and methods 1. Cell culture, lentivirus infection, and cell transfection A549 cells and BEAS-2B cells were cultured in DMEM/F12 medium (Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, USA). NCI-H460 cells and NCI-H1975 cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, USA) supplemented with 10% FBS. Calu-3 cells were cultured in DMEM (Gibco, Carlsbad, USA) supplemented with 10% FBS. All the cells were maintained at 37°C in a humidified atmosphere with 5% CO2. NCI-H1975, Calu-3, and BEAS-2B cells were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China), whereas A549 and NCI-H460 cells were obtained from Shanghai Fuheng Biotechnology Co., Ltd. The human CD276-C-3×Flag Lentiviral vector was generously provided by Dr. Lei Cao (The First Affiliated Hospital of Soochow University, Suzhou, China). The empty backbone vector pLenti-GII-CMV-PGK-Puro was used as a control. B7-H3 sgRNAs (Suppl Table 1) were constructed in the Lenti-CRISPR V2 vector. Lentiviruses were generated by cotransfection of HEK293T cells with one of the above Lenti-vectors and packaging plasmids (pVSV.G and pΔR8.9) using Lipofectamine™ 3000 (Invitrogen, CA, USA), the lentivirus containing medium was harvested at 36 hours post transfection. A549 cells were infected with lentivirus, and stable cell lines were cultured in medium containing 1 µg/mL puromycin. HK2 siRNA, B7-H3 siRNA (Suppl Table 1), and NC siRNA were purchased from GenePharma Co. Ltd. (Suzhou, China). The cells were transfected with the siRNAs via Lipofectamine 3000 according to the manufacturer’s instructions. A549 cells were treated with 12.5 nM rapamycin (HY-10219, MCE, Shanghai, China) and assessed after 24 hours. 2. Specimens and follow-up Eight patients with primary NSCLC who underwent surgical resection at Aoyang Hospital were included in this study. Lung cancer tissues and adjacent normal lung tissues were removed aseptically and frozen in liquid nitrogen until further use. In addition, a total of 140 lung cancer patients were hospitalized and 122 healthy volunteers were enrolled in this study from August 2020 to August 2021. 2 mL peripheral blood samples were collected from both healthy individuals and lung cancer patients using ethylenediaminetetraacetic acid (EDTA) tubes. The samples were then centrifuged (1,500 ×g, 4°C, 10 minutes) to obtain plasma, which was stored at -80°C. The follow-up involved a combination of outpatient visits and telephone calls. Each patient was monitored every three to six months, with the maximum follow-up period being four years. The follow-up continued until January 2024. During this period, the survival of the patients was recorded, and the 4-year survival rate was calculated. Overall survival (OS) was defined as the duration between the initiation of data collection and either the patient’s death or the last follow-up visit. 3. Bioinformatics analysis UALCAN, which includes 31 different types of cancer with RNA-seq data from The Cancer Genome Atlas (TCGA) and proteomics data from the Clinical Proteomic Tumour Analysis Consortium (CPTAC), provides an accessible and interactive online tool for cancer OMICS data analysis. We compared the differential expression of B7-H3 and HK2 in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) samples with that in healthy samples from the UALCAN website ( https://ualcan.path.uab.edu/index.html ). 4. Western blot studies Total protein from the tissues and cells was extracted via a total protein extraction kit (Sangon Biotech, Shanghai, China) and quantified via a BCA protein assay kit (UUBIO, Suzhou, China). Western blot analysis of target proteins was conducted using the appropriate primary antibodies, followed by probing with the corresponding conjugated secondary antibody. The reactive bands were visualized using the Tanon 5500 imaging system (Tanon, Shanghai, China) with ECL Plus reagents (Tanon, Shanghai, China). The relative intensity of each band was analysed via ImageJ software (NIH, Bethesda, MD, USA). The antibodies used for western blotting are listed in Suppl Table 2. 5. RNA preparation and qRT-PCR Total RNA was isolated using a TRIzol kit (Life Technology, CA, USA), and 1 µg of RNA was reverse transcribed into cDNA using reverse transcription kits (Takara, Shiga, Japan). qRT-PCR was performed on a CFX96 Touch™ real-time PCR system (Bio-Rad, CA, US) using SYBR Green Master Mix (Takara, Shiga, Japan) according to the manufacturer’s instructions. All genes were normalized to β-actin in the same sample. Relative expression levels were calculated according to the standard 2 −ΔΔCT method. The primer sequences utilized are presented in Suppl Table 1. 6. Flow cytometry analysis A total of 1 × 10 6 cells were incubated with 0.1 µg/µL PE-conjugated anti-human B7-H3 (Bright Scistar, Suzhou, China) in the dark for 30 minutes at 4°C. After being washed with PBS twice, the cells were resuspended in 100 µl of PBS and detected via flow cytometry (Beckman DxFLEX), and all the data were analysed using FlowJo software (version 10, USA). 7. CCK8 assay The cells were plated in 96-well plates at a density of 5 × 10 3 cells/mL and cultured for 4, 24, or 48 hours. CCK‐8 solution(Dojindo, Kumamoto, Japan) was introduced to each well, followed by incubation at 37°C for 2 hours. The OD value at 450 nm was measured using a microwell reader (Thermo Fisher Scientific, Vantaa, Finland). 8. Wound healing The cells were seeded in 6-well plates and allowed to grow until they reached 90% confluence. The monolayer of cells was then scratched via sterile micropipette tips to create a wound. The cells transferred from the edge of the wound and the distance between the gaps were counted under a microscope. The migration ability was assessed by calculating the ratio of the gap distance at 24 hours and 48 hours to that at 0 hours. 9. Glucose consumption and lactic acid assays The cells were seeded and cultured at a density of 2×10 5 /well in 500 µL of medium in 24-well microplates (Corning, USA). After treatment with 6 mM 2-DG (HY-13966, MCE, Shanghai, China) for 18 or 48 hours, the supernatant was collected and centrifuged at 1000 rpm for 5 minutes. The glucose concentration was tested via a glucose assay kit (Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s protocol. Glucose consumption was calculated by subtracting the amount of glucose present in the cell culture medium without any cells. Lactic acid produced in the medium was quantified via a lactic acid assay kit (Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s protocol. 10. ELISA Soluble B7-H3 in the supernatant of A549 cells and plasma samples was assayed in duplicate via an ELISA kit (Bright Scistar Antibody Biotech, Suzhou, China). All the experiments were conducted according to the manufacturer’s instructions( 16 ). 11. Statistical analysis Statistical analysis was performed via GraphPad Prism 8.0 (GraphPad Software, Inc.) and SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation (SD) or as the median with a range of lower and upper quartiles (25th and 75th percentiles). Comparisons between groups were analysed via the Mann-Whitney U test, two-tailed Student’s t-test or one-way analysis of variance (ANOVA). The Spearman rank correlation test was used for correlation analysis. The receiver operating characteristic (ROC) analysis was conducted to ascertain the area under the curve (AUC) and the sensitivity and specificity at specified points on the curve.Survival analysis was performed via the Kaplan-Meier method with a log-rank (Mantel-Cox) comparison of survival curves. A P -value < 0.05 indicated a statistically significant difference (* P < 0.05, ** P < 0.01, *** P < 0.001). Results 1. B7-H3 promotes the proliferation and migration of lung cancer cells We examined the expression levels of B7-H3 in adenocarcinoma and squamous cell carcinoma, as well as in matched normal tissues, using the UALCAN database, and the results revealed that B7-H3 is highly expressed at both the mRNA and protein levels in lung cancer tissues (Fig.S1A). Among the eight lung cancer patients who were examined, five had significantly higher expression levels of B7-H3 in primary tumour lesions than in corresponding adjacent normal tissues (Fig.1A). This result is consistent with previous research (17). In addition, we detected the expression of B7-H3 in lung cancer cell lines, including A549, NCI-H460, NCI-H1975, and Calu-3, and found that the levels of B7-H3 were elevated in all lung cancer cell lines compared with those in the normal lung epithelial cell line BEAS-2B (Fig. S1B). To further study the function of B7-H3, we knocked down B7-H3 expression by transfecting H1975 cells with a B7-H3-specific siRNA, which resulted in a decrease in a decrease in B7-H3 expression to less than 60% of that of the NC control. (Fig. S2A-B). Our results indicated that reducing B7-H3 levels in H1975 cells led to a decrease in cell proliferation (Fig. S2C). Moreover, B7-H3 knockdown decreased proliferating cell nuclear antigen (PCNA), suggesting that B7-H3 could be involved in the proliferation of lung cancer cells. (Fig. S2D). To further validate the biological functions of B7-H3, we established stable B7-H3 -overexpressing and -knockout A549 cell lines. The efficiency of overexpression and knockout was validated (Fig.1B-D). Compared with the control, the overexpression of B7-H3 in A549 cells promoted cell proliferation and migration, whereas the knockout of B7-H3 inhibited cell proliferation and migration (Fig. 1E-F). Furthermore, B7-H3 overexpression led to an increase in PCNA expression， whereas, B7-H3 knockout cells presented downregulated PCNA expression (Fig.1G). Collectively, these data demonstrate that B7-H3 promoted the proliferation and migration of lung cancer cells. 2. B7-H3 modulates glucose uptake and lactate generation via HK2 Increased aerobic glycolysis is now widely considered a hallmark of many cancers(18). We found that B7-H3 is associated with the metabolism of glucose compared with the control group, cells overexpressing B7-H3 showed a significant increase in glucose consumption and lactate production, while knockout of B7-H3 significantly reduced glucose consumption and lactate production (Fig. 2A). These results suggest that B7-H3 may be involved in aerobic glycolysis in lung cancer cells. We examined the expression levels of various key glycolysis-related genes, such as hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), and phosphofructokinase (PFKP), in B7-H3-overexpressing and B7-H3-knockout A549 cells (Fig. 2C). The results showed that overexpression of B7-H3 promoted HK2, PFK, and PKM2 mRNA levels, while knockout of B7-H3 reduced the mRNA levels of HK2 but does not affect the expression of PFK or PKM2. We obtained datasets on the expression of HK2 in adenocarcinoma, squamous cell carcinoma and paired normal tissues from the CPTAC online database, and the results showed that HK2 is highly expressed at the protein level in lung cancer tissues (Fig. S3). In addition, B7-H3 overexpression led to a notable rise in HK2 protein levels, while the knockout of B7-H3 significantly decreased HK2 protein expression in A549 cells (Fig. 2D); similar results were validated in H1975 B7-H3 knockdown cells (Fig. S4A-D). Thus, our results suggest that B7-H3 promotes glycolysis by regulating the expression of the key glycolytic enzyme HK2. To verify that B7-H3 affects lung cancer cell glycolysis through HK2, we employed HK2 siRNA to downregulate HK2 expression. (Fig. 2E-F). Furthermore, the application of 2-deoxy-D-glucose (2-DG), an inhibitor of HK2, completely eliminated the rise in glucose uptake and lactate production induced by B7-H3 (Fig. 2G-H). These results support the conclusion that the B7-H3-mediated enhancement of aerobic glycolysis is dependent on HK2. 3. B7-H3 regulates the PI3K/AKT/mTOR pathway through HK2 Prior reports have indicated that B7-H3 promotes aerobic glycolysis in oral squamous cell carcinoma through the activation of the PI3K/Akt/mTOR pathway. Our findings corroborate these observations, demonstrating that B7-H3 overexpression significantly increased levels of phosphorylated PI3K (p-PI3K), AKT (p-AKT), and mTOR (p-mTOR), while B7-H3 knockout resulted in diminished levels of these phosphorylated proteins (Fig.3A). However, HK2 expression did not significantly change in response to treatment with rapamycin, an mTOR inhibitor (Fig. 3B). Moreover, the lactate and glucose consumption levels did not significantly change (Fig. 3C). Interestingly, we detected a marked decrease in the levels of p-PI3K, p-AKT, and p-mTOR following the knockdown of HK2, which effectively countered the elevation of the PI3K/AKT/mTOR signaling pathways induced by B7-H3 overexpression in A549 cells. (Fig.3D). Similar results were observed in A549 cells treated with 2-DG, an HK2 inhibitor (Fig. 3E). These findings suggest that B7-H3 activates the PI3K/AKT/mTOR signalling pathway through the modulation of HK2, resulting in increased tumour cell proliferation. 4. The plasma sB7-H3 level is positively correlated with the lactate level and is associated with clinical prognosis The soluble form of B7-H3, sB7-H3, is generated by the cleavage of the variable region of the B7-H3 protein. It can be released into the extracellular fluid under physiological or pathological conditions, both in vivo and in vitro(19). Our results indicated that the level of sB7-H3 in the supernatant of A549 cells overexpressing B7-H3 was greater compared to control cells. In contrast, knocking out B7-H3 resulted in a reduction of sB7-H3 expression (Fig. 4A). Additionally, sB7-H3 levels in plasma were positively correlated with the protein levels of B7-H3 in the cancer tissues of lung cancer patients (Fig. 4B). We analysed the plasma level of sB7-H in patients with lung cancer compared with healthy controls. The results revealed that the plasma level of sB7-H3 in lung cancer patients was significantly greater than that in healthy controls. Mann-Whitney U test analyses revealed a significant correlation between sB7-H3 levels and age, TNM stage, and metastasis, whereas no significant correlation was detected with gender or type of pathology (Fig. 4C and Table 1). The ROC curve was used to assess the diagnostic value of plasma sB7-H3 expression levels in patients with lung cancer. The results revealed the AUC for sB7-H3 in the prediction of lung cancer was 0.8352. The sensitivity was 0.75, while the specificity was 0.793. These findings indicate that sB7-H3 provides valuable information for lung cancer diagnosis. (Fig. 4D). Mann-Whitney U test analyses revealed a significant correlation between sB7-H3 levels and TNM stage and metastasis, while no significant correlation was detected with gender or age (Suppl table 3). Kaplan-Meier survival curve analysis indicated that patients exhibiting elevated levels of sB7-H3 experienced lower overall survival rates compared to those with reduced levels of sB7-H3 (Fig. 4E and Table 2). In addition, we analysed the plasma lactate levels of patients with lung cancer and healthy controls. The findings indicated that lung cancer patients had significantly higher plasma lactate levels compared to healthy controls. Furthermore, we observed a positive correlation between sB7-H3 levels and lactate levels in the plasma (Fig. 4F-G). Table 1. The relationship between the levels of sB7-H3 in plasma of patients with LC and clinicopathological characteristics Lung cancer sB7-H3 expression Healthy sB7-H3 expression Median (Min-Max) P25~P75 U P Median (Min-Max) P25~P75 U P Gender 2278 0.8011 1579 0.4176 Male 85 14.75(5.415-32.36) 11.85-17.82 77 9.617(3.322-17.86) 7.925-11.22 Female 55 14.32(7.130-32.08) 11.79-32.08 45 10.04(3.164-16.36) 7.933-12.02 Age 1578 0.2783 1047 0.9262 ≤ 60 34 13.58(5.415-32.36) 10.62-17.21 101 9.817(3.164-17.86) 7.404-11.46 >60 106 14.73(7.130-32.08) 12.27-18.28 21 9.672(4.909-16.36) 8.217-11.30 TNM 1618 0.0261 Ⅰ+Ⅱ 44 13.14(5.415-24.33) 10.22-16.21 NA NA NA Ⅲ+Ⅳ 96 14.89(7.427-32.36) 12.42-18.40 NA NA NA Metastasis 1308 0.0241 No 33 12.73(5.415-22.25) 10.36-16.12 NA NA NA Yes 107 14.83(7.340-32.36) 12.34-18.41 NA NA NA Type of pathology 0.8237 Adenocarcinoma 90 14.1(5.41-32.4) 11.3-18.0 NA NA NA Squamous carcinoma 32 14.7(7.43-27.1) 13-16.7 NA NA NA Small cell lung cancer 15 15.5(7.9-23) 10.9-18.4 NA NA NA Other 3 13.5(12.3-15.9) 12.3-15.9 NA NA NA Table 2. Univariate and multivariate analysis of overall survival factors Univariable analysis Multivariate analysis HR (95% CI) p HR (95% CI) p Gender (Male vs Female) 2.481(1.555-3.957) 0.0004 2.129(1.199-3.783) 0.01 Age (>60 vs ≤ 60) 1.693(0.9939-2.885) 0.1056 TNM (Ⅲ + Ⅳ vs Ⅰ + Ⅱ) 11.38(7.084-18.28) <0.001 4.25(1.318-13.703) 0.015 Metastasis (Yes vs No) 31.55(19.09-52.16) <0.001 7.858(0.811-76.127) 0.075 sB7-H3 (High vs Low) 1.735(1.084-2.777) 0.0162 1.753(1.074-2.862) 0.025 5. The mechanism by which B7-H3 promotes glycolysis and its prognostic value in lung cancer B7-H3 enhances PI3K/AKT/mTOR signalling pathway activation by upregulating the key glycolytic enzyme HK2, thereby participating in glycolysis. The activated mTOR pathway regulates crucial cellular processes including proliferation and migration. In tumour cells, glucose is metabolized through glycolysis, producing ATP and lactate. The accumulation of lactate acidifies the tumour microenvironment, facilitating tumour growth, invasion, and migration. sB7-H3 is a soluble form generated by alternative splicing of the variable region of the B7-H3 protein. The presence of sB7-H3 in peripheral blood holds clinical value in the prognosis of lung cancer. Discussion Previous studies have shown that B7-H3 is a negative costimulatory molecule that inhibits immune cells through negative signals, deactivates the functions of T cells and NK cells, and then leads to tumour immune escape(11,20,21). In this study, we explored the role of B7-H3 in lung cancer and discovered that it was significantly elevated in lung cancer tissues. Moreover, increased levels of B7-H3 were associated with enhanced proliferation and migration of lung cancer cells. Furthermore, our research indicated that B7-H3 enhances glycolysis through HK2 and modulates the PI3K/Akt/mTOR pathway, which in turn boosts the proliferation and migration of lung cancer cells. We also observed a positive relationship between B7-H3 expression in cell lines and lung cancer tissues, as well as with the levels of soluble B7-H3 (sB7-H3) in the corresponding supernatant and serum. Importantly, the concentration of sB7-H3 in the plasma of lung cancer patients was higher than that in healthy controls and showed a positive correlation with lactate levels. The role of anaerobic glycolysis in cancer has long been well known. With the extensive application of immunotherapy in the clinic, an increasing number of scholars have linked immunotherapy with aerobic glycolysis. Dussol suggested that regulating macrophage metabolism is a new cancer immunotherapy strategy(22). Hu Z suggested that glycolysis drives STING signalling to promote the antitumour function of dendritic cells(23), Zhang L found that STING can limit aerobic glycolysis without relying on its innate immune function by directly inhibiting hexokinase activity through targeting HK2、As a result, STING reduces aerobic glycolysis in tumors and enhances antitumor immunity in vivo (24). Jing Y reported that sting directly affects the activity of PI3K in the study of actin reorganization during the regulation of BCR activation by sting(25). Shi T’s study indicated that B7-H3 could influence glucose metabolism and contribute to chemotherapy resistance in colorectal cancer cells by modulating the expression of HK2 (13). Zuo J study found that B7-H3 interacts with the glycolytic enzyme ENO1, thus affecting glycolysis in cervical cancer cells (26). A Nunes-Xavier CE study revealed that B7-H3 can increase the glycolytic ability and drug resistance of inhibitors of the PI3K/AKT/mTOR pathway in triple negative breast cancer cells(27). As the first key enzyme in the glycolytic pathway, HK2 may act through the NF-κBp65(28), ROS/PI3K/AKT/HIF-1α(9), and AKT-GLUT1(29) signalling pathways, but it is more closely related to PI3K/AKT/mTOR(14,15). In order to better apply B7-H3 in the clinic, we first studied the relationship between B7-H3 and the prognosis of lung adenocarcinoma patients in the TCGA found that high expression of B7-H3 was related to poor prognosis of lung adenocarcinoma patients(30,31){Darshan Shimoga, 2022 #479}. Secondly, we found that soluble B7-H3 (sB7-H3) in the supernatant of lung cancer cells was consistent with the expression of B7-H3 mRNA and protein, and we also found the same results in the plasma and tissues of the lung cancer patients we collected. These results suggest that the plasma level of sB7-H3 may serve as a prognostic indicator for lung cancer patients and help inform their clinical treatment decisions.sB7-H3 is produced by monocytes, dendritic cells, activated T cells and B7-H3 + tumour cells(32). Studies have indicated that serum levels of sB7-H3 are significantly elevated in patients with non-small cell lung cancer. This increase is strongly associated with tumor stage, size, and lymph node metastasis, but shows no correlation with age, gender, or pathological classification(33). As a tumour biomarker, sB7-H3 is superior to CA125, CA153, CA199 and CEA(34). In addition, high expression of sB7-H3 in the serum of patients with intestinal cancer is negatively correlated with the number of T lymphocytes infiltrated by intestinal cancer(35). Soluble B7-H3 secreted by pancreatic cancer cells can promote tumour invasion and metastasis through the TLR4/NF-κB pathway(36). Therefore, as a potential biomarker for tumour diagnosis and prognosis, sB7-H3 is attracting increasing attention (19). Research indicates that higher levels of B7-H3 expression are associated with increased release of sB7-H3. sB7-H3 can spread to other tissues through the circulatory system and play an important biological role. Therefore, the positive correlation between B7-H3 and sB7-H3 may play an important role in tumour growth and immune regulation(37). However, there is no research on whether sB7-H3 is involved in the glycolysis of tumour cells at present, and further research can be conducted on whether sB7-H3 affects the glycolysis of immune cells and tumour cells. In summary, this study provides a theoretical basis for the influence of B7-H3 on the proliferation and migration of lung cancer cells through aerobic glycolysis, provides evidence for the treatment of lung cancer patients through targeting B7-H3, and provides a new idea for the application of sB7-H3 in clinical Prognosis. Abbreviations HK2: hexokinase 2 PKM2: pyruvate kinase M2 PFKP: phosphofructokinase sB7-H3: soluble B7-H3 PD-L1: programmed death receptor-1 TCGA: The Cancer Genome Atlas CPTAC: Clinical Proteomic Tumour Analysis Consortium LUAD: lung adenocarcinoma LUSC: lung squamous cell carcinoma ROC: receiver operating characteristic AUC: area under the curve PCNA: proliferating cell nuclear antigen Declarations Ethics approval and consent to participate The study was approved by the Ethics Committee of Jiangsu University Affiliated Aoyang Hospital (Approval No. 2021-015). Prior to the commencement of the study, all patients were provided with detailed information about the purpose and methodology of the research. They were fully informed about the study and its potential risks and benefits, and they voluntarily signed written informed consent forms. Funding This work was supported by grants from the Suzhou Science and Technology Development Program (SKJYD2021005), the Suzhou Science and Technology Program (SYSD2020011), the Clinical Medical Science and Technology Development Fund of Jiangsu University (JLY20180145), and the Zhangjiagang Health Youth Science and Technology Project (ZJGQNKJ202018, ZJGQNKJ202136). Authorship contribution statement Shiyang Han: conceptualization, data curation, investigation, writing – original draft, methodology. Yuantao Wu: Writing – original draft, Funding acquisition. Yan Zhou: Data curation. Qiuyan Xia: Data curation, Funding acquisition. Yan Zhang, Yi Wu, Zhanguo Liu and Jing Yang: Data curation. Ye Shen: Supervision. Shuru Zhou: Project administration, writing, review, editing and funding acquisition. Shiyang Han and Yuantao Wu contributed equally to this work. Declaration of competing interest The authors declare no competing interests. Data availability All the data supporting the findings of this study are available within the article and its Supplementary information files and from the corresponding authors upon reasonable request. Acknowledgment The authors acknowledge Dr. Jiajia Jiang and Dr. Shijie Tang from the Affiliated Aoyang Hospital of Jiangsu University for their review and feedback on this manuscript, which is highly important for further improving our research. References Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024;74:229-63. http://10.3322/caac.21834. J Saller J, Boyle TJCSHpim. 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B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression. 2020;11:55. http://10.1038/s41419-020-2252-3. Zhang T, Zhu X, Wu H, et al. Targeting the ROS/PI3K/AKT/HIF-1α/HK2 axis of breast cancer cells: Combined administration of Polydatin and 2-Deoxy-d-glucose. 2019;23:3711-23. http://10.1111/jcmm.14276. Yue G, Tang J, Zhang L, et al. CD276 suppresses CAR-T cell function by promoting tumor cell glycolysis in esophageal squamous cell carcinoma. 2021;12:38-51. http://10.21037/jgo-21-50. Zhang H, Zhu M, Zhao A, et al. B7-H3 regulates anti-tumor immunity and promotes tumor development in colorectal cancer. 2024;1879:189031. http://10.1016/j.bbcan.2023.189031. Li Z, Liu J, Que L, et al. The immunoregulatory protein B7-H3 promotes aerobic glycolysis in oral squamous carcinoma via PI3K/Akt/mTOR pathway. J Cancer 2019;10:5770-84. http://10.7150/jca.29838. Shi T, Ma Y, Cao L, et al. B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2. 2019;10:308. http://10.1038/s41419-019-1549-6. Liu S, Chen Q, Wang YJHc. MiR-125b-5p suppresses the bladder cancer progression via targeting HK2 and suppressing PI3K/AKT pathway. 2020;33:185-94. http://10.1007/s13577-019-00285-x. Wang P, Cong M, Liu T, et al. FoxA2 inhibits the proliferation of hepatic progenitor cells by reducing PI3K/Akt/HK2-mediated glycolysis. 2020;235:9524-37. http://10.1002/jcp.29759. Han S, Zhang Y, Yuan J, et al. sPD-L1 and sPD-L2 in plasma of patients with lung cancer and their clinical significance. Cytokine 2024;176:156532. http://10.1016/j.cyto.2024.156532. Sun Y, Wang Y, Zhao J, et al. B7-H3 and B7-H4 expression in non-small-cell lung cancer. Lung Cancer 2006;53:143-51. http://10.1016/j.lungcan.2006.05.012. Martinez-Outschoorn UE, Peiris-Pages M, Pestell RG, et al. Cancer metabolism: a therapeutic perspective. Nat Rev Clin Oncol 2017;14:113. http://10.1038/nrclinonc.2017.1. Chen W, Liu P, Wang Y, et al. Characterization of a soluble B7-H3 (sB7-H3) spliced from the intron and analysis of sB7-H3 in the sera of patients with hepatocellular carcinoma. PLoS One 2013;8:e76965. http://10.1371/journal.pone.0076965. Zhang D, Huang H, Gao X, et al. High expression of B7-H3 on monocyte/macrophages in tumor microenvironment promotes lung cancer progression by inhibiting apoptosis. 2024;41:101874. http://10.1016/j.tranon.2023.101874. Liu H, Du H, Khabibullin D, et al. mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion. 2023;14:1214. http://10.1038/s41467-023-36881-7. Dussold C, Zilinger K, Turunen J, et al. Modulation of macrophage metabolism as an emerging immunotherapy strategy for cancer. 2024;134. http://10.1172/jci175445. Hu Z, Yu X, Ding R, et al. Glycolysis drives STING signaling to facilitate dendritic cell antitumor function. 2023;133. http://10.1172/jci166031. Zhang L, Jiang C, Zhong Y, et al. STING is a cell-intrinsic metabolic checkpoint restricting aerobic glycolysis by targeting HK2. 2023;25:1208-22. http://10.1038/s41556-023-01185-x. Jing Y, Dai X, Yang L, et al. STING couples with PI3K to regulate actin reorganization during BCR activation. Sci Adv 2020;6:eaax9455. http://10.1126/sciadv.aax9455. Zuo J, Wang B, Long M, et al. The type 1 transmembrane glycoprotein B7-H3 interacts with the glycolytic enzyme ENO1 to promote malignancy and glycolysis in HeLa cells. 2018;592:2476-88. http://10.1002/1873-3468.13164. Nunes-Xavier C, Karlsen K, Tekle C, et al. Decreased expression of B7-H3 reduces the glycolytic capacity and sensitizes breast cancer cells to AKT/mTOR inhibitors. 2016;7:6891-901. http://10.18632/oncotarget.6902. Chen L, Lin X, Lei Y, et al. Aerobic glycolysis enhances HBx-initiated hepatocellular carcinogenesis via NF-κBp65/HK2 signalling. 2022;41:329. http://10.1186/s13046-022-02531-x. Sun T, Du B, Diao Y, et al. ATAD2 expression increases [18F]Fluorodeoxyglucose uptake value in lung adenocarcinoma via AKT-GLUT1/HK2 pathway. 2019;52:457-62. http://10.5483/BMBRep.2019.52.7.042. Chandrashekar DS, Karthikeyan SK, Korla PK, et al. UALCAN: An update to the integrated cancer data analysis platform. Neoplasia 2022;25:18-27. http://10.1016/j.neo.2022.01.001. Chandrashekar DS, Bashel B, Balasubramanya SAH, et al. UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses. Neoplasia 2017;19:649-58. http://10.1016/j.neo.2017.05.002. Zhang G, Hou J, Shi J, et al. Soluble CD276 (B7-H3) is released from monocytes, dendritic cells and activated T cells and is detectable in normal human serum. Immunology 2008;123:538-46. http://10.1111/j.1365-2567.2007.02723.x. Li Y, Xu L, Li J, et al. Diagnostic and prognostic value of serum soluble B7-H3 in nonsmall cell lung cancer. Anticancer Drugs 2024;35:426-32. http://10.1097/CAD.0000000000001577. Zhang G, Xu Y, Lu X, et al. Diagnosis value of serum B7-H3 expression in non-small cell lung cancer. Lung Cancer 2009;66:245-9. http://10.1016/j.lungcan.2009.01.017. Sun J, Chen LJ, Zhang GB, et al. Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma. Cancer Immunol Immunother 2010;59:1163-71. http://10.1007/s00262-010-0841-1. Xie C, Liu D, Chen Q, et al. Soluble B7-H3 promotes the invasion and metastasis of pancreatic carcinoma cells through the TLR4/NF-kappaB pathway. Sci Rep 2016;6:27528. http://10.1038/srep27528. Huang L, Zhou Y, Sun Q, et al. Evaluation of the role of soluble B7-H3 in association with membrane B7-H3 expression in gastric adenocarcinoma. Cancer Biomark 2022;33:123-9. http://10.3233/CBM-210178. Additional Declarations No competing interests reported. Supplementary Files B7H3OriginalWB.pptx Appendix.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5738861","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":396145138,"identity":"a6672803-31db-4189-8f2a-fc4bfae8f723","order_by":0,"name":"Shiyang Han","email":"","orcid":"","institution":"The Aoyang Cancer Institute, The Affilated Aoyang Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Shiyang","middleName":"","lastName":"Han","suffix":""},{"id":396145139,"identity":"1036adb8-85c1-44d9-919e-285ba12abe70","order_by":1,"name":"Yuantao Wu","email":"","orcid":"","institution":"The Affiliated Aoyang Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Yuantao","middleName":"","lastName":"Wu","suffix":""},{"id":396145140,"identity":"72499fa2-a8b6-4778-81ba-4ccd0ba8f9c9","order_by":2,"name":"Yan Zhang","email":"","orcid":"","institution":"The Aoyang Cancer Institute, The Affilated Aoyang Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhang","suffix":""},{"id":396145141,"identity":"c1c4cdb5-87ae-4818-94a3-28df623effd8","order_by":3,"name":"Yan Zhou","email":"","orcid":"","institution":"The Affiliated Aoyang Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhou","suffix":""},{"id":396145142,"identity":"d79a79a3-8d21-4e76-8641-c99c111bfa33","order_by":4,"name":"Qiuyan Xia","email":"","orcid":"","institution":"The Affiliated Aoyang Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Qiuyan","middleName":"","lastName":"Xia","suffix":""},{"id":396145143,"identity":"8b245606-5af9-4b71-9558-5ac7895bc022","order_by":5,"name":"Yi Wu","email":"","orcid":"","institution":"The Affiliated Aoyang Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Wu","suffix":""},{"id":396145144,"identity":"3420d6c4-aa30-4637-a941-950dffd20d77","order_by":6,"name":"Zhanguo Liu","email":"","orcid":"","institution":"The Affiliated Aoyang Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Zhanguo","middleName":"","lastName":"Liu","suffix":""},{"id":396145145,"identity":"4f337623-99d9-4f01-8701-ef8d403b4506","order_by":7,"name":"Jing Yang","email":"","orcid":"","institution":"The Affiliated Aoyang Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yang","suffix":""},{"id":396145146,"identity":"c7e251f1-8175-489b-8773-2dd6bcb26003","order_by":8,"name":"Ye Shen","email":"","orcid":"","institution":"The Affiliated Aoyang Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Shen","suffix":""},{"id":396145147,"identity":"eacc466a-3c71-4388-b0ec-311b261397e2","order_by":9,"name":"Shuru Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYDACCSBmbAAS7D1gPg8f8Vp4zjAwHABSbMRrkcgBa2EgqEV+dvMxyZ87Ducb3Hx78PHHHDsZNgbmh49u4NHCOOdYmjTvmcOWG27nJRsc3JYMdBibsXEOHi3MEjlm0oxthw0MbueYSRzcxgzUwsMmjU8LG1CL5E+QlptnQFrqCWvhAWqR4AVpucED0nKYsBYJibRka962dAPJMznGBme3HedhYybgF/kZyQdv/myzNuA7fsbwQeW2ant+9uaHj/FpwQKYSVM+CkbBKBgFowALAAAfrUOuULtvBQAAAABJRU5ErkJggg==","orcid":"","institution":"The Affiliated Aoyang Hospital of Jiangsu University","correspondingAuthor":true,"prefix":"","firstName":"Shuru","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-12-31 03:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5738861/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5738861/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72795674,"identity":"659c27c6-4a9b-4012-8735-0e749718ea02","added_by":"auto","created_at":"2025-01-02 08:57:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":289969,"visible":true,"origin":"","legend":"\u003cp\u003eB7-H3 promotes the proliferation and migration of lung cancer cells.\u003c/p\u003e\n\u003cp\u003eA: The expression levels of B7-H3 protein in lung cancer tissues from eight patients were evaluated through western blotting (N: normal tissues; LC: lung cancer tissues). B-D: Western blotting, flow cytometry, and qRT-PCR were used to verify the efficiency of B7-H3 overexpression (OE) using vector as the control and knockout (KO) using mock as the control in A549 cells. E: Cell proliferation was assessed via the CCK-8 assay. F: Representative micrographs of the wound healing assay. G: The expression of PCNA in A549 cells was detected by western blotting.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-5738861/v1/4e81183f1c776210f22dd0c7.png"},{"id":72796537,"identity":"39d62622-0273-47ef-82cd-6c6cfaf6ed45","added_by":"auto","created_at":"2025-01-02 09:05:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133787,"visible":true,"origin":"","legend":"\u003cp\u003eB7-H3 promotes aerobic glycolysis through HK2 in lung cancer cells.\u003c/p\u003e\n\u003cp\u003eA-B: Glucose uptake and lactate generation were assessed in A549 cells with either overexpression or knockout. C: The expression of glycolysis-related genes was measured by qRT-PCR in B7-H3 overexpressing or knockout A549 cells. D: The protein levels of B7-H3 in B7-H3 overexpressing and B7-H3 knockout cells were analysed via western blotting. E: The protein expression of HK2 in B7-H3-overexpressing A549 cells was assessed using western blotting following transfection with either negative control (NC) siRNA or HK2 siRNA. F Lactate levels and glucose uptake were assessed in B7-H3-overexpressing A549 cells that were transfected with either negative control (NC) or HK2 siRNA. G: The protein expression of HK2 in B7-H3-overexpressing A549 cells following treatment with PBS or 2-DG was assessed using western blot analysis. H: Lactate production and glucose consumption were measured in B7-H3-overexpressing A549 cells treated with PBS or 2-DG.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-5738861/v1/e6948bce99bde90a527ad176.png"},{"id":72799164,"identity":"fbc93add-272e-4d20-82f7-46b20b2b0a67","added_by":"auto","created_at":"2025-01-02 09:21:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":147117,"visible":true,"origin":"","legend":"\u003cp\u003eB7-H3 regulates the expression of the PI3K/AKT/mTOR pathway through HK2\u003c/p\u003e\n\u003cp\u003eA: Western blot analysis was conducted to evaluate the protein levels of the PI3K/AKT/mTOR pathway in cells with either B7-H3 overexpression or B7-H3 knockout. B: The protein levels of mTOR and HK2 in mock or B7-H3-overexpressing A549 cells after treatment with DMSO or rapamycin(12.5 mM) were analysed via western blotting. C: Lactate production and glucose consumption in mock or B7-H3-overexpressing A549 cells after treatment with DMSO or rapamycin (12.5 mM). D: The protein levels of the PI3K/AKT/mTOR pathway were assessed in B7-H3-overexpressing A549 cells after transfection with siHK2 via Western blot analysis. E:\u003c/p\u003e\n\u003cp\u003eWestern blotting was used to analyze the protein levels of the PI3K/AKT/mTOR pathway in A549 cells overexpressing B7-H3, following treatment with either PBS or 2-DG.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-5738861/v1/09d33011be09157a337be930.png"},{"id":72795680,"identity":"9b3291de-1414-48b6-aac4-e1940fa0d4dd","added_by":"auto","created_at":"2025-01-02 08:57:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81146,"visible":true,"origin":"","legend":"\u003cp\u003esB7-H3 may act as a promising biomarker for lung cancer.\u003c/p\u003e\n\u003cp\u003eA: The concentrations of sB7-H3 in the supernatants from A549 cells, including both B7-H3-overexpressing and B7-H3 knockout variants, were assessed using ELISA. B: A correlation analysis was conducted comparing the plasma levels of sB7-H3 (via ELISA, n=8) with the protein levels of sB7-H3 in lung cancer tissues (via Western blot, n=8). C: The levels of sB7-H3 were evaluated by ELISA in the plasma samples from lung cancer patients (n=140) compared with those in healthy plasma samples (n=122). D: ROC curves of plasma sB7-H3 levels in patients with lung cancer. E Kaplan-Meier curves representing the overall survival of plasma sB7-H3 levels in lung cancer patients, n (sB7-H3 high) =77, n (sB7-H3 low) =63. F: The levels of lactate in the plasma samples from lung cancer patients(n=110) were compared with those in healthy plasma samples(n=90). G: Correlation analysis was conducted to examine the relationship between sB7-H3 levels and plasma lactate levels.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-5738861/v1/858a6dde139bff4b36b17856.png"},{"id":72795677,"identity":"d82167f2-de7e-4a99-8eb6-d29f352b5008","added_by":"auto","created_at":"2025-01-02 08:57:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":92029,"visible":true,"origin":"","legend":"\u003cp\u003eThe role of B7-H3 in enhancing glycolysis and its potential as a prognosis in lung cancer.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-5738861/v1/57a3757c32950310039093cb.png"},{"id":78078984,"identity":"94aa8cde-1cc4-4bc7-b4e8-b82ed278deae","added_by":"auto","created_at":"2025-03-09 11:08:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1388431,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5738861/v1/d04b1428-dcca-46de-aa90-e7cbf1c92657.pdf"},{"id":72795691,"identity":"a1f0c7ef-f4e2-476a-81c7-7c0d53368a13","added_by":"auto","created_at":"2025-01-02 08:57:28","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19726536,"visible":true,"origin":"","legend":"","description":"","filename":"B7H3OriginalWB.pptx","url":"https://assets-eu.researchsquare.com/files/rs-5738861/v1/5376e697548e1c69e90688c0.pptx"},{"id":72795675,"identity":"65eaca10-cd2d-42f2-97fd-49bcc3822221","added_by":"auto","created_at":"2025-01-02 08:57:27","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1560652,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-5738861/v1/81824a775903d74e34f93bee.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"B7-H3 regulates HK2 expression and promotes the PI3K/AKT/mTOR pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is one of the most common malignant tumours in the world, and it is also the most common cancer type (20.8%) and the main cause of cancer related death in China(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Non-small cell lung cancer, accounting for approximately 85% of the pathological types of lung cancer, is divided into lung adenocarcinoma and lung squamous cell carcinoma(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Immune checkpoint inhibitors (PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies) have become the standard first-line therapy(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eB7-H3, also known as CD276, is a membrane protein of the B7-CD28 family of immunomodulatory proteins, and is a type I membrane protein similar to the extracellular domain sequence of programmed death receptor-1(PD-L1)(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). As a negative costimulatory molecule, B7-H3 inhibits immune cells through negative signals, which inactivates the functions of T cells and NK cells and then leads to tumour immune escape (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). On the other hand, many studies have shown that B7-H3 can directly act on tumour cells without depending on the immune system, including the NF-κB pathway, STAT-3 pathway and ROS-1 pathway, to promote the proliferation, invasion and metastasis of tumour cells(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). At present, studies have shown that B7-H3 can promote glycolysis in colon cancer, oral squamous cell carcinoma and breast cancer cells(\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA century ago, Otto Warburg reported that tumour tissue slices in vitro use glucose to produce a large amount of lactic acid, which is referred to as aerobic glycolysis. Some studies have shown that targeting key glycolytic enzymes can alleviate and treat tumours. At present, the targets under study are GLUT, SGLT, HK, GK, and PK (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Among these key enzymes in glycolysis, HK is the first key enzyme in glycolysis, and HK2 is highly expressed in many tumour cells(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). By studying the relationship between B7-H3 and HK2 in lung cancer and their influence on glycolysis, this study elucidates the mechanism of inhibiting the proliferation of lung cancer cells by targeting B7-H3.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e1. Cell culture, lentivirus infection, and cell transfection\u003c/p\u003e \u003cp\u003eA549 cells and BEAS-2B cells were cultured in DMEM/F12 medium (Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, USA). NCI-H460 cells and NCI-H1975 cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, USA) supplemented with 10% FBS. Calu-3 cells were cultured in DMEM (Gibco, Carlsbad, USA) supplemented with 10% FBS. All the cells were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO2. NCI-H1975, Calu-3, and BEAS-2B cells were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China), whereas A549 and NCI-H460 cells were obtained from Shanghai Fuheng Biotechnology Co., Ltd. The human CD276-C-3\u0026times;Flag Lentiviral vector was generously provided by Dr. Lei Cao (The First Affiliated Hospital of Soochow University, Suzhou, China). The empty backbone vector pLenti-GII-CMV-PGK-Puro was used as a control. B7-H3 sgRNAs (Suppl Table\u0026nbsp;1) were constructed in the Lenti-CRISPR V2 vector. Lentiviruses were generated by cotransfection of HEK293T cells with one of the above Lenti-vectors and packaging plasmids (pVSV.G and pΔR8.9) using Lipofectamine\u0026trade; 3000 (Invitrogen, CA, USA), the lentivirus containing medium was harvested at 36 hours post transfection.\u003c/p\u003e \u003cp\u003eA549 cells were infected with lentivirus, and stable cell lines were cultured in medium containing 1 \u0026micro;g/mL puromycin. HK2 siRNA, B7-H3 siRNA (Suppl Table\u0026nbsp;1), and NC siRNA were purchased from GenePharma Co. Ltd. (Suzhou, China). The cells were transfected with the siRNAs via Lipofectamine 3000 according to the manufacturer\u0026rsquo;s instructions. A549 cells were treated with 12.5 nM rapamycin (HY-10219, MCE, Shanghai, China) and assessed after 24 hours.\u003c/p\u003e \u003cp\u003e2. Specimens and follow-up\u003c/p\u003e \u003cp\u003eEight patients with primary NSCLC who underwent surgical resection at Aoyang Hospital were included in this study. Lung cancer tissues and adjacent normal lung tissues were removed aseptically and frozen in liquid nitrogen until further use. In addition, a total of 140 lung cancer patients were hospitalized and 122 healthy volunteers were enrolled in this study from August 2020 to August 2021. 2 mL peripheral blood samples were collected from both healthy individuals and lung cancer patients using ethylenediaminetetraacetic acid (EDTA) tubes. The samples were then centrifuged (1,500 \u0026times;g, 4\u0026deg;C, 10 minutes) to obtain plasma, which was stored at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe follow-up involved a combination of outpatient visits and telephone calls. Each patient was monitored every three to six months, with the maximum follow-up period being four years. The follow-up continued until January 2024. During this period, the survival of the patients was recorded, and the 4-year survival rate was calculated. Overall survival (OS) was defined as the duration between the initiation of data collection and either the patient\u0026rsquo;s death or the last follow-up visit.\u003c/p\u003e \u003cp\u003e3. Bioinformatics analysis\u003c/p\u003e \u003cp\u003eUALCAN, which includes 31 different types of cancer with RNA-seq data from The Cancer Genome Atlas (TCGA) and proteomics data from the Clinical Proteomic Tumour Analysis Consortium (CPTAC), provides an accessible and interactive online tool for cancer OMICS data analysis. We compared the differential expression of B7-H3 and HK2 in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) samples with that in healthy samples from the UALCAN website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ualcan.path.uab.edu/index.html\u003c/span\u003e\u003cspan address=\"https://ualcan.path.uab.edu/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003cp\u003e4. Western blot studies\u003c/p\u003e \u003cp\u003eTotal protein from the tissues and cells was extracted via a total protein extraction kit (Sangon Biotech, Shanghai, China) and quantified via a BCA protein assay kit (UUBIO, Suzhou, China). Western blot analysis of target proteins was conducted using the appropriate primary antibodies, followed by probing with the corresponding conjugated secondary antibody. The reactive bands were visualized using the Tanon 5500 imaging system (Tanon, Shanghai, China) with ECL Plus reagents (Tanon, Shanghai, China). The relative intensity of each band was analysed via ImageJ software (NIH, Bethesda, MD, USA). The antibodies used for western blotting are listed in Suppl Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e5. RNA preparation and qRT-PCR\u003c/p\u003e \u003cp\u003eTotal RNA was isolated using a TRIzol kit (Life Technology, CA, USA), and 1 \u0026micro;g of RNA was reverse transcribed into cDNA using reverse transcription kits (Takara, Shiga, Japan). qRT-PCR was performed on a CFX96 Touch\u0026trade; real-time PCR system (Bio-Rad, CA, US) using SYBR Green Master Mix (Takara, Shiga, Japan) according to the manufacturer\u0026rsquo;s instructions. All genes were normalized to β-actin in the same sample. Relative expression levels were calculated according to the standard 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The primer sequences utilized are presented in Suppl Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e6. Flow cytometry analysis\u003c/p\u003e \u003cp\u003eA total of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells were incubated with 0.1 \u0026micro;g/\u0026micro;L PE-conjugated anti-human B7-H3 (Bright Scistar, Suzhou, China) in the dark for 30 minutes at 4\u0026deg;C. After being washed with PBS twice, the cells were resuspended in 100 \u0026micro;l of PBS and detected via flow cytometry (Beckman DxFLEX), and all the data were analysed using FlowJo software (version 10, USA).\u003c/p\u003e \u003cp\u003e7. CCK8 assay\u003c/p\u003e \u003cp\u003eThe cells were plated in 96-well plates at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/mL and cultured for 4, 24, or 48 hours. CCK‐8 solution(Dojindo, Kumamoto, Japan) was introduced to each well, followed by incubation at 37\u0026deg;C for 2 hours. The OD value at 450 nm was measured using a microwell reader (Thermo Fisher Scientific, Vantaa, Finland).\u003c/p\u003e \u003cp\u003e8. Wound healing\u003c/p\u003e \u003cp\u003eThe cells were seeded in 6-well plates and allowed to grow until they reached 90% confluence. The monolayer of cells was then scratched via sterile micropipette tips to create a wound. The cells transferred from the edge of the wound and the distance between the gaps were counted under a microscope. The migration ability was assessed by calculating the ratio of the gap distance at 24 hours and 48 hours to that at 0 hours.\u003c/p\u003e \u003cp\u003e9. Glucose consumption and lactic acid assays\u003c/p\u003e \u003cp\u003eThe cells were seeded and cultured at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e /well in 500 \u0026micro;L of medium in 24-well microplates (Corning, USA). After treatment with 6 mM 2-DG (HY-13966, MCE, Shanghai, China) for 18 or 48 hours, the supernatant was collected and centrifuged at 1000 rpm for 5 minutes. The glucose concentration was tested via a glucose assay kit (Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer\u0026rsquo;s protocol. Glucose consumption was calculated by subtracting the amount of glucose present in the cell culture medium without any cells. Lactic acid produced in the medium was quantified via a lactic acid assay kit (Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e10. ELISA\u003c/h2\u003e \u003cp\u003eSoluble B7-H3 in the supernatant of A549 cells and plasma samples was assayed in duplicate via an ELISA kit (Bright Scistar Antibody Biotech, Suzhou, China). All the experiments were conducted according to the manufacturer\u0026rsquo;s instructions(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e11. Statistical analysis\u003c/p\u003e \u003cp\u003eStatistical analysis was performed via GraphPad Prism 8.0 (GraphPad Software, Inc.) and SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) or as the median with a range of lower and upper quartiles (25th and 75th percentiles). Comparisons between groups were analysed via the Mann-Whitney U test, two-tailed Student\u0026rsquo;s t-test or one-way analysis of variance (ANOVA). The Spearman rank correlation test was used for correlation analysis. The receiver operating characteristic (ROC) analysis was conducted to ascertain the area under the curve (AUC) and the sensitivity and specificity at specified points on the curve.Survival analysis was performed via the Kaplan-Meier method with a log-rank (Mantel-Cox) comparison of survival curves. A \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated a statistically significant difference (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e1. B7-H3 promotes the proliferation and migration of lung cancer cells\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We examined the expression levels of B7-H3 in adenocarcinoma and squamous cell carcinoma, as well as in matched normal tissues, using the UALCAN database, and the results revealed that B7-H3 is highly expressed at both the mRNA and protein levels in lung cancer tissues (Fig.S1A). Among the eight lung cancer patients who were examined, five had significantly higher expression levels of B7-H3 in primary tumour lesions than in corresponding adjacent normal tissues (Fig.1A). This result is consistent with previous research\u0026nbsp;(17). In addition, we detected the expression of B7-H3 in lung cancer cell lines, including A549, NCI-H460, NCI-H1975, and Calu-3, and found that the levels of B7-H3 were elevated in all lung cancer cell lines compared with those in the normal lung epithelial cell line BEAS-2B (Fig. S1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further study the function of B7-H3, we knocked down B7-H3 expression by transfecting H1975 cells with a B7-H3-specific siRNA, which resulted in a decrease in a decrease in B7-H3 expression to less than 60% of that of the NC control. (Fig. S2A-B). Our results indicated that reducing B7-H3 levels in H1975 cells led to a decrease in cell proliferation (Fig. S2C). Moreover, B7-H3 knockdown decreased proliferating cell nuclear antigen (PCNA), suggesting that B7-H3 could be involved in the proliferation of lung cancer cells. (Fig. S2D). To further validate the biological functions of B7-H3, we established stable B7-H3 -overexpressing and -knockout A549 cell lines. The efficiency of overexpression and knockout was validated (Fig.1B-D). Compared with the control, the overexpression of B7-H3 in A549 cells promoted cell proliferation and migration, whereas the knockout of B7-H3 inhibited cell proliferation and migration (Fig. 1E-F). Furthermore, B7-H3 overexpression led to an increase in PCNA expression， whereas, B7-H3 knockout cells presented downregulated PCNA expression (Fig.1G). Collectively, these data demonstrate that B7-H3 promoted the proliferation and migration of lung cancer cells.\u003c/p\u003e\n\u003cp\u003e2. B7-H3 modulates glucose uptake and lactate generation via HK2\u003c/p\u003e\n\u003cp\u003eIncreased aerobic glycolysis is now widely considered a hallmark of many cancers(18). We found that B7-H3 is associated with the metabolism of glucose compared with the control group, cells overexpressing B7-H3 showed a significant increase in glucose consumption and lactate production, while knockout of B7-H3 significantly reduced glucose consumption and lactate production (Fig. 2A). These results suggest that B7-H3 may be involved in aerobic glycolysis in lung cancer cells. We examined the expression levels of various key glycolysis-related genes, such as hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), and phosphofructokinase (PFKP), in B7-H3-overexpressing and B7-H3-knockout A549 cells (Fig. 2C). The results showed that overexpression of B7-H3 promoted HK2, PFK, and PKM2 mRNA levels, while knockout of B7-H3 reduced the mRNA levels of HK2 but does not affect the expression of PFK or PKM2. We obtained datasets on the expression of HK2 in adenocarcinoma, squamous cell carcinoma and paired normal tissues from the CPTAC online database, and the results showed that HK2 is highly expressed at the protein level in lung cancer tissues (Fig. S3). In addition, B7-H3 overexpression led to a notable rise in HK2 protein levels, while the knockout of B7-H3 significantly decreased HK2 protein expression in A549 cells (Fig. 2D); similar results were validated in H1975 B7-H3 knockdown cells (Fig. S4A-D). Thus, our results suggest that B7-H3 promotes glycolysis by regulating the expression of the key glycolytic enzyme HK2. To verify that B7-H3 affects lung cancer cell glycolysis through HK2, we employed HK2 siRNA to downregulate HK2 expression. (Fig. 2E-F). Furthermore, the application of 2-deoxy-D-glucose (2-DG), an inhibitor of HK2, completely eliminated the rise in glucose uptake and lactate production induced by B7-H3 (Fig. 2G-H). These results support the conclusion that the B7-H3-mediated enhancement of aerobic glycolysis is dependent on HK2.\u003c/p\u003e\n\u003cp\u003e3. B7-H3 regulates the PI3K/AKT/mTOR pathway through HK2\u003c/p\u003e\n\u003cp\u003ePrior reports have indicated that B7-H3 promotes aerobic glycolysis in oral squamous cell carcinoma through the activation of the PI3K/Akt/mTOR pathway. \u0026nbsp; \u0026nbsp;Our findings corroborate these observations, demonstrating that B7-H3 overexpression significantly increased levels of phosphorylated PI3K (p-PI3K), AKT (p-AKT), and mTOR (p-mTOR), while B7-H3 knockout resulted in diminished levels of these phosphorylated proteins (Fig.3A). However, HK2 expression did not significantly change in response to treatment with rapamycin, an mTOR inhibitor (Fig. 3B). \u0026nbsp; Moreover, the lactate and glucose consumption levels did not significantly change (Fig. 3C). Interestingly, we detected a marked decrease in the levels of p-PI3K, p-AKT, and p-mTOR following the knockdown of HK2, which effectively countered the elevation of the PI3K/AKT/mTOR signaling pathways induced by B7-H3 overexpression in A549 cells. (Fig.3D). Similar results were observed in A549 cells treated with 2-DG, an HK2 inhibitor (Fig. 3E). These findings suggest that B7-H3 activates the PI3K/AKT/mTOR signalling pathway through the modulation of HK2, resulting in increased tumour cell proliferation.\u003c/p\u003e\n\u003cp\u003e4. The plasma sB7-H3 level is positively correlated with the lactate level and is associated with clinical prognosis\u003c/p\u003e\n\u003cp\u003eThe soluble form of B7-H3, sB7-H3, is generated by the cleavage of the variable region of the B7-H3 protein. It can be released into the extracellular fluid under physiological or pathological conditions, both in vivo and in vitro(19). Our results indicated that the level of sB7-H3 in the supernatant of A549 cells overexpressing B7-H3 was greater compared to control cells. In contrast, knocking out B7-H3 resulted in a reduction of sB7-H3 expression (Fig. 4A). Additionally, sB7-H3 levels in plasma were positively correlated with the protein levels of B7-H3 in the cancer tissues of lung cancer patients (Fig. 4B). We analysed the plasma level of sB7-H in patients with lung cancer\u0026nbsp;compared with healthy controls. The results revealed that the plasma level of sB7-H3 in lung cancer patients was significantly greater than that in healthy controls. Mann-Whitney U test analyses revealed a significant correlation between sB7-H3 levels and age, TNM stage, and metastasis, whereas no significant correlation was detected with gender or type of pathology (Fig. 4C and Table 1). The ROC curve was used to assess the diagnostic value of plasma sB7-H3 expression levels in patients with lung cancer. The results revealed the AUC for sB7-H3 in the prediction of lung cancer was 0.8352. The sensitivity was 0.75, while the specificity was 0.793. These findings indicate that sB7-H3 provides valuable information for lung cancer diagnosis. (Fig. 4D). Mann-Whitney U test analyses revealed a significant correlation between sB7-H3 levels and TNM stage and metastasis, while no significant correlation was detected with gender or age (Suppl table 3). Kaplan-Meier survival curve analysis indicated that patients exhibiting elevated levels of sB7-H3 experienced lower overall survival rates compared to those with reduced levels of sB7-H3\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Fig. 4E and Table 2). In addition, we analysed the plasma lactate levels of patients with lung cancer and healthy controls. The findings indicated that lung cancer patients had significantly higher plasma lactate levels compared to healthy controls. Furthermore, we observed a positive correlation between sB7-H3 levels and lactate levels in the plasma (Fig. 4F-G).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"12\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1. The relationship between the levels of sB7-H3 in plasma of patients with LC and clinicopathological characteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\"\u003e\n \u003cp\u003eLung cancer sB7-H3 expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"bottom\"\u003e\n \u003cp\u003eHealthy sB7-H3 expression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eMedian (Min-Max)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eP25~P75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eMedian (Min-Max)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eP25~P75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2278\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.8011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1579\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.4176\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e14.75(5.415-32.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e11.85-17.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e9.617(3.322-17.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e7.925-11.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e14.32(7.130-32.08)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e11.79-32.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10.04(3.164-16.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e7.933-12.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.2783\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.9262\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026le; 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e13.58(5.415-32.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10.62-17.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e9.817(3.164-17.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e7.404-11.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026gt;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e14.73(7.130-32.08)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12.27-18.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e9.672(4.909-16.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e8.217-11.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTNM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0261\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eⅠ+Ⅱ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e13.14(5.415-24.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10.22-16.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; Ⅲ+Ⅳ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e14.89(7.427-32.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12.42-18.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eMetastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0241\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12.73(5.415-22.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10.36-16.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; Yes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e14.83(7.340-32.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12.34-18.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eType of pathology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.8237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAdenocarcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e14.1(5.41-32.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e11.3-18.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eSquamous carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e14.7(7.43-27.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e13-16.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eSmall cell lung cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e15.5(7.9-23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10.9-18.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eOther\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e13.5(12.3-15.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12.3-15.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2. Univariate and multivariate analysis of overall survival factors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 28px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003eUnivariable analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 2px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003eMultivariate analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003eHR (95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u0026nbsp;p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003eHR (95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u0026nbsp;p\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 28px;\"\u003e\n \u003cp\u003eGender (Male vs Female)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003e2.481(1.555-3.957)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0004\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003e2.129(1.199-3.783)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 28px;\"\u003e\n \u003cp\u003eAge (\u0026gt;60 vs\u0026nbsp;\u0026le;\u0026nbsp;60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003e1.693(0.9939-2.885)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.1056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 28px;\"\u003e\n \u003cp\u003eTNM (Ⅲ\u0026nbsp;+\u0026nbsp;Ⅳ\u0026nbsp;vs\u0026nbsp;Ⅰ\u0026nbsp;+\u0026nbsp;Ⅱ)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003e11.38(7.084-18.28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003e4.25(1.318-13.703)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.015\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 28px;\"\u003e\n \u003cp\u003eMetastasis (Yes vs No)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003e31.55(19.09-52.16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003e7.858(0.811-76.127)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.075\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 28px;\"\u003e\n \u003cp\u003esB7-H3 (High vs Low)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003e1.735(1.084-2.777)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0162\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 25px;\"\u003e\n \u003cp\u003e1.753(1.074-2.862)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.025\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e5. The mechanism by which B7-H3 promotes glycolysis and its prognostic value in lung cancer\u003c/p\u003e\n\u003cp\u003eB7-H3 enhances PI3K/AKT/mTOR signalling pathway activation by upregulating the key glycolytic enzyme HK2, thereby participating in glycolysis. The activated mTOR pathway regulates crucial cellular processes including proliferation and migration. In tumour cells, glucose is metabolized through glycolysis, producing ATP and lactate. The accumulation of lactate acidifies the tumour microenvironment, facilitating tumour growth, invasion, and migration. sB7-H3 is a soluble form generated by alternative splicing of the variable region of the B7-H3 protein. The presence of sB7-H3 in peripheral blood holds clinical value in the prognosis of lung cancer.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have shown that B7-H3 is a negative costimulatory molecule that inhibits immune cells through negative signals, deactivates the functions of T cells and NK cells, and then leads to tumour immune escape(11,20,21). In this study, we explored the role of B7-H3 in lung cancer and discovered that it was significantly elevated in lung cancer tissues. Moreover, increased levels of B7-H3 were associated with enhanced proliferation and migration of lung cancer cells. Furthermore,\u0026nbsp;our research indicated that B7-H3 enhances glycolysis through HK2 and modulates the PI3K/Akt/mTOR pathway, which in turn boosts the proliferation and migration of lung cancer cells. We also observed a positive relationship between B7-H3 expression in cell lines and lung cancer tissues, as well as with the levels of soluble B7-H3 (sB7-H3) in the corresponding supernatant and serum. Importantly, the concentration of sB7-H3 in the plasma of lung cancer patients was higher than that in healthy controls and showed a positive correlation with lactate levels.\u003c/p\u003e\n\u003cp\u003eThe role of anaerobic glycolysis in cancer has long been well known. With the extensive application of immunotherapy in the clinic, an increasing number of scholars have linked immunotherapy with aerobic glycolysis. Dussol suggested that regulating macrophage metabolism is a new cancer immunotherapy strategy(22). Hu Z suggested that glycolysis drives STING signalling to promote the antitumour function of dendritic cells(23), Zhang L found that STING can limit aerobic glycolysis without relying on its innate immune function by directly inhibiting hexokinase activity through targeting HK2、As a result, STING reduces aerobic glycolysis in tumors and enhances antitumor immunity in vivo (24). Jing Y reported that sting directly affects the activity of PI3K in the study of actin reorganization during the regulation of BCR activation by sting(25). Shi T\u0026rsquo;s study indicated that B7-H3 could influence glucose metabolism and contribute to chemotherapy resistance in colorectal cancer cells by modulating the expression of HK2 (13). Zuo J study found that B7-H3 interacts with the glycolytic enzyme ENO1, thus affecting glycolysis in cervical cancer cells (26). A Nunes-Xavier CE study revealed that B7-H3 can increase the glycolytic ability and drug resistance of inhibitors of the PI3K/AKT/mTOR pathway in triple negative breast cancer cells(27). As the first key enzyme in the glycolytic pathway, HK2 may act through the NF-\u0026kappa;Bp65(28), ROS/PI3K/AKT/HIF-1\u0026alpha;(9), and AKT-GLUT1(29) signalling pathways, but it is more closely related to PI3K/AKT/mTOR(14,15).\u003c/p\u003e\n\u003cp\u003eIn order to better apply B7-H3 in the clinic, we first studied the relationship between B7-H3 and the prognosis of lung adenocarcinoma patients in the TCGA found that high expression of B7-H3 was related to poor prognosis of lung adenocarcinoma patients(30,31){Darshan Shimoga, 2022 #479}. Secondly, we found that soluble B7-H3 (sB7-H3) in the supernatant of lung cancer cells was consistent with the expression of B7-H3 mRNA and protein, and we also found the same results in the plasma and tissues of the lung cancer patients we collected. These results suggest that the plasma level of sB7-H3 may serve as a prognostic indicator for lung cancer patients and help inform their clinical treatment decisions.sB7-H3 is produced by monocytes, dendritic cells, activated T cells and B7-H3\u003csup\u003e+\u003c/sup\u003e tumour cells(32). Studies have indicated that serum levels of sB7-H3 are significantly elevated in patients with non-small cell lung cancer. This increase is strongly associated with tumor stage, size, and lymph node metastasis, but shows no correlation with age, gender, or pathological classification(33). As a tumour biomarker, sB7-H3 is superior to CA125, CA153, CA199 and CEA(34). In addition, high expression of sB7-H3 in the serum of patients with intestinal cancer is negatively correlated with the number of T lymphocytes infiltrated by intestinal cancer(35). Soluble B7-H3 secreted by pancreatic cancer cells can promote tumour invasion and metastasis through the TLR4/NF-\u0026kappa;B pathway(36). Therefore, as a potential biomarker for tumour diagnosis and prognosis, sB7-H3 is attracting increasing attention (19). Research indicates that higher levels of B7-H3 expression are associated with increased release of sB7-H3. sB7-H3 can spread to other tissues through the circulatory system and play an important biological role. Therefore, the positive correlation between B7-H3 and sB7-H3 may play an important role in tumour growth and immune regulation(37). However, there is no research on whether sB7-H3 is involved in the glycolysis of tumour cells at present, and further research can be conducted on whether sB7-H3 affects the glycolysis of immune cells and tumour cells.\u003c/p\u003e\n\u003cp\u003eIn summary, this study provides a theoretical basis for the influence of B7-H3 on the proliferation and migration of lung cancer cells through aerobic glycolysis, provides evidence for the treatment of lung cancer patients through targeting B7-H3, and provides a new idea for the application of sB7-H3 in clinical Prognosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHK2: hexokinase 2\u003c/p\u003e\n\u003cp\u003ePKM2: pyruvate kinase M2\u003c/p\u003e\n\u003cp\u003ePFKP: phosphofructokinase\u003c/p\u003e\n\u003cp\u003esB7-H3: soluble B7-H3\u003c/p\u003e\n\u003cp\u003ePD-L1:\u0026nbsp; \u0026nbsp;\u0026nbsp;programmed death receptor-1\u003c/p\u003e\n\u003cp\u003eTCGA:\u0026nbsp; \u0026nbsp;\u0026nbsp;The Cancer Genome Atlas\u003c/p\u003e\n\u003cp\u003eCPTAC: Clinical Proteomic Tumour Analysis Consortium\u003c/p\u003e\n\u003cp\u003eLUAD:\u0026nbsp; \u0026nbsp;\u0026nbsp;lung adenocarcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLUSC:\u0026nbsp; \u0026nbsp; \u0026nbsp;lung squamous cell carcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROC: receiver operating characteristic\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAUC: area under the curve\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCNA: \u0026nbsp; \u0026nbsp;proliferating cell nuclear antigen\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of Jiangsu University Affiliated Aoyang Hospital (Approval No. 2021-015). Prior to the commencement of the study, all patients were provided with detailed information about the purpose and methodology of the research. They were fully informed about the study and its potential risks and benefits, and they voluntarily signed written informed consent forms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Suzhou Science and Technology Development Program (SKJYD2021005), the Suzhou Science and Technology Program (SYSD2020011), the Clinical Medical Science and Technology Development Fund of Jiangsu University (JLY20180145), and the Zhangjiagang Health Youth Science and Technology Project (ZJGQNKJ202018, ZJGQNKJ202136).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShiyang Han: conceptualization, data curation, investigation, writing \u0026ndash; original draft, methodology. Yuantao Wu: Writing \u0026ndash; original draft, Funding acquisition. Yan Zhou: Data curation. Qiuyan Xia: Data curation, Funding acquisition. Yan Zhang, Yi Wu, Zhanguo Liu and Jing Yang: Data curation. Ye Shen: Supervision. Shuru Zhou: Project administration, writing, review, editing and funding acquisition. Shiyang Han and Yuantao Wu contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data supporting the findings of this study are available within the article and its Supplementary information files and from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge Dr. Jiajia Jiang and Dr. Shijie Tang from the Affiliated Aoyang Hospital of Jiangsu University for their review and feedback on this manuscript, which is highly important for further improving our research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Laversanne M, Sung H, et al. 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Cancer Biomark 2022;33:123-9. http://10.3233/CBM-210178.\u003c/li\u003e\n\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":"B7-H3, glycolysis, HK2, lung cancer, PI3K/AKT/mTOR signalling pathway","lastPublishedDoi":"10.21203/rs.3.rs-5738861/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5738861/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLung cancer is the foremost cause of cancer-related mortality globally. B7-H3, an immune checkpoint, not only has immunomodulatory functions but also promotes tumor cell proliferation, drug resistance, metastasis, and aerobic glycolysis. The specific role of B7-H3 in aerobic glycolysis in lung cancer has not yet been elucidated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eB7-H3 expression was validated in lung cancer tissues and lung cancer cell lines. Overexpression and knockout experiments were conducted in lung cancer cell lines to assess cell proliferation, migration, lactate production, and glucose consumption. The expression of hexokinase 2 (HK2) and the PI3K/Akt/mTOR signaling pathway were analyzed. Additionally, soluble B7-H3 (sB7-H3) along with lactate levels in the plasma of lung cancer patients were measured, and the clinical characteristics were analysed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eB7-H3 was highly expressed in lung cance tissue and lung cancer cell lines, and enhanced the proliferation and migration, along with increased lactate production and glucose consumption rates. Our study also demonstrated that B7-H3 regulates the expression of HK2, thereby promoting the proliferation and migration of lung cancer cells through the PI3K/Akt/mTOR signaling pathway. Furthermore, sB7-H3 levels was postively correlated with the lacte levels in the plasma of lung cancer patients, and high sB7-H3 levels were associated with a poorer prognosis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe research suggests that B7-H3 affects lung cancer cell proliferation and migration by enhancing aerobic glycolysis via HK2, providing a new perspective for the application of B7-H3 in clinical prognosis and treatment.\u003c/p\u003e","manuscriptTitle":"B7-H3 regulates HK2 expression and promotes the PI3K/AKT/mTOR pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-02 08:57:23","doi":"10.21203/rs.3.rs-5738861/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":"868f71dc-4e03-44ae-ac74-e2cea926afa6","owner":[],"postedDate":"January 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-09T11:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-02 08:57:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5738861","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5738861","identity":"rs-5738861","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}