{"paper_id":"0e520bb5-078a-4702-958a-883690122be8","body_text":"BACE2 is a prognostic biomarker and induced macrophage M2 polarization in Non-Small Cell Lung Cancer | 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 BACE2 is a prognostic biomarker and induced macrophage M2 polarization in Non-Small Cell Lung Cancer Yuanyuan Feng, Xiaohan Zhou, Kaifan Yang, Ziyan Zhu, Shuyi Liu, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5406902/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 BACE2 has gained attention for its upregulation in various human cancers, suggesting relevance in cancer biology. However, its specific role in tumor growth and the tumor immune microenvironment (TIME) remains underexplored. We conducted bioinformatics analyses using pan-cancer datasets to assess BACE2 expression patterns and its correlation with patient outcomes. Additionally, we investigated associations with immune-related genes and immune cell infiltrations in tumor microenvironments. A BACE2 knockdown lung cancer model was established to evaluate its impact on Sorafenib efficacy and PD-L1 inhibitor-induced antitumor immune responses. BACE2 was significantly upregulated in multiple cancers compared to normal tissues and associated with poorer patient outcomes. It played a critical role in modulating immune-related gene expression and promoting immune cell infiltrations within tumors. In vitro and in vivo experiments showed that BACE2 knockdown inhibited M2 macrophage polarization, suppressed lung tumor progression, and enhanced Sorafenib efficacy by increasing activated immune cell infiltrations. Similarly, BACE2 knockdown potentiated PD-L1 inhibitor-induced antitumor immune responses in lung cancer models. This study underscores BACE2's pivotal role in lung cancer tumorigenesis and its influence on the tumor immune microenvironment. Inhibiting BACE2 could enhance current treatment effectiveness in lung cancer therapy, offering promising avenues for improving patient outcomes. BACE2 Tumor immune microvironment Macrophage NSCLC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Lung cancer has surpassed the breast cancer is the most commonly occurring cancer worldwide with 2.5 million new cases accounting for 12.4% of total new cases. Lung cancer remained the first leading cause of cancer death, with an estimated 9.74 million(18.7%) 1 . In recent years, immune checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have been proven its significant efficacy on advanced non-small cell lung cancer (NSCLC) 2 . However, the efficiency of immunotherapy (mainly immune checkpoint inhibitors) in unselected populations is relatively low, and only a small proportion of patients can benefit from immunotherapy, because of a lack of optimal biomarkers. Further research is needed to identify more biomarkers to aid in patients selection to improve immunotherapy respone rate 3 . BACE2 (β-site amyloid precursor protein cleaving enzyme 2) is a gene encoding the β-amyloid precursor protein cleaving enzyme that has garnered widespread research interest in recent years 4 . Initially, BACE2 was primarily associated with the nervous system and Alzheimer's disease 5 . However, recent studies have shown that BACE2 plays a significant role in key biological processes of tumor proliferation, invasion, and metastasis 6 – 8 . In the last decade, a very active field in cancer research has been the study of tumor microenvironment. The tumor microenvironment is composed of immune cells, such as macrophages, lymphocytes and neutrophils, of endothelial and stromal cells, of the extracellular matrix (ECM) and of soluble factors.Inside the microenvironment, the soluble factors, secreted by tumor cells, are able to recruit all these different types of cells, that, educated by the tumor itself, produce a pro-tumoral environment 9 . BACE2, thanks to their ability of shedding polypeptides in the extracellular space, can be considered key players that orchestrate the crosstalk between the tumor and the surrounding cells 10 . Therefore, gaining a deeper understanding of the relationship between the BACE2 gene and cancer is of paramount importance for developing new cancer treatment strategies. In this study, we first conducted bioinformatics analysis to investigate the expression of BACE2 in various cancers. We also analyzed the impact of BACE2 on survival prognosis and the immune microenvironment. The results indicate that BACE2 can serve as a biological marker for prognostic assessment and M2 macrophages infiltration. Additionally, the effect of BACE2 in lung cancer were verified by in vivo and in vitro experiments. we validated the role of BACE2 in the tumor immune microenvironment, revealing its influence on the infiltration of T cells and macrophages in tumor tissues. Furthermore, knocking down BACE2 not only slows tumor growth but also enhances the efficacy of immunotherapy and sorafenib treatment in lung cancer model.Collectively, the obtained result indicate that BACE2 may be a potiential biomarker for lung cancer therapies. Meterials and methods Data collection We retrieved and downloaded mRNA expression profiles and related clinical data for 33 types of cancer from The Cancer Genome Atlas (TCGA) database ( https://portal.gdc.cancer.gov/ ), and mRNA expression profiles for normal human tissues from the Genotype-Tissue Expression (GTEx) database ( https://www.gtexportal.org/home/ ). Cell line gene expression substrates of tumors were obtained from the Cancer Cell Line Encyclopedia dataset (CCLE, https://portals.broadinstitute.org/ccle/about ). Pan-cancer differential expression and prognostic value of BACE2 test was used to detect the expression differences between tumors and normal tissues; statistical analysis was performed using R software (version 4.0.2), and the R package \"ggplot2\" was used to draw box plots. Similar data processing methods were used to study the correlation between BACE2 and tumor stage. p < 0.05 was considered statistically significant. The GTEx database was used to analyze the expression of BACE2 in normal tissues, and the TCGA database was used to analyze the expression of BACE2 in pan-cancer tissues. The expression profiles of TCGA and GTEx were integrated to compare the expression differences of BACE2 in tumor and normal tissues. The expression level of BACE2 in tumor cell lines was analyzed using CCLE dataset. Radar plots were then drawn based on the median expression and expression differences of BACE2 in each of the 33 cancer types or in each tissue. After ranking the BACE2 mRNA expression levels, the data from 33 cancers in the TCGA pan-cancer database were categorized into a low BACE2 expression group and a high BACE2 expression group based on the median BACE2 expression level. Thereafter, to investigate the effect of BACE2 on survival in pan-cancer patients, we performed univariate Cox regression (uniCox) using the R packages \"survminer\" and \"survival\" to derive p-values and risk ratios (HR, 95% confidence intervals 38 ) for the Kaplan-Meier curves. According to the analysis results (Kaplan- Meier curves), the clinical prognosis and outcomes (represented by the overall survival (OS) of patients in both groups (P < 0.05)) were compared between the patients in the BACE2 high-expression group and those in the low-expression group using the R software package, \"survival\" (P < 0.05). Gene Set Enrichment Analysis (GSEA) related to BACE2 To explore the potential biological processes and molecular functions affected by BACE, we performed gene set enrichment analysis using GSEA software (v.4.2.3), which was performed using the R package \"clusterprofiler\". An adjusted P value of < 0.05 was considered statistically significant. The steps were: 1) Integrate the gene expression data of BACE2 low and high expression groups and submit them to GSEA software; 2) Download the \"gmt\" file of Hallmark pathway gene set from the Molecular Signature Database (MSigDB); 3) Calculate the normalized enrichment score (NES) and the false discovery rate (FDR) for each bioprocess; 4) Apply R package \"Ggplot2\" to visualize the results. Correlation analysis of BACE2 expression with immune-related genes The correlation between BACE2 expression and immune-related genes was analyzed, including immune-activating genes, cytokines, immune-suppressing genes, cytokine receptors, and genes for the major histocompatibility complex (MHC). Pearson correlation analysis was performed to show the association between BACE2 mRNA and the expression of immune-related genes, which were obtained from the TCGA pan-cancer data (p < 0.05 was considered significant), and the results of the analysis are presented in heat maps. Relationship Between BACE2 Expression With Immune Cell Infiltration A total of three different methods were used in this study to assess the association between BACE2 expression levels and immune cell infiltration in pan-cancer. The Tumor Immune Estimation Resource (TIMER) can be used to analyze immune cell infiltration in different cancers using various algorithms. Therefore, first we searched the TIMER 2.0 database ( http://timer . comp-genomics.org/) to download BACE2-associated immune cell infiltration correlations in the TCGA pan-cancer project, which included various types of T cell and macrophage infiltration. In the second, we obtained data on immune cell infiltration in TCGA pan-cancer patients from previous published works and analyzed them for correlation. In the third, we downloaded the immune cell infiltration scores of TCGA pan-cancer patients on the ImmuCellAI platform ( http://bioinfo.life.hust.edu.cn/web/ImmuCellAI/ ) and processed the data by similarly correlating them. Then we visualized the correlation between the expression of BACE2 and the level of tumor infiltration of different immune cells, and the results were presented in heat maps. Cell lines and cell culture The human lung cancer cell line (A549) and murine lung cancer cell line (LLC) were obtained from Guang Zhou Jennio Biotech Co.,Ltd. All cell lines were validated by short tandem repeat analysis. All cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), and were cultured in a humidified incubator set at 37℃ with a 5% CO2 environment. qRT-PCR Initially, the total cellular RNA of LLC and A549 were extracted using the RNA isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China). Then RNA was reverse transcribed into complementary DNA (cDNA) with HiScript II Q RT SuperMix for qPCR (+ g DNA wiper) (Vazyme) according to the manufacturer’s introduction. qRT-PCR was conducted using SYBR® Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, Changsha, China). Standardized with GAPDH expression level, the levels of target were calculated using 2-ΔΔCT method. Cell counting kit-8 proliferation assay (CCK-8) LLC and A549 cells were seeded in 96-well plates at a concentration of 1000 cells/well with 5 repetitions in each group. The culture medium was replaced with CCK-8 reagent(Fdbio science, Hangzhou, China), and plates were incubated at 37℃ for 2h. Absorbance was measured at 450 nm. Transwell assay LLC cells and A549 cells(6 ×10 4 ) in 300µL of FBS-free medium were respectively seeded into the upper chamber of the 24-well transwell system (8 µm pore size) (Falcon, USA), while 700µL of 10% FBS was added to the lower chamber. The system was incubated at 37℃ for 24h. The cells in the upper chamber were removed, while the cells in the lower chamber were fixed with methanol and stained with 5% crystal violet. The migrating cells were photographed and counted under microscope from at least five random fields. Animal experiments All experiments were approved by the Animal Ethics Committee of Nanfang Hospital and conducted according to the international guidelines for animal care and use. Female C57BL/6J mice of 3–4 weeks old were purchased from Risemice Biotechnology (China). LLC cells (1×10 6 ) resuspended in 100µL PBS were subcutaneously injected into the right flank of the mice. Sorafenib(30mg/kg) (Glpbio, USA) and PD-L1 inhibitor(100µL) (Glpbio) were administered to the experimental group mice via gavage and intraperitoneally injection every 2 days respectively. Bi-weekly tumor measurements were performed every 2 days when the tumor volume reached 200mm 3 , calculated as 0.5×length×width 2 . Flow cytometry analysis Tumors were dissociated into single-cell suspensions with collagenase. Then the dissociated cells were filter through a 40µm cell strainers (Biologix, Jinan, China) and resuspended in PBS. After blocking with anti-mouse CD16/CD32(BD Biosciences, US) macrophages were incubated with antibodies APC anti-mouse CD86 and FITC anti-mouse CD206, while T cells were stained with antibodies FITC anti-mouse CD3, PE-Cy7 anti-mouse CD8, APC-Cy7 anti-mouse CD4 and PE anti-mouse FOXP3 (all from 4A Biotech, Suzhou, China) for 30 minutes in dark. Washed by flow buffer, the samples were then analyzed using flow cytometry (BD Biosciences, US). As for the measurement of intracellular ROS level, A549 cells were harvested and stained with DCFH-DA (Solarbio, Beijing, China). RESULTS BACE2 is upregulated in multiple cancer tissues By retrieving BACE2 expression through GTEx and TCGA mRNA expression datasets, it is evident that BACE2 gene level is upregulated in a wide range of tumors, including Adrenal cortical carcinoma (ACC), cholangiocarcinoma (CHOL), colorectal adenocarcinoma (COAD), lymphomatous diffuse large B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), renal chromotropic disorder (KICH), renal form cell carcinoma (KIRP), acute myeloid leukemia (LAML), brain low-grade glioma (LGG), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), ovarian serous cystadenocarcinoma (OV), pancreatic cancer (PAAD), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), skin cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid cancer (THYM), Uterine body endometrial cancer (UCEC) while down-regulated expression of this gene can be seen in Clear cell carcinoma of kidney (KIRC) and uterine carcinosarcoma (UCS), compare to normal tissues ( Fig. 1 A ) .Then we made three graphs showing the profiles of BACE2 expression in normal tissues, pan-carcinoma tissues, and tumor cell lines respectively, indicating the average expression level of BACE2, and the differences in expression level in various tissues, and the expression level is arranged in the order from low to high ( Fig. 1 B-D ) . The expression of BACE2 was found to be significantly upregulated in KICH, LUAD, LUSC, COAD, CHOL, STAD, READ, PRAD, and ESCA paired tumors relative to adjacent normal tissues in the TCGA dataset. Except for ESCA, the expression of BACE2 in other tumors and adjacent tissues was statistically significant (Fig. 2A-I) . Moreover, BACE2 expression was up-regulated in different stages, high levels of BACE2 expression had worse clinical stage,espically in LIHC, STAD, and PAAD tumors (Fig. 2J-L) Figure 2 Relationship between BACE2 expression level and tumorigenesis and development (A-I) Patterns of BACE2 expression levels in tumor as well as adjacent normal tissues based on a TCGA dataset. (J-L) Correlation between BACE2 expression levels and the stages of three different tumors(LIHC\\STAD\\PAAD). * P < 0.05, ** P < 0.01, **** P < 0.0001, ns, not significant. BACE2 expression correlated with outcomes of patients in multiple cancer types The forest plot demonstrates the overall survival (OS) of BACE2 levels in TCGA pan-cancer patients obtained after analysis using Cox proportional risk model. The results showed that in LGG ( P < 0.001), CESC ( P = 0.001), GBM ( P = 0.005), PAAD ( P = 0.017), KIRC ( P = 0.021), LIHC ( P = 0.004), BRCA ( P = 0.035), LUAD ( P = 0.039) and SKCM ( P = 0.049) patients, high BACE2 levels were significantly linked to poorer OS ( Fig. 3 A ) . Moreover, the expression level of BACE2 significantly affected the DSS, DFI and PFI of patients in multiple cancers (Fig. S1 A-C) . In addition, Kaplan-Meier curves found that in CESC ( p < 0.0001), KIRC ( p < 0.0001), UVM ( p = 0.00053), HNSC ( p = 0.0039), GBM ( p = 0.0016), SKCM ( p = 0.00043), LUAD ( p = 0.0074), BRCA ( p = 0.0043) patients, high levels of BACE2 expression were associated with shorter OS times Similarly, the patients with higher level of BACE2 showed poorer DSS and DFI in multiple cancers (Fig. S2-3) . BACE2 played an important role in cancer immune response To investigate the possible biological processes regulated by BACE2 in pan-cancer, we performed gene set enrichment analysis (GSEA) using R packages \"clusterprofiler\" in pan-cancer subjects, then we finally selected six tumors (UCS, LUSC, OV, PAAD, STAD, THCA) with similar results. The results showed that the enriched pathways had positive normalized enrichment scores (NES), suggesting that most of the potential biological pathways regulated by BACE2 are associated with immune regulatory mechanisms, such as neutrophil activation, myeloid cell activation and cell activation involved in immune response, cytokine production in the immune system, as well as adaptive and innate immune systems ( Fig. 4 A-F ) . This suggests that BACE2 plays an important role in tumor immune response, especailly in tumor immune micro-environment. Relationship between BACE2 and that of immune-related genes Next, we performed gene co-expression analysis in order to further elucidate the relationship between BACE2 expression and immune-related genes in pan-cellular carcinomas, which revealed that BACE2 expression was positively correlated with most immune-suppressing genes (Fig. 5C) chemokines (Fig. 5B) and chemokine receptors (Fig. 5D) , and was negatively associated with many immune-activating genes (Fig. 5A) and MHC genes (Fig. 5E) in a variety of tumor types. The above results suggested that BACE2 expression was strongly linked to immune checkpoint genes in most tumor types. Among these immune marker genes, IL10RB, IL-10, and TGFBR1 were found to be significantly associated with BACE2 expression in most tumor types. And we have learned that there is a significant correlation between TGFBR1, IL-10 and IL-10RB expression and tumor-associated macrophage(TAMs), which are closely involved in tumorigenesis infiltration and metastasis 11 , 12 . Moveover, we observed that the expression of BACE2 is positively correlated with the expression of CCR2, CXCL1/CXCL8-CXCR2, CXCL12-CXCR4, and CCR8 in multiple cancer types. These chemokines and their receptors have been demonstrated to be associated with the recruitment of tumor-associated immunosuppressive cells 13 – 16 . In addition, we observed that BACE2 expression was associated with HAVCR2, PDCD1,CTLA4,TIGIT and LAG3,the markers of exhaustion in CD8 + T cells 17 , which implied BACE2 highly expressed patients would be benefited form immune checkpoint therapy. BACE2 was associated with immune cell infiltrations Previous studies have shown that tumor-infiltrating immune cells usually function abnormally and promote immune escape of tumor cells, i.e., they fail to inhibit tumor growth and even promote its progression 18 , 19 . The Estimate algorithm was used to predict the relationship between BACE2 expression and immune infiltration. BACE2 was negatively associated with the stromal score, immune score, and ESTIMATE score in LUAD, UCEC, CESC, SKCM, and KICH, indicating that BACE2 significantly correlated with poor immune cell infiltration in certain types of cancer (Fig.S4A) .In the tumor microenvironment, tumorigenesis and progression are closely linked to the enrichment of tumor-associated macrophages and T cells. Therefore, we evaluated the correlation between BACE2 expression and TAMs and T-cell infiltration using the TIMER2 database. The assessment showed that the expression levels of most T cell types were negatively correlated with BACE2 expression in most tumor types, but the level of T cells in the tumor immune microenvironment were positively correlated with BACE2 expression. And the level of TAMs infiltration was positively correlated with BACE2 expression, especially with M2-type macrophages. ( Fig. 6 A ) .We then evaluated the correlation between BACE2 levels and immune scores (representing immune cell infiltration in tumor tissues) in 33 cancers by calculating BACE2 levels and performing pan-cancer analyses of different immune cell tumor infiltrations with BACE2 expression using published work and the ImmuCellAI database. Clustering heatmaps showed a positive correlation between BACE2 expression and macrophages in most tumor types, consistent with TIMER2 results ( Fig. 6 B-C ) . We could also observe that BACE2 was significantly positively correlated with Treg cells and negatively correlated with CD8 + T cells in a variety of cancers ( Fig. 6 B-C ) . Consistently, correlation analysis also showed that the expression of BACE2 was negatively correlated with the infiltration of CD8 + T cells and B cells (Fig.S4B-C) . BACE2 is involved in the progression of lung cancer and macrophage M2 polarization. To investigate the impact of BACE2 on cancer cell proliferation and migration, we initially constructed LLC and A549 cell lines with BACE2 knockdown. qRT-PCR confirmed the successful suppression of BACE2 in both LLC and A549 cell lines ( Fig. 7 A-B ) . The absence of BACE2 significantly impeded the proliferation and migration of LLC and A549 cells, as evidenced by Cell Counting Kit-8 proliferation assay (CCK8) and Transwell Assay results ( Fig. 7 C-H ) .Moreover, we implanted lung cancer cells into mice to establish subcutaneous tumors. In vivo experiments revealed that tumors with BACE2 knockdown exhibited smaller size, lighter weight, and slower growth rate compared to the control group. Mice bearing tumors with BACE2 knockdown also demonstrated a prolonged survival time (Fig .7I-L). Additionally, we explored whether the expression level of BACE2 is associated with macrophage infiltration in the tumor microenvironment. Flow cytometry analysis illustrated that the downregulation of BACE2 did not significantly affect the infiltration of M1 macrophages (CD86 + CD206 − ), but a reduced presence of M2 macrophages (CD86 − CD206 + ) was observed in tumors with low expression of BACE2 ( Fig. 7 M-N ). In summary, all the aforementioned findings indicate that the deletion of BACE2 exhibits antitumor effect. BACE2 knockdown enhances the efficacy of Sorafenib, increasing the infiltration of activated immune cells in lung cancer. Through co-expression analysis, we identified a positive correlation between BACE2 expression and numerous ferroptosis-related genes and autophagy-related genes across various cancer types (Fig. S5A-B) . Flow cytometry analysis revealed that A549 cells with lower BACE2 expression exhibited elevated levels of reactive oxygen species (ROS) ( Fig. 8 A- 8 C ) . Consequently, we investigated whether BACE2 could influence the therapeutic efficacy of a ferroptosis inducer in mice. Mice, each with tumor inoculation of stable BACE2 expression and BACE2 knockdown LLC cells, were respectively assigned to the control and treatment groups. Sorafenib, a classical ferroptosis inducer, was administered to the treatment group ( Fig. 8 B ). In comparison to the control group, mice receiving Sorafenib treatment and exhibiting lower BACE2 expression displayed prolonged overall survival ( Fig. 8 D ) . Tumor growth was significantly inhibited by Sorafenib in mice bearing BACE2-downregulated cells compared to the control cells ( Fig. 8 E-I ). Furthermore, the previously results mentioned the relationship between BACE2 and immunity, and flow cytometry analysis demonstrated that compared to the control group, mice bearing BACE2-knockdown cells combined sorafenib treatment presented a higher proportion of macrophages with M1 phenotype. Sorafenib reduced the number of macrophage M2 on BACE2 control group.but the decrease in macrophage M2 polarization did not show a significant difference on BACE2-silent cells ( Fig. 8 G-H ). Moreover, upon treatment with Sorafenib, a greater infiltration of CD3 + T cells and CD8 + T cells, along with a reduced presence of Treg cells (CD4 + FOXP3 + ), was observed in the tumor microenvironment of mice with low BACE2 levels compared to the control ( Fig. 8 J-M ) . These findings collectively suggest that a low expression level of BACE2 enhances the efficacy of Sorafenib, thereby suppressing the progression of lung cancer through the promotion of ferroptosis. BACE2 knockdown enhances the antitumor immune response elicited by the PD-L1 inhibitor in lung cancer. As a prototypical immune checkpoint inhibitor, the PD-L1 inhibitor has found application in the therapeutic landscape of numerous tumors 20 . Considering the intricate interplay between BACE2 and the tumor immune microenvironment, our focus shifted to investigating whether the downregulation of BACE2 could enhance the antitumor immune response when combined with the PD-L1 inhibitor. We generated subcutaneous tumour xenograft models as delineated in (Fig. 9 A ) . Crucially, compared to the control group, the synergistic application of BACE2 downregulation and PD-L1 inhibitor yielded a remarkable suppression of tumor growth and weight, with an extended overall survival ( Fig. 9 B-D ) . Strikingly, when treated with the PD-L1 inhibitor, the mice bearing BACE2 knockdown tumors exhibited an increased presence of M1 macrophages, CD3 + cells, and CD8 + cells, along with a reduction in Treg cells ( Fig. 9 E-K ). These findings suggest that a diminished expression level of BACE2 enhances the antitumor immune response elicited by the PD-L1 inhibitor. Discussion Previous studies have shown that BACE2 plays a significant role in key biological processes of tumor proliferation, invasion, and metastasis. Howerver, little is know about the possible role of BACE2 in tumor microenvironment.In this study, results indicat that BACE2 is upregulated in many tumor types and Kaplan-Meier survival curves of prognostic value in pan-cancer revealed that its upregulation was markedly linked to worse OS.Further, BACE2 levels correlate with infiltrating immune cells in pan-cancer,.The expression levels of most T cell types were negatively correlated with BACE2 expression in various tumor types, but positive correlation with TAM infiltration, particularly with M2-type macrophages, underscores the potential involvement of BACE2 in promoting immunosuppressive and pro-tumorigenic microenvironmental conditions.Futhermore,the role of BACE2 in lung cancer progression and tumor immune environment is elucidated in our study through a series of experiments. Knockdown BACE2 expression significantly inhibits the proliferation and migration of both LLC and A549 lung cancer cell lines. Furthermore, in vivo experiments show that tumors with BACE2 knockdown exhibit reduced size, weight, and growth rate compared to control tumors. we explores the impact of BACE2 expression on macrophage and T cell infiltration in the tumor microenvironment. Flow cytometry analysis reveals that while downregulation of BACE2 does not significantly affect the infiltration of M1 macrophages, it leads to a reduced presence of M2 macrophages and elevated CD8 + T proportion in tumors with low BACE2 expression. And BACE2 knockdown enhances the efficacy of Sorafenib and PD-L1 inhibitor, provided new therapy strategies for lung cancer. BACE1 and BACE2 are two aspartic proteases involved in the cleavage of different substrates within distinct extracellular domains 21 . Despite their high degree of homology, they exhibit divergent functions depending on the cellular context. BACE1 primarily acts within the central nervous system 22 , while BACE2 is primarily involved in the maintenance of β cells and pigment deposition in melanocytes 23 . These differential roles reflect their tissue distribution, with BACE1 being highly expressed in various regions of the nervous system and BACE2 being more widely distributed in peripheral tissues.Although discovered simultaneously, BACE1 initially garnered scientific attention primarily due to its regulation of cytotoxic Aβ peptide formation in Alzheimer's disease (AD). Consequently, BACE1 inhibitors have been developed to counteract the production of toxic amyloid-like β peptides. Some of these drugs have been investigated in clinical trials 24 .However, despite reducing Aβ peptide production, they have not ameliorated cognitive deficits in AD patients 25 , 26 . Previous studies have indicated an upregulation of BACE2 across various tumors, while BACE1 appears to remain unaltered in cancer. Elevated BACE2 expression is associated with poorer prognosis in melanoma, pancreatic cancer, and gliomas, suggesting a potential active role for this protease in cancer progression 6 – 8 , 27 . Conversely, the expression of BACE1 is not correlated with disease prognosis, indicating that BACE2, rather than BACE1, appears to be directly or indirectly involved in cancer pathogenesis or progression. Additionally, BACE2, due to their ability to release peptides into the extracellular space, can be considered key players in coordinating the interplay between tumors and surrounding cells, highlighting its versatility as a potential biomarker or therapeutic target. Ferroptosis is a distinct form of programmed cell death characterised by the involvement of intracellular iron and excessive lipid peroxidation, morphologically and mechanistically separating from other forms of cell death 28 Our results showed than sorafenib treatment presented a higher proportion of macrophages with M1 phenotype and reduced the number of macrophage M2. This phenomenon may also be related to the increased generation of ROS due to the low expression of BACE2, thereby enhancing the efficacy of sorafenib 29 , 30 .Futhermore, BACE2 expression is correlated with ferroptosis-related genes,indicated BACE2 may enhances ferroptosis. Previous research findings suggest that induces intracellular iron overload in macrophages, promoting their polarization towards the M1 phenotype, increasing the production of inflammatory factors, and inhibiting tissue repair and immune regulation abilities 31 – 33 . Both M1 and M2 macrophages exhibit distinct iron metabolism statuses and sensitivities to ferroptosis. High iron concentrations stimulate macrophages to secrete pro-inflammatory cytokines, thereby making them more prone to polarize into M1 macrophages. Conversely, low iron concentrations can promote polarization towards M2 macrophages 34 , 35 . Apart from regulating macrophage polarization through ferroptosis, interventions in the cellular environment and modulation of signaling pathways can also influence macrophage polarization. For instance, extracellular cytokines, and light, upon binding to macrophage receptors, can activate relevant signaling pathways, thereby affecting macrophage polarization 36 , 37 . In conclusion, current research indicates a certain connection between macrophages and ferroptosis, although the precise regulatory mechanisms of their interaction remain incompletely understood and warrant further investigation. However, elucidating the interplay between ferroptosis and macrophages is crucial for understanding the specific mechanisms underlying various diseases, particularly tumor pathogenesis, and is essential for developing innovative therapeutic strategies and evaluating their efficacy. However, our data primarily originate from public databases, which possess inherent limitations, and certain results cannot be validated through clinical specimens from patients. Furthermore, while BACE2 exhibits varying degrees of upregulation in most cancers, the precise molecular mechanisms underlying its promotion of tumor growth remain elusive. BACE2 influences the immune microenvironment and enhances the efficacy of sorafenib and PD-L1 inhibitors, the specific molecular mechanisms require further experimental exploration.Overall, our findings underscore the importance of BACE2 in cancer progression and immune regulation, highlighting its potential as a prognostic biomarker and therapeutic target. Declarations Funding This work was supported by the National Natural Science Foundation of China (Grant No.82102926 and No.82372970), the China Postdoctoral Science Foundation (Grant No.2022M711511), Guangdong Basic and Applied Basic Research Foundation (Grant No.2022A1515010083, 2023A1515110363 and 2023A1515010492), Ganzhou Municipal Science and Technology Project(Grant No.2022—RC1348) and President Foundation of Nanfang Hospital, Southern Medical University (Grant No. 2023A048). Competing interests The authors declare no competing interests. Authors' contributions LC and YL conceived the study and analysed the results.FY and YK wrote the manuscript. ZX designed and performed the experiments. JL, LS, ZZ,XZ ,and HL were involved in analyses and discussions of the data. ZJ,DW and SM collected the clinical data. WH,LC and YL revised the manuscript and provided scientific directions. All authors read and approved the final manuscript. Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The animal procedures in this study were approved by the Ethical Com- mittee for Animal Research of Southern Medical University (Guangzhou, China). Consent for publication Not applicable. Acknowledgements Not applicable. References Bray F; Laversanne M; Sung H; Ferlay J; Siegel RL; Soerjomataram I; A., J. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024 . Reck, M.; Remon, J.; Hellmann, M. D. First-Line Immunotherapy for Non-Small-Cell Lung Cancer. J Clin Oncol 2022 , 40 (6), 586-597. From NLM http://doi.org/10.1200/jco.21.01497. Tostes, K.; Siqueira, A. P.; Reis, R. M.; Leal, L. F.; Arantes, L. Biomarkers for Immune Checkpoint Inhibitor Response in NSCLC: Current Developments and Applicability. 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From NLM http://doi.org/10.1038/s41419-022-04775-z. Zhou, Z.; Xu, B.; Hu, N.; Guo, Z.; Bao, W.; Shao, B.; Yang, W. Targeting the Macrophage-Ferroptosis Crosstalk: A Novel Insight into Tumor Immunotherapy. Front Biosci (Landmark Ed) 2022 , 27 (7), 203. From NLM http://doi.org/10.31083/j.fbl2707203. Ni, S.; Yuan, Y.; Kuang, Y.; Li, X. Iron Metabolism and Immune Regulation. Front Immunol 2022 , 13 , 816282. From NLM http://doi.org/10.3389/fimmu.2022.816282. Vogt, A. S.; Arsiwala, T.; Mohsen, M.; Vogel, M.; Manolova, V.; Bachmann, M. F. On Iron Metabolism and Its Regulation. Int J Mol Sci 2021 , 22 (9). From NLM http://doi.org/10.3390/ijms22094591. Shapouri-Moghaddam, A.; Mohammadian, S.; Vazini, H.; Taghadosi, M.; Esmaeili, S. A.; Mardani, F.; Seifi, B.; Mohammadi, A.; Afshari, J. T.; Sahebkar, A. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol 2018 , 233 (9), 6425-6440. From NLM http://doi.org/10.1002/jcp.26429. Yang, H. C.; Park, H. C.; Quan, H.; Kim, Y. Immunomodulation of Biomaterials by Controlling Macrophage Polarization. Adv Exp Med Biol 2018 , 1064 , 197-2 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.pdf 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-5406902\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":382188086,\"identity\":\"7a19d883-cb32-4a39-b950-6c7b96ac4265\",\"order_by\":0,\"name\":\"Yuanyuan Feng\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanfang Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuanyuan\",\"middleName\":\"\",\"lastName\":\"Feng\",\"suffix\":\"\"},{\"id\":382188087,\"identity\":\"8bfbb489-87f0-4404-885e-02be43d638ca\",\"order_by\":1,\"name\":\"Xiaohan 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(D)\\u003c/strong\\u003eexpression of BACE2 in tumor cell lines. *\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ****\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001, ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/b08b5530afdc9b721584fb38.png\"},{\"id\":71071871,\"identity\":\"8075afc5-3a4a-4265-a8fb-e9d22ca7293f\",\"added_by\":\"auto\",\"created_at\":\"2024-12-10 22:02:04\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1006512,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eRelationship between BACE2 expression level and tumorigenesis and development\\u003c/strong\\u003e \\u003cstrong\\u003e(A-I)\\u003c/strong\\u003e Patterns of BACE2 expression levels in tumor as well as adjacent normal tissues based on a TCGA dataset.\\u003cstrong\\u003e (J-L)\\u003c/strong\\u003e Correlation between BACE2 expression levels and the stages of three different tumors(LIHC\\\\STAD\\\\PAAD). *\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ****\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001, ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/632ca6876510bbe6127b337e.png\"},{\"id\":71071874,\"identity\":\"56d5f487-15d2-46ed-9524-256c7a7634b6\",\"added_by\":\"auto\",\"created_at\":\"2024-12-10 22:02:04\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":248707,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eRelationship between BACE2 expression level and survival of pan-cancer patients\\u003c/strong\\u003e 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UCS(carcinosarcoma); LUSC(lung squamous cell carcinoma) ; OV(ovarian serous cystadenocarcinoma); PAAD(pancreatic cancer); STAD(gastric adenocarcinoma); THCA(thyroid cancer).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/e90c3416229c07b12b2245a5.png\"},{\"id\":71071870,\"identity\":\"2b13eb27-5c34-4e24-971c-5b7a28ecff99\",\"added_by\":\"auto\",\"created_at\":\"2024-12-10 22:02:04\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":584979,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eRelationship between BACE2 expression and that of immune-related genes (A)\\u003c/strong\\u003eImmune-activating gene.\\u003cstrong\\u003e(B)\\u003c/strong\\u003eCytokines. \\u003cstrong\\u003e(C) \\u003c/strong\\u003eImmunosuppressive genes. \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Cytokine receptors \\u003cstrong\\u003e(E)\\u003c/strong\\u003e MHC genes. Red box represents positive correlation, blue box represents negative correlation, and the darker the color, the higher the relevance.\\u003cstrong\\u003e \\u003c/strong\\u003e*\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ****\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001, ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/1e1a269cbc15882ebb7986a7.png\"},{\"id\":71072317,\"identity\":\"0d30a94d-7fbe-4212-ac09-080f0dd176d1\",\"added_by\":\"auto\",\"created_at\":\"2024-12-10 22:10:04\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":484467,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eRelationship between BACE2 expression and tumor infiltration of different immune cells in PCa\\u003c/strong\\u003e \\u003cstrong\\u003e(A) \\u003c/strong\\u003eCorrelation of BACE2 expression levels in the TIMER2 database with macrophage and T cell infiltration. \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Correlation of BACE2 expression with different immune cells in previously published studies.\\u003cstrong\\u003e(C)\\u003c/strong\\u003e Correlation of BACE2 expression with different immune cells in the ImmuCellAI database. Red box represents positive correlation, blue box represents negative correlation, and the darker the color, the higher the relevance. *\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ****\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001, ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/917bebd45c65b2d71f5d6ebf.png\"},{\"id\":71071873,\"identity\":\"e6476540-8760-43ec-b2f3-f9f0e7a7f3d7\",\"added_by\":\"auto\",\"created_at\":\"2024-12-10 22:02:04\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":298979,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eBACE2 promotes lung cancer progression and TAMs infiltration in vitro and in vivo\\u003c/strong\\u003e (\\u003cstrong\\u003eA-B\\u003c/strong\\u003e) The expression of BACE2 was quantified by qPCR in murine lung cancer cells (LLC) and human lung cancer cells (A549). (\\u003cstrong\\u003eC-D\\u003c/strong\\u003e) The proliferation of BACE2-knockdown and control cells in LLC and A549 was assessed using the CCK8 assay. (\\u003cstrong\\u003eE-F\\u003c/strong\\u003e) Migration and invasion assays were conducted on BACE2-knockdown LLCs using the Transwell assay and followed by statistical analysis .(\\u003cstrong\\u003eG-H\\u003c/strong\\u003e) Migration and invasion assays were conducted on BACE2-knockdown A549 cells using the Transwell assay and followed by statistical analysis . (\\u003cstrong\\u003eI-K\\u003c/strong\\u003e) Subcutaneous tumor size tumor weight,survival curves,and tumor growth curves were evaluated in mouse models. (\\u003cstrong\\u003eM-O\\u003c/strong\\u003e) Flow cytometry analysis of M1 macrophages (CD86+CD206-)) and M2 macrophages (CD86-CD206+) in subcutaneous tumors (n=4). Data are presented as mean ± SD. *P \\u0026lt; 0.05, **P \\u0026lt; 0.01, ****P \\u0026lt; 0.0001, ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/62edd655804976409cc6be56.png\"},{\"id\":71071876,\"identity\":\"e9cfa965-fe67-494c-a7d7-c79b86a202e5\",\"added_by\":\"auto\",\"created_at\":\"2024-12-10 22:02:04\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":447337,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eBACE2 knockdown enhances the efficacy of Sorafenib, increasing the infiltration of activated immune cells in lung cancer \\u003c/strong\\u003e(\\u003cstrong\\u003eA-C\\u003c/strong\\u003e) Intracellular ROS levels were detected using flow cytometry analysis with DCFH-DA staining in control and BACE2-knockdown A549 cells and followed by statistical analysis.(\\u003cstrong\\u003eB\\u003c/strong\\u003e)The experimental scheme of the animal model. (\\u003cstrong\\u003eD-E\\u003c/strong\\u003e) Survival curves,tumor growth curves. (\\u003cstrong\\u003eF-H\\u003c/strong\\u003e) Flow cytometry analysis was respectively applied to demonstrate the infiltration of macrophages (CD86+CD206-for M1, CD86-CD206+ for M2) of mice tumors (n=6) and followed by statistical analysis. (\\u003cstrong\\u003eI\\u003c/strong\\u003e)tumor weight .(\\u003cstrong\\u003eJ-M\\u003c/strong\\u003e) Flow cytometry analysis was respectively applied to demonstrate the infiltration of CD3+ CD4+T cells and Treg of mice tumors and followed by statistical analysis. *P \\u0026lt; 0.05, **P \\u0026lt; 0.01, ****P \\u0026lt; 0.0001, ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/e429a8a5fac5fafb98861702.png\"},{\"id\":71071877,\"identity\":\"3830675f-7f49-4d75-9d38-f12db9e8ce39\",\"added_by\":\"auto\",\"created_at\":\"2024-12-10 22:02:04\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":438824,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eBACE2 knockdown enhances the antitumor immune response in lung cancer \\u003c/strong\\u003e(\\u003cstrong\\u003eA\\u003c/strong\\u003e) The experimental scheme of the animal model. (\\u003cstrong\\u003eB-D\\u003c/strong\\u003e) Tumor growth curves, survival curves, and tumor weight were evaluated in mouse models. (\\u003cstrong\\u003eE-G\\u003c/strong\\u003e) Flow cytometry analysis was respectively applied to demonstrate the infiltration of macrophages (CD86\\u003csup\\u003e+\\u003c/sup\\u003eCD206\\u003csup\\u003e-\\u003c/sup\\u003efor M1, CD86\\u003csup\\u003e-\\u003c/sup\\u003eCD206\\u003csup\\u003e+\\u003c/sup\\u003e for M2) of mice tumors (n=6), followed by statistical analysis. (\\u003cstrong\\u003eH-K\\u003c/strong\\u003e) Flow cytometry analysis was respectively applied to demonstrate the infiltration of CD3\\u003csup\\u003e+\\u003c/sup\\u003e CD4\\u003csup\\u003e+\\u003c/sup\\u003eT cells and Treg of mice tumors, followed by statistical analysis. *\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ****\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001, ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/885c0fcc4a7a1ddcc82efb98.png\"},{\"id\":71072623,\"identity\":\"bb53f281-6e32-4fda-b699-873bc742a7fb\",\"added_by\":\"auto\",\"created_at\":\"2024-12-10 22:26:08\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":5352507,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/0d9e13a3-d30a-4b73-97e1-fb2215060eb6.pdf\"},{\"id\":71071878,\"identity\":\"3450b931-e553-4f60-b9ab-3f77174f779d\",\"added_by\":\"auto\",\"created_at\":\"2024-12-10 22:02:04\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":2422490,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarymaterial.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5406902/v1/6699ecbc4b00817b59e8df30.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"BACE2 is a prognostic biomarker and induced macrophage M2 polarization in Non-Small Cell Lung Cancer\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eLung cancer has surpassed the breast cancer is the most commonly occurring cancer worldwide with 2.5\\u0026nbsp;million new cases accounting for 12.4% of total new cases. Lung cancer remained the first leading cause of cancer death, with an estimated 9.74\\u0026nbsp;million(18.7%)\\u003csup\\u003e1\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eIn recent years, immune checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have been proven its significant efficacy on advanced non-small cell lung cancer (NSCLC)\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. However, the efficiency of immunotherapy (mainly immune checkpoint inhibitors) in unselected populations is relatively low, and only a small proportion of patients can benefit from immunotherapy, because of a lack of optimal biomarkers. Further research is needed to identify more biomarkers to aid in patients selection to improve immunotherapy respone rate\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eBACE2 (β-site amyloid precursor protein cleaving enzyme 2) is a gene encoding the β-amyloid precursor protein cleaving enzyme that has garnered widespread research interest in recent years\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. Initially, BACE2 was primarily associated with the nervous system and Alzheimer's disease\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. However, recent studies have shown that BACE2 plays a significant role in key biological processes of tumor proliferation, invasion, and metastasis\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR7\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. In the last decade, a very active field in cancer research has been the study of tumor microenvironment. The tumor microenvironment is composed of immune cells, such as macrophages, lymphocytes and neutrophils, of endothelial and stromal cells, of the extracellular matrix (ECM) and of soluble factors.Inside the microenvironment, the soluble factors, secreted by tumor cells, are able to recruit all these different types of cells, that, educated by the tumor itself, produce a pro-tumoral environment\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e. BACE2, thanks to their ability of shedding polypeptides in the extracellular space, can be considered key players that orchestrate the crosstalk between the tumor and the surrounding cells\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, gaining a deeper understanding of the relationship between the BACE2 gene and cancer is of paramount importance for developing new cancer treatment strategies.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we first conducted bioinformatics analysis to investigate the expression of BACE2 in various cancers. We also analyzed the impact of BACE2 on survival prognosis and the immune microenvironment. The results indicate that BACE2 can serve as a biological marker for prognostic assessment and M2 macrophages infiltration. Additionally, the effect of BACE2 in lung cancer were verified by in vivo and in vitro experiments. we validated the role of BACE2 in the tumor immune microenvironment, revealing its influence on the infiltration of T cells and macrophages in tumor tissues. Furthermore, knocking down BACE2 not only slows tumor growth but also enhances the efficacy of immunotherapy and sorafenib treatment in lung cancer model.Collectively, the obtained result indicate that BACE2 may be a potiential biomarker for lung cancer therapies.\\u003c/p\\u003e\"},{\"header\":\"Meterials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eData collection\\u003c/h2\\u003e \\u003cp\\u003eWe retrieved and downloaded mRNA expression profiles and related clinical data for 33 types of cancer from The Cancer Genome Atlas (TCGA) database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://portal.gdc.cancer.gov/\\u003c/span\\u003e\\u003cspan address=\\\"https://portal.gdc.cancer.gov/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), and mRNA expression profiles for normal human tissues from the Genotype-Tissue Expression (GTEx) database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.gtexportal.org/home/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.gtexportal.org/home/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Cell line gene expression substrates of tumors were obtained from the Cancer Cell Line Encyclopedia dataset (CCLE, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://portals.broadinstitute.org/ccle/about\\u003c/span\\u003e\\u003cspan address=\\\"https://portals.broadinstitute.org/ccle/about\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003ePan-cancer differential expression and prognostic value of BACE2\\u003c/h3\\u003e\\n\\u003cp\\u003etest was used to detect the expression differences between tumors and normal tissues; statistical analysis was performed using R software (version 4.0.2), and the R package \\\"ggplot2\\\" was used to draw box plots. Similar data processing methods were used to study the correlation between BACE2 and tumor stage. p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered statistically significant. The GTEx database was used to analyze the expression of BACE2 in normal tissues, and the TCGA database was used to analyze the expression of BACE2 in pan-cancer tissues. The expression profiles of TCGA and GTEx were integrated to compare the expression differences of BACE2 in tumor and normal tissues. The expression level of BACE2 in tumor cell lines was analyzed using CCLE dataset. Radar plots were then drawn based on the median expression and expression differences of BACE2 in each of the 33 cancer types or in each tissue.\\u003c/p\\u003e \\u003cp\\u003eAfter ranking the BACE2 mRNA expression levels, the data from 33 cancers in the TCGA pan-cancer database were categorized into a low BACE2 expression group and a high BACE2 expression group based on the median BACE2 expression level. Thereafter, to investigate the effect of BACE2 on survival in pan-cancer patients, we performed univariate Cox regression (uniCox) using the R packages \\\"survminer\\\" and \\\"survival\\\" to derive p-values and risk ratios (HR, 95% confidence intervals \\u003csup\\u003e38\\u003c/sup\\u003e) for the Kaplan-Meier curves. According to the analysis results (Kaplan- Meier curves), the clinical prognosis and outcomes (represented by the overall survival (OS) of patients in both groups (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05)) were compared between the patients in the BACE2 high-expression group and those in the low-expression group using the R software package, \\\"survival\\\" (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05).\\u003c/p\\u003e\\n\\u003ch3\\u003eGene Set Enrichment Analysis (GSEA) related to BACE2\\u003c/h3\\u003e\\n\\u003cp\\u003eTo explore the potential biological processes and molecular functions affected by BACE, we performed gene set enrichment analysis using GSEA software (v.4.2.3), which was performed using the R package \\\"clusterprofiler\\\". An adjusted P value of \\u0026lt;\\u0026thinsp;0.05 was considered statistically significant.\\u003c/p\\u003e \\u003cp\\u003eThe steps were: 1) Integrate the gene expression data of BACE2 low and high expression groups and submit them to GSEA software; 2) Download the \\\"gmt\\\" file of Hallmark pathway gene set from the Molecular Signature Database (MSigDB); 3) Calculate the normalized enrichment score (NES) and the false discovery rate (FDR) for each bioprocess; 4) Apply R package \\\"Ggplot2\\\" to visualize the results.\\u003c/p\\u003e\\n\\u003ch3\\u003eCorrelation analysis of BACE2 expression with immune-related genes\\u003c/h3\\u003e\\n\\u003cp\\u003eThe correlation between BACE2 expression and immune-related genes was analyzed, including immune-activating genes, cytokines, immune-suppressing genes, cytokine receptors, and genes for the major histocompatibility complex (MHC). Pearson correlation analysis was performed to show the association between BACE2 mRNA and the expression of immune-related genes, which were obtained from the TCGA pan-cancer data (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered significant), and the results of the analysis are presented in heat maps.\\u003c/p\\u003e\\n\\u003ch3\\u003eRelationship Between BACE2 Expression With Immune Cell Infiltration\\u003c/h3\\u003e\\n\\u003cp\\u003eA total of three different methods were used in this study to assess the association between BACE2 expression levels and immune cell infiltration in pan-cancer. The Tumor Immune Estimation Resource (TIMER) can be used to analyze immune cell infiltration in different cancers using various algorithms. Therefore, first we searched the TIMER 2.0 database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://timer\\u003c/span\\u003e\\u003cspan address=\\\"http://timer\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. comp-genomics.org/) to download BACE2-associated immune cell infiltration correlations in the TCGA pan-cancer project, which included various types of T cell and macrophage infiltration. In the second, we obtained data on immune cell infiltration in TCGA pan-cancer patients from previous published works and analyzed them for correlation. In the third, we downloaded the immune cell infiltration scores of TCGA pan-cancer patients on the ImmuCellAI platform (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://bioinfo.life.hust.edu.cn/web/ImmuCellAI/\\u003c/span\\u003e\\u003cspan address=\\\"http://bioinfo.life.hust.edu.cn/web/ImmuCellAI/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) and processed the data by similarly correlating them. Then we visualized the correlation between the expression of BACE2 and the level of tumor infiltration of different immune cells, and the results were presented in heat maps.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell lines and cell culture\\u003c/h2\\u003e \\u003cp\\u003eThe human lung cancer cell line (A549) and murine lung cancer cell line (LLC) were obtained from Guang Zhou Jennio Biotech Co.,Ltd. All cell lines were validated by short tandem repeat analysis. All cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), and were cultured in a humidified incubator set at 37℃ with a 5% CO2 environment.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eqRT-PCR\\u003c/h3\\u003e\\n\\u003cp\\u003eInitially, the total cellular RNA of LLC and A549 were extracted using the RNA isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China). Then RNA was reverse transcribed into complementary DNA (cDNA) with HiScript II Q RT SuperMix for qPCR (+\\u0026thinsp;g DNA wiper) (Vazyme) according to the manufacturer\\u0026rsquo;s introduction. qRT-PCR was conducted using SYBR\\u0026reg; Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, Changsha, China). Standardized with GAPDH expression level, the levels of target were calculated using 2-ΔΔCT method.\\u003c/p\\u003e\\n\\u003ch3\\u003eCell counting kit-8 proliferation assay (CCK-8)\\u003c/h3\\u003e\\n\\u003cp\\u003eLLC and A549 cells were seeded in 96-well plates at a concentration of 1000 cells/well with 5 repetitions in each group. The culture medium was replaced with CCK-8 reagent(Fdbio science, Hangzhou, China), and plates were incubated at 37℃ for 2h. Absorbance was measured at 450 nm.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTranswell assay\\u003c/h2\\u003e \\u003cp\\u003eLLC cells and A549 cells(6 \\u0026times;10\\u003csup\\u003e4\\u003c/sup\\u003e) in 300\\u0026micro;L of FBS-free medium were respectively seeded into the upper chamber of the 24-well transwell system (8 \\u0026micro;m pore size) (Falcon, USA), while 700\\u0026micro;L of 10% FBS was added to the lower chamber. The system was incubated at 37℃ for 24h. The cells in the upper chamber were removed, while the cells in the lower chamber were fixed with methanol and stained with 5% crystal violet. The migrating cells were photographed and counted under microscope from at least five random fields.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnimal experiments\\u003c/h2\\u003e \\u003cp\\u003e All experiments were approved by the Animal Ethics Committee of Nanfang Hospital and conducted according to the international guidelines for animal care and use. Female C57BL/6J mice of 3\\u0026ndash;4 weeks old were purchased from Risemice Biotechnology (China). LLC cells (1\\u0026times;10\\u003csup\\u003e6\\u003c/sup\\u003e) resuspended in 100\\u0026micro;L PBS were subcutaneously injected into the right flank of the mice. Sorafenib(30mg/kg) (Glpbio, USA) and PD-L1 inhibitor(100\\u0026micro;L) (Glpbio) were administered to the experimental group mice via gavage and intraperitoneally injection every 2 days respectively. Bi-weekly tumor measurements were performed every 2 days when the tumor volume reached 200mm\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e, calculated as 0.5\\u0026times;length\\u0026times;width\\u003csup\\u003e2\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFlow cytometry analysis\\u003c/h2\\u003e \\u003cp\\u003eTumors were dissociated into single-cell suspensions with collagenase. Then the dissociated cells were filter through a 40\\u0026micro;m cell strainers (Biologix, Jinan, China) and resuspended in PBS. After blocking with anti-mouse CD16/CD32(BD Biosciences, US) macrophages were incubated with antibodies APC anti-mouse CD86 and FITC anti-mouse CD206, while T cells were stained with antibodies FITC anti-mouse CD3, PE-Cy7 anti-mouse CD8, APC-Cy7 anti-mouse CD4 and PE anti-mouse FOXP3 (all from 4A Biotech, Suzhou, China) for 30 minutes in dark. Washed by flow buffer, the samples were then analyzed using flow cytometry (BD Biosciences, US). As for the measurement of intracellular ROS level, A549 cells were harvested and stained with DCFH-DA (Solarbio, Beijing, China).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBACE2 is upregulated in multiple cancer tissues\\u003c/h2\\u003e \\u003cp\\u003eBy retrieving BACE2 expression through GTEx and TCGA mRNA expression datasets, it is evident that BACE2 gene level is upregulated in a wide range of tumors, including Adrenal cortical carcinoma (ACC), cholangiocarcinoma (CHOL), colorectal adenocarcinoma (COAD), lymphomatous diffuse large B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), renal chromotropic disorder (KICH), renal form cell carcinoma (KIRP), acute myeloid leukemia (LAML), brain low-grade glioma (LGG), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), ovarian serous cystadenocarcinoma (OV), pancreatic cancer (PAAD), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), skin cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid cancer (THYM), Uterine body endometrial cancer (UCEC) while down-regulated expression of this gene can be seen in Clear cell carcinoma of kidney (KIRC) and uterine carcinosarcoma (UCS), compare to normal tissues \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e.Then we made three graphs showing the profiles of BACE2 expression in normal tissues, pan-carcinoma tissues, and tumor cell lines respectively, indicating the average expression level of BACE2, and the differences in expression level in various tissues, and the expression level is arranged in the order from low to high \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB-D\\u003cb\\u003e)\\u003c/b\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe expression of BACE2 was found to be significantly upregulated in KICH, LUAD, LUSC, COAD, CHOL, STAD, READ, PRAD, and ESCA paired tumors relative to adjacent normal tissues in the TCGA dataset. Except for ESCA, the expression of BACE2 in other tumors and adjacent tissues was statistically significant \\u003cb\\u003e(Fig.\\u0026nbsp;2A-I)\\u003c/b\\u003e. Moreover, BACE2 expression was up-regulated in different stages, high levels of BACE2 expression had worse clinical stage,espically in LIHC, STAD, and PAAD tumors \\u003cb\\u003e(Fig.\\u0026nbsp;2J-L)\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eFigure\\u0026nbsp;2 Relationship between BACE2 expression level and tumorigenesis and development (A-I)\\u003c/b\\u003e Patterns of BACE2 expression levels in tumor as well as adjacent normal tissues based on a TCGA dataset. \\u003cb\\u003e(J-L)\\u003c/b\\u003e Correlation between BACE2 expression levels and the stages of three different tumors(LIHC\\\\STAD\\\\PAAD). *\\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;0.0001, ns, not significant.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBACE2 expression correlated with outcomes of patients in multiple cancer types\\u003c/h2\\u003e \\u003cp\\u003eThe forest plot demonstrates the overall survival (OS) of BACE2 levels in TCGA pan-cancer patients obtained after analysis using Cox proportional risk model. The results showed that in LGG (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001), CESC (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.001), GBM (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.005), PAAD (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.017), KIRC (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.021), LIHC (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.004), BRCA (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.035), LUAD (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.039) and SKCM (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.049) patients, high BACE2 levels were significantly linked to poorer OS \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e. Moreover, the expression level of BACE2 significantly affected the DSS, DFI and PFI of patients in multiple cancers\\u003cb\\u003e(Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eA-C)\\u003c/b\\u003e. In addition, Kaplan-Meier curves found that in CESC (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001), KIRC (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001), UVM (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.00053), HNSC (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.0039), GBM (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.0016), SKCM (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.00043), LUAD (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.0074), BRCA (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.0043) patients, high levels of BACE2 expression were associated with shorter OS times Similarly, the patients with higher level of BACE2 showed poorer DSS and DFI in multiple cancers \\u003cb\\u003e(Fig. S2-3)\\u003c/b\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBACE2 played an important role in cancer immune response\\u003c/h2\\u003e \\u003cp\\u003eTo investigate the possible biological processes regulated by BACE2 in pan-cancer, we performed gene set enrichment analysis (GSEA) using R packages \\\"clusterprofiler\\\" in pan-cancer subjects, then we finally selected six tumors (UCS, LUSC, OV, PAAD, STAD, THCA) with similar results. The results showed that the enriched pathways had positive normalized enrichment scores (NES), suggesting that most of the potential biological pathways regulated by BACE2 are associated with immune regulatory mechanisms, such as neutrophil activation, myeloid cell activation and cell activation involved in immune response, cytokine production in the immune system, as well as adaptive and innate immune systems\\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA-F\\u003cb\\u003e)\\u003c/b\\u003e. This suggests that BACE2 plays an important role in tumor immune response, especailly in tumor immune micro-environment.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eRelationship between BACE2 and that of immune-related genes\\u003c/h2\\u003e \\u003cp\\u003eNext, we performed gene co-expression analysis in order to further elucidate the relationship between BACE2 expression and immune-related genes in pan-cellular carcinomas, which revealed that BACE2 expression was positively correlated with most immune-suppressing genes \\u003cb\\u003e(Fig.\\u0026nbsp;5C)\\u003c/b\\u003e chemokines \\u003cb\\u003e(Fig.\\u0026nbsp;5B)\\u003c/b\\u003e and chemokine receptors \\u003cb\\u003e(Fig.\\u0026nbsp;5D)\\u003c/b\\u003e, and was negatively associated with many immune-activating genes \\u003cb\\u003e(Fig.\\u0026nbsp;5A)\\u003c/b\\u003e and MHC genes \\u003cb\\u003e(Fig.\\u0026nbsp;5E)\\u003c/b\\u003e in a variety of tumor types. The above results suggested that BACE2 expression was strongly linked to immune checkpoint genes in most tumor types. Among these immune marker genes, IL10RB, IL-10, and TGFBR1 were found to be significantly associated with BACE2 expression in most tumor types. And we have learned that there is a significant correlation between TGFBR1, IL-10 and IL-10RB expression and tumor-associated macrophage(TAMs), which are closely involved in tumorigenesis infiltration and metastasis\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. Moveover, we observed that the expression of BACE2 is positively correlated with the expression of CCR2, CXCL1/CXCL8-CXCR2, CXCL12-CXCR4, and CCR8 in multiple cancer types. These chemokines and their receptors have been demonstrated to be associated with the recruitment of tumor-associated immunosuppressive cells\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR14 CR15\\\" citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e. In addition, we observed that BACE2 expression was associated with HAVCR2, PDCD1,CTLA4,TIGIT and LAG3,the markers of exhaustion in CD8\\u003csup\\u003e+\\u003c/sup\\u003eT cells\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e, which implied BACE2 highly expressed patients would be benefited form immune checkpoint therapy.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBACE2 was associated with immune cell infiltrations\\u003c/h2\\u003e \\u003cp\\u003ePrevious studies have shown that tumor-infiltrating immune cells usually function abnormally and promote immune escape of tumor cells, i.e., they fail to inhibit tumor growth and even promote its progression\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. The Estimate algorithm was used to predict the relationship between BACE2 expression and immune infiltration. BACE2 was negatively associated with the stromal score, immune score, and ESTIMATE score in LUAD, UCEC, CESC, SKCM, and KICH, indicating that BACE2 significantly correlated with poor immune cell infiltration in certain types of cancer\\u003cb\\u003e(Fig.S4A)\\u003c/b\\u003e.In the tumor microenvironment, tumorigenesis and progression are closely linked to the enrichment of tumor-associated macrophages and T cells. Therefore, we evaluated the correlation between BACE2 expression and TAMs and T-cell infiltration using the TIMER2 database.\\u003c/p\\u003e \\u003cp\\u003eThe assessment showed that the expression levels of most T cell types were negatively correlated with BACE2 expression in most tumor types, but the level of T cells in the tumor immune microenvironment were positively correlated with BACE2 expression. And the level of TAMs infiltration was positively correlated with BACE2 expression, especially with M2-type macrophages. \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e.We then evaluated the correlation between BACE2 levels and immune scores (representing immune cell infiltration in tumor tissues) in 33 cancers by calculating BACE2 levels and performing pan-cancer analyses of different immune cell tumor infiltrations with BACE2 expression using published work and the ImmuCellAI database. Clustering heatmaps showed a positive correlation between BACE2 expression and macrophages in most tumor types, consistent with TIMER2 results\\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB-C\\u003cb\\u003e)\\u003c/b\\u003e. We could also observe that BACE2 was significantly positively correlated with Treg cells and negatively correlated with CD8\\u0026thinsp;+\\u0026thinsp;T cells in a variety of cancers \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB-C\\u003cb\\u003e)\\u003c/b\\u003e. Consistently, correlation analysis also showed that the expression of BACE2 was negatively correlated with the infiltration of CD8\\u0026thinsp;+\\u0026thinsp;T cells and B cells\\u003cb\\u003e(Fig.S4B-C)\\u003c/b\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eBACE2 is involved in the progression of lung cancer and macrophage M2 polarization.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo investigate the impact of BACE2 on cancer cell proliferation and migration, we initially constructed LLC and A549 cell lines with BACE2 knockdown. qRT-PCR confirmed the successful suppression of BACE2 in both LLC and A549 cell lines \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA-B\\u003cb\\u003e)\\u003c/b\\u003e. The absence of BACE2 significantly impeded the proliferation and migration of LLC and A549 cells, as evidenced by Cell Counting Kit-8 proliferation assay (CCK8) and Transwell Assay results \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eC-H\\u003cb\\u003e)\\u003c/b\\u003e.Moreover, we implanted lung cancer cells into mice to establish subcutaneous tumors. \\u003cem\\u003eIn vivo\\u003c/em\\u003e experiments revealed that tumors with BACE2 knockdown exhibited smaller size, lighter weight, and slower growth rate compared to the control group. Mice bearing tumors with BACE2 knockdown also demonstrated a prolonged survival time \\u003cb\\u003e(Fig .7I-L).\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eAdditionally, we explored whether the expression level of BACE2 is associated with macrophage infiltration in the tumor microenvironment. Flow cytometry analysis illustrated that the downregulation of BACE2 did not significantly affect the infiltration of M1 macrophages (CD86\\u003csup\\u003e+\\u003c/sup\\u003eCD206\\u003csup\\u003e\\u0026minus;\\u003c/sup\\u003e), but a reduced presence of M2 macrophages (CD86\\u003csup\\u003e\\u0026minus;\\u003c/sup\\u003eCD206\\u003csup\\u003e+\\u003c/sup\\u003e) was observed in tumors with low expression of BACE2 \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eM-N\\u003cb\\u003e).\\u003c/b\\u003eIn summary, all the aforementioned findings indicate that the deletion of BACE2 exhibits antitumor effect.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eBACE2 knockdown enhances the efficacy of Sorafenib, increasing the infiltration of activated immune cells in lung cancer.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThrough co-expression analysis, we identified a positive correlation between BACE2 expression and numerous ferroptosis-related genes and autophagy-related genes across various cancer types \\u003cb\\u003e(Fig. S5A-B)\\u003c/b\\u003e. Flow cytometry analysis revealed that A549 cells with lower BACE2 expression exhibited elevated levels of reactive oxygen species (ROS) \\u003cb\\u003e(\\u003c/b\\u003e Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eA- \\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eC\\u003cb\\u003e)\\u003c/b\\u003e. Consequently, we investigated whether BACE2 could influence the therapeutic efficacy of a ferroptosis inducer in mice. Mice, each with tumor inoculation of stable BACE2 expression and BACE2 knockdown LLC cells, were respectively assigned to the control and treatment groups. Sorafenib, a classical ferroptosis inducer, was administered to the treatment group \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eB\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eIn comparison to the control group, mice receiving Sorafenib treatment and exhibiting lower BACE2 expression displayed prolonged overall survival \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eD\\u003cb\\u003e)\\u003c/b\\u003e. Tumor growth was significantly inhibited by Sorafenib in mice bearing BACE2-downregulated cells compared to the control cells \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eE-I\\u003cb\\u003e).\\u003c/b\\u003e Furthermore, the previously results mentioned the relationship between BACE2 and immunity, and flow cytometry analysis demonstrated that compared to the control group, mice bearing BACE2-knockdown cells combined sorafenib treatment presented a higher proportion of macrophages with M1 phenotype. Sorafenib reduced the number of macrophage M2 on BACE2 control group.but the decrease in macrophage M2 polarization did not show a significant difference on BACE2-silent cells \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eG-H\\u003cb\\u003e).\\u003c/b\\u003eMoreover, upon treatment with Sorafenib, a greater infiltration of CD3\\u003csup\\u003e+\\u003c/sup\\u003e T cells and CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells, along with a reduced presence of Treg cells (CD4\\u003csup\\u003e+\\u003c/sup\\u003eFOXP3\\u003csup\\u003e+\\u003c/sup\\u003e), was observed in the tumor microenvironment of mice with low BACE2 levels compared to the control \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eJ-M\\u003cb\\u003e)\\u003c/b\\u003e. These findings collectively suggest that a low expression level of BACE2 enhances the efficacy of Sorafenib, thereby suppressing the progression of lung cancer through the promotion of ferroptosis.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eBACE2 knockdown enhances the antitumor immune response elicited by the PD-L1 inhibitor in lung cancer.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eAs a prototypical immune checkpoint inhibitor, the PD-L1 inhibitor has found application in the therapeutic landscape of numerous tumors\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. Considering the intricate interplay between BACE2 and the tumor immune microenvironment, our focus shifted to investigating whether the downregulation of BACE2 could enhance the antitumor immune response when combined with the PD-L1 inhibitor. We generated subcutaneous tumour xenograft models as delineated in (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e.\\u003c/p\\u003e \\u003cp\\u003eCrucially, compared to the control group, the synergistic application of BACE2 downregulation and PD-L1 inhibitor yielded a remarkable suppression of tumor growth and weight, with an extended overall survival \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eB-D\\u003cb\\u003e)\\u003c/b\\u003e. Strikingly, when treated with the PD-L1 inhibitor, the mice bearing BACE2 knockdown tumors exhibited an increased presence of M1 macrophages, CD3\\u003csup\\u003e+\\u003c/sup\\u003e cells, and CD8\\u003csup\\u003e+\\u003c/sup\\u003e cells, along with a reduction in Treg cells \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eE-K\\u003cb\\u003e).\\u003c/b\\u003e These findings suggest that a diminished expression level of BACE2 enhances the antitumor immune response elicited by the PD-L1 inhibitor.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003ePrevious studies have shown that BACE2 plays a significant role in key biological processes of tumor proliferation, invasion, and metastasis.\\u003c/p\\u003e \\u003cp\\u003eHowerver, little is know about the possible role of BACE2 in tumor microenvironment.In this study, results indicat that BACE2 is upregulated in many tumor types and Kaplan-Meier survival curves of prognostic value in pan-cancer revealed that its upregulation was markedly linked to worse OS.Further, BACE2 levels correlate with infiltrating immune cells in pan-cancer,.The expression levels of most T cell types were negatively correlated with BACE2 expression in various tumor types, but positive correlation with TAM infiltration, particularly with M2-type macrophages, underscores the potential involvement of BACE2 in promoting immunosuppressive and pro-tumorigenic microenvironmental conditions.Futhermore,the role of BACE2 in lung cancer progression and tumor immune environment is elucidated in our study through a series of experiments. Knockdown BACE2 expression significantly inhibits the proliferation and migration of both LLC and A549 lung cancer cell lines. Furthermore, in vivo experiments show that tumors with BACE2 knockdown exhibit reduced size, weight, and growth rate compared to control tumors. we explores the impact of BACE2 expression on macrophage and T cell infiltration in the tumor microenvironment. Flow cytometry analysis reveals that while downregulation of BACE2 does not significantly affect the infiltration of M1 macrophages, it leads to a reduced presence of M2 macrophages and elevated CD8\\u003csup\\u003e+\\u003c/sup\\u003eT proportion in tumors with low BACE2 expression. And BACE2 knockdown enhances the efficacy of Sorafenib and PD-L1 inhibitor, provided new therapy strategies for lung cancer.\\u003c/p\\u003e \\u003cp\\u003eBACE1 and BACE2 are two aspartic proteases involved in the cleavage of different substrates within distinct extracellular domains\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. Despite their high degree of homology, they exhibit divergent functions depending on the cellular context. BACE1 primarily acts within the central nervous system\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e, while BACE2 is primarily involved in the maintenance of β cells and pigment deposition in melanocytes\\u003csup\\u003e\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. These differential roles reflect their tissue distribution, with BACE1 being highly expressed in various regions of the nervous system and BACE2 being more widely distributed in peripheral tissues.Although discovered simultaneously, BACE1 initially garnered scientific attention primarily due to its regulation of cytotoxic Aβ peptide formation in Alzheimer's disease (AD). Consequently, BACE1 inhibitors have been developed to counteract the production of toxic amyloid-like β peptides. Some of these drugs have been investigated in clinical trials\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e.However, despite reducing Aβ peptide production, they have not ameliorated cognitive deficits in AD patients\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. Previous studies have indicated an upregulation of BACE2 across various tumors, while BACE1 appears to remain unaltered in cancer. Elevated BACE2 expression is associated with poorer prognosis in melanoma, pancreatic cancer, and gliomas, suggesting a potential active role for this protease in cancer progression\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR7\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. Conversely, the expression of BACE1 is not correlated with disease prognosis, indicating that BACE2, rather than BACE1, appears to be directly or indirectly involved in cancer pathogenesis or progression. Additionally, BACE2, due to their ability to release peptides into the extracellular space, can be considered key players in coordinating the interplay between tumors and surrounding cells, highlighting its versatility as a potential biomarker or therapeutic target.\\u003c/p\\u003e \\u003cp\\u003eFerroptosis is a distinct form of programmed cell death characterised by the involvement of intracellular iron and excessive lipid peroxidation, morphologically and mechanistically separating from other forms of cell death\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e Our results showed than sorafenib treatment presented a higher proportion of macrophages with M1 phenotype and reduced the number of macrophage M2. This phenomenon may also be related to the increased generation of ROS due to the low expression of BACE2, thereby enhancing the efficacy of sorafenib\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e.Futhermore, BACE2 expression is correlated with ferroptosis-related genes,indicated BACE2 may enhances ferroptosis. Previous research findings suggest that induces intracellular iron overload in macrophages, promoting their polarization towards the M1 phenotype, increasing the production of inflammatory factors, and inhibiting tissue repair and immune regulation abilities\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR32\\\" citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. Both M1 and M2 macrophages exhibit distinct iron metabolism statuses and sensitivities to ferroptosis. High iron concentrations stimulate macrophages to secrete pro-inflammatory cytokines, thereby making them more prone to polarize into M1 macrophages. Conversely, low iron concentrations can promote polarization towards M2 macrophages\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e. Apart from regulating macrophage polarization through ferroptosis, interventions in the cellular environment and modulation of signaling pathways can also influence macrophage polarization. For instance, extracellular cytokines, and light, upon binding to macrophage receptors, can activate relevant signaling pathways, thereby affecting macrophage polarization\\u003csup\\u003e\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e. In conclusion, current research indicates a certain connection between macrophages and ferroptosis, although the precise regulatory mechanisms of their interaction remain incompletely understood and warrant further investigation. However, elucidating the interplay between ferroptosis and macrophages is crucial for understanding the specific mechanisms underlying various diseases, particularly tumor pathogenesis, and is essential for developing innovative therapeutic strategies and evaluating their efficacy.\\u003c/p\\u003e \\u003cp\\u003eHowever, our data primarily originate from public databases, which possess inherent limitations, and certain results cannot be validated through clinical specimens from patients. Furthermore, while BACE2 exhibits varying degrees of upregulation in most cancers, the precise molecular mechanisms underlying its promotion of tumor growth remain elusive. BACE2 influences the immune microenvironment and enhances the efficacy of sorafenib and PD-L1 inhibitors, the specific molecular mechanisms require further experimental exploration.Overall, our findings underscore the importance of BACE2 in cancer progression and immune regulation, highlighting its potential as a prognostic biomarker and therapeutic target.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the National Natural Science\\u0026nbsp;Foundation of China (Grant\\u0026nbsp;No.82102926 and No.82372970), the China Postdoctoral Science Foundation (Grant\\u0026nbsp;No.2022M711511), Guangdong Basic and Applied Basic Research Foundation (Grant\\u0026nbsp;No.2022A1515010083, 2023A1515110363 and 2023A1515010492), Ganzhou Municipal Science and Technology Project(Grant No.2022\\u0026mdash;RC1348) and President Foundation of\\u0026nbsp;Nanfang\\u0026nbsp;Hospital, Southern Medical University (Grant No. 2023A048).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026apos; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eLC and YL conceived the study and analysed the results.FY and YK wrote the manuscript. ZX designed and performed the experiments. JL, LS, ZZ,XZ ,and HL were involved in analyses and discussions of the data. ZJ,DW and SM collected the clinical data. WH,LC and YL revised the manuscript and provided scientific directions. All authors read and approved the final manuscript.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe animal procedures in this study were approved by the Ethical Com- mittee for Animal Research of Southern Medical University (Guangzhou, China).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eBray F; Laversanne M; Sung H; Ferlay J; Siegel RL; Soerjomataram I; A., J. 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From NLM http://doi.org/10.3390/ijms22094591.\\u003c/li\\u003e\\n\\u003cli\\u003eShapouri-Moghaddam, A.; Mohammadian, S.; Vazini, H.; Taghadosi, M.; Esmaeili, S. A.; Mardani, F.; Seifi, B.; Mohammadi, A.; Afshari, J. T.; Sahebkar, A. Macrophage plasticity, polarization, and function in health and disease. \\u003cem\\u003eJ Cell Physiol \\u003c/em\\u003e\\u003cstrong\\u003e2018\\u003c/strong\\u003e, \\u003cem\\u003e233\\u003c/em\\u003e (9), 6425-6440. From NLM http://doi.org/10.1002/jcp.26429.\\u003c/li\\u003e\\n\\u003cli\\u003eYang, H. C.; Park, H. C.; Quan, H.; Kim, Y. Immunomodulation of Biomaterials by Controlling Macrophage Polarization. \\u003cem\\u003eAdv Exp Med Biol \\u003c/em\\u003e\\u003cstrong\\u003e2018\\u003c/strong\\u003e, \\u003cem\\u003e1064\\u003c/em\\u003e, 197-2\\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\":\"info@researchsquare.com\",\"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\":\"BACE2, Tumor immune microvironment, Macrophage, NSCLC\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5406902/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5406902/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eBACE2 has gained attention for its upregulation in various human cancers, suggesting relevance in cancer biology. However, its specific role in tumor growth and the tumor immune microenvironment (TIME) remains underexplored. We conducted bioinformatics analyses using pan-cancer datasets to assess BACE2 expression patterns and its correlation with patient outcomes. Additionally, we investigated associations with immune-related genes and immune cell infiltrations in tumor microenvironments. A BACE2 knockdown lung cancer model was established to evaluate its impact on Sorafenib efficacy and PD-L1 inhibitor-induced antitumor immune responses. BACE2 was significantly upregulated in multiple cancers compared to normal tissues and associated with poorer patient outcomes. It played a critical role in modulating immune-related gene expression and promoting immune cell infiltrations within tumors. In vitro and in vivo experiments showed that BACE2 knockdown inhibited M2 macrophage polarization, suppressed lung tumor progression, and enhanced Sorafenib efficacy by increasing activated immune cell infiltrations. Similarly, BACE2 knockdown potentiated PD-L1 inhibitor-induced antitumor immune responses in lung cancer models. This study underscores BACE2's pivotal role in lung cancer tumorigenesis and its influence on the tumor immune microenvironment. Inhibiting BACE2 could enhance current treatment effectiveness in lung cancer therapy, offering promising avenues for improving patient outcomes.\\u003c/p\\u003e\",\"manuscriptTitle\":\"BACE2 is a prognostic biomarker and induced macrophage M2 polarization in Non-Small Cell Lung Cancer\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-12-10 22:01:59\",\"doi\":\"10.21203/rs.3.rs-5406902/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"b3608bde-e496-4079-8b7a-139c1b97d026\",\"owner\":[],\"postedDate\":\"December 10th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-12-10T22:02:01+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-12-10 22:01:59\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5406902\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5406902\",\"identity\":\"rs-5406902\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}