DAPP1 Elevated Expression Influences Macrophages and Predicts Poor Prognosis in Breast Cancer

DAPP1 Elevated Expression Influences Macrophages and Predicts Poor Prognosis in Breast 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 DAPP1 Elevated Expression Influences Macrophages and Predicts Poor Prognosis in Breast Cancer Qiming Gu, Jiali Shan, Yan Li, Zhengyi Lian, Tang Liu, Lei Yang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7220359/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 Purpose The main purpose of this study is to clarify the intricate regulatory pathways in the breast cancer tumor microenvironment, with a specific focus on revealing the immunological relevance and prognostic significance of the adaptor protein DAPP1 (dual adaptor of phosphotyrosine and 3-phosphoinositides 1) in the breast cancer microenvironment. The core research question is to determine how DAPP1 influences breast cancer progression, particularly its association with immune-related factors and patient outcomes. Methods This study adopted an integrated research approach, including bioinformatics analysis, clinical tissue microarray verification, and molecular experiments. Multiplex immunohistochemistry was used to quantitatively assess the expression level of DAPP1 and analyze its correlation with tumor-infiltrating immune cells (such as specific macrophage subsets) and immune checkpoints. Western blotting was employed to identify the protein expression of DAPP1 in cell lines, and stable knockdown and overexpression models of DAPP1 were constructed. Functional validation was performed using Cell Counting Kit-8 assay to detect cell proliferation ability and Transwell assay to detect migration and invasion abilities. Results The study found that DAPP1 was significantly highly expressed in breast cancer tissues and independently associated with advanced clinical stages and poor overall survival. In addition, the expression level of DAPP1 was positively correlated with the infiltration of CD68⁺CD86⁺ macrophages and the expression of CTLA4. Functional analyses further confirmed that DAPP1 could enhance the proliferation, migration, and invasion abilities of breast cancer cells. Conclusion These findings indicate that DAPP1 promotes the development of breast cancer by enhancing the malignant phenotypes of tumor cells and creating an immunosuppressive microenvironment. Therefore, DAPP1 can serve as a valuable prognostic indicator and a potential therapeutic target. Breast cancer DAPP1 prognosis tumor-infiltrating immune cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Breast cancer (BC) has emerged as the most prevalent malignant neoplasm among women globally, representing a significant threat to female life and health [ 1 ] . Alongside advancements in contemporary medical technologies and an increasingly profound comprehension of BC pathogenesis, treatment approaches have transitioned from a singular surgical focus to an integrated multidisciplinary therapeutic framework. Despite notable progress achieved in diagnostic and therapeutic techniques, a considerable proportion of patients continue to suffer from tumor recurrence and distant metastasis, while the prognosis for those at advanced stages remains unsatisfactory [ 2 ] . Consequently, establishing more effective therapeutic approaches remains an urgent priority to enhance patient outcomes. The progression and dissemination of BC are influenced not only by the intrinsic malignancy of cancer cells but also by regulatory cues originating from the tumor microenvironment (TME) [ 3 ] . The TME comprises immune cells, stromal components, extracellular matrix structures, and soluble mediators [ 4 ] . Among these constituents, tumor-associated macrophages (TAMs) contribute to tumor neovascularization and the invasion–metastasis cascade through the secretion of cytokines, including vascular endothelial growth factor and matrix metalloproteinases [ 5 ] . Cancer-associated fibroblasts facilitate the migratory potential of tumor cells by modulating the rigidity of the extracellular matrix [ 6 ] , whereas the dysregulated expression of immune checkpoint proteins like programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) enables immune evasion by impairing T cell activation [ 7 ] . A strong correlation has been observed between elevated TAM infiltration and lymph node metastasis, chemoresistance, and poor clinical outcomes in BC patients [ 8 ] , while individuals expressing PD-L1 positivity tend to exhibit enhanced responsiveness to immunotherapy [ 9 ] , indicating that TME components hold promise as predictive biomarkers of therapeutic efficacy. Nevertheless, the upstream molecular pathways governing the TME remain incompletely characterized, and the identification of pivotal regulatory molecules has emerged as a critical objective in the pursuit of precision BC therapy. Dual adaptor of phosphotyrosine and 3-phosphoinositides 1 (DAPP1, also referred to as BAM32 or B lymphocyte adaptor protein) is an adaptor molecule that becomes translocated to the plasma membrane in a phosphoinositide 3-kinase-dependent fashion upon B cell receptor (BCR) crosslinking [ 10 ] , and exhibits significant involvement in modulating cellular proliferation, apoptosis, and immune responses through the recruitment of downstream signaling proteins [ 11 ] . Previous investigations have indicated that DAPP1 might be involved in modulating tumor cell growth and dissemination, with alterations in its expression levels potentially correlating with tumor aggressiveness and clinical prognosis. For instance, silencing of DAPP1 has been shown to markedly suppress the proliferation of epidermal growth factor receptor-mutant lung adenocarcinoma cells [ 12 ] . While current findings suggest a possible role for DAPP1 in tumor development and advancement, its specific function in BC remains incompletely understood. Therefore, this investigation sought to examine the immune-associated relevance of DAPP1 within the BC TME and to assess its clinical prognostic implications. Throughout the investigation, a comprehensive approach incorporating bioinformatics analyses and multivariate immunohistochemistry (mIHC) was adopted to systematically characterize DAPP1 protein expression in BC tissue samples, as well as its associations with tumor-infiltrating immune cells (TIICs) and immune checkpoint markers. By integrating clinicopathological parameters, Cox regression modeling was applied to rigorously evaluate the link between DAPP1 expression patterns and the clinicopathological features of individuals with BC. Moreover, in vitro functional assays involving cell migration, invasion, and proliferation were conducted in conjunction with DAPP1 overexpression regulatory interventions, with the aim of further elucidating the intrinsic linkages between DAPP1, the BC TME, and malignant cellular phenotypes. The findings indicated that the DAPP1 level was markedly elevated in BC patients and exhibited a significant association with aggressive tumor features, thereby offering novel insights and a theoretical framework for advancing precision therapeutic strategies in BC. 2. Materials and methods 2.1 Bioinformatics analysis Expression profiling data for the ENSG00000070190 (DAPP1) gene were procured from the UCSC Xena platform ( https://xenabrowser.net/ ), encompassing 1,092 BC samples and 292 adjacent non-tumor tissue specimens. RNA-Seq datasets derived from The Cancer Genome Atlas for BC patients were also accessed, comprising 2,032 tumor tissue entries, with 1,012 classified as high-expression cases and 1,020 as low-expression cases. The comparative examination of the DAPP1 level between cancerous and corresponding normal specimens was executed through the “limma” package in the R programming environment. The significance criteria were developed as log 2 (fold change) > 1.5 and P < 0.05. Associations between DAPP1 mRNA expression levels and overall survival (OS) in individuals with BC were evaluated by Kaplan–Meier survival curve analysis, while group-based survival distinctions were analyzed utilizing the log-rank test, with P -values below 0.05 interpreted as statistically significant. 2.2 Clinical sample and data collection Tumor specimens from 242 BC patients pathologically confirmed between 2010 and 2017, along with 113 adjacent normal tissues, were harvested and subsequently paraffin-embedded to generate tissue microarrays (TMA). The TMA was constructed by organizing the specimens into 2 mm cores. Concurrently, comprehensive clinicopathological information was obtained and documented, encompassing variables such as age, sex, molecular subtype, tumor size, lymph node metastasis status, tumor-node-metastasis (TNM) classification, and follow-up outcomes. Samples from individuals who had undergone preoperative neoadjuvant therapy were excluded from the analysis. This study was reviewed and sanctioned by the institutional ethics board (2018-K020), and informed consent was procured from all enrolled participants. 2.3 Multiplex immunofluorescence staining BC TMA sections were subjected to baking, deparaffinization, and rehydration. Antigen retrieval was executed utilizing a 10 mM sodium citrate buffer (pH 6.0), succeeded by overnight incubation with a validated and specific anti-DAPP1 primary antibody. The staining procedure was conducted with a commercially available multiplex immunofluorescence kit (PANOVUE). Each antibody pairing was processed through repeated staining cycles, and nuclear counterstaining was subsequently performed using DAPI (Sigma, USA). Multispectral images were acquired through the high-resolution Vectra 3.0 system (PerkinElmer, USA), and quantitative evaluation of DAPP1-positive cell proportions was performed within histologically verified tumor regions and stromal compartments using the integrated image analysis software InForm. The following primary antibodies were utilized in this investigation: anti-DAPP1 (1:400, 14722-1-AP, Proteintech, USA); anti-cytokeratin (1:4000, orb69073, Biorbyt, UK); anti-CD3 (1:800, 85061s, CST, USA); anti-CD4 (1:400, ab133616, Novus, USA); anti-CD8 (1:800, 85336s, CST, USA); anti-CD20 (1:400, ab78237, Abcam, USA); anti-CD66b (1:400, arg66287, Arigo, China); anti-CD68 (1:400, 76437s, CST, USA); anti-CD86 (1:400, orb388891, Biorbyt, UK); anti-CD163 (1:200, 93498s, CST, USA); anti-LAMP3 (1:400, orb1433531, Biorbyt, USA); anti-PD-1 (1:200, 13684T, CST, USA); anti-PD-L1 (1:200, 18616s, CST, USA); and anti-CTLA-4 (1:200, orb527271, Biorbyt, UK). 2.4 BC cell lines and cell culture Human BC cell lines MDA-MB-468, MDA-MB-231, SKBR-3, MCF-7, and BT474 were procured from ATCC (Nanjing, China). MDA-MB-468, MDA-MB-231, and MCF-7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) comprising 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. SKBR-3 cells received cultivation in a 5A medium comprising 10% FBS and 1% penicillin-streptomycin. BT474 cells underwent incubation in DMEM comprising 10% FBS, 1% penicillin-streptomycin, and 0.2% 474Advance (Cobioer, Nanjing). All cell lines received cultivation in humidified conditions at 37°C with 5% CO₂. 2.5 Western blotting (WB) analysis Total proteins were procured from MDA-MB-468, MDA-MB-231, SKBR-3, MCF-7, and BT474 cells using a protease inhibitor (NCM Biotech, Suzhou, China) combined with pre-chilled radioimmunoprecipitation assay buffer (NCM Biotech, Suzhou, China) at a dilution ratio of 1:100. The protein extracts underwent denaturation through heat treatment for 10 min and were separated via 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Ya-Enzyme, Shanghai, China), before transferring to 0.45 µm polyvinylidene fluoride membranes (Millipore, USA). The membranes underwent blocking in Tris-buffered saline containing Tween-20 (TBST) with 5% non-fat milk for 2 h, followed by overnight incubation with anti-DAPP1 (1:3000) at 4°C, triple washing with TBST, and subsequent exposure to secondary antibody for 1 h at ambient conditions. Detection of DAPP1 protein expression in BC cell lines was accomplished using an enhanced chemiluminescence kit (Biosharp, Shanghai, China) and documented via Bio-Rad system (USA). β-actin (1:10000, 66009-1-IG, Proteintech, USA) functioned as the loading control. The secondary antibody used was goat anti-rabbit (1:5000, LF102, EpiZyme, Shanghai, China). Relative DAPP1s were quantified using ImageJ software based on the gray value ratio (DAPP1/β-Actin), and all experiments were independently conducted in triplicate or more. 2.6 Cell transfection Per the supplier’s protocol (OBiO Technology, Shanghai), cells were placed into 6-well plates at 2 × 10⁵ cells per well 24 h before transfection. The next day, the growth medium was exchanged with serum- and antibiotic-free medium, and the cells were incubated for 8 h prior to viral transduction, which was facilitated using polybrene plus. The constructs shDAPP1#1 (TargetSeq: GATGAGTGGATCAAGATATTA), shDAPP1#2(TargetSeq:AGCTGTACAATTCGATTATTC), and a non-coding shRNA control (shNC, TargetSeq: CCTAAGGTTAAGTCGCCCTCG) were introduced into MDA-MB-468 and SKBR-3 cells, while DAPP1 overexpression (OE-DAPP1) plasmids were delivered into MDA-MB-231 and MCF-7 cells. For generating stable expression lines, cells with successful transduction were isolated utilizing 2.0 µg/mL puromycin (Solarbio) selection 48 h after infection. The efficiency of viral infection was validated through WB analysis. 2.7 Detection of cell proliferation activity by Cell Counting Kit-8 (CCK-8) assay The transfected cells underwent placement in 96-well plates, with each well containing 5 × 10³ cells. During specific time points (6/24/48/72 h), the addition of CCK-8 reagent solution occurred, succeeded by a 2-hour incubation period at 37°C in an environment with 5% CO₂. A Multiskan Go microplate reader (Thermo Fisher Scientific) measured the absorbance at 450 nm wavelength, which indicated the number of viable cells and their metabolic functions. Each group contained five technical replicates, and the entire experiment was independently performed in triplicate. 2.8 Transwell assay The evaluation of cellular motility and invasiveness utilized the Transwell chamber methodology. For motility examination, the experimental setup involved placing cells in a medium containing 3% bovine serum albumin, with 1 × 10⁵ cells introduced into the Transwell insert’s upper portion in 200 µl total volume, while the bottom compartment received 600 µl of medium enriched with 10% FBS. The experimental plate underwent incubation at 37°C in moisture-controlled conditions with 5% CO₂ for 24 h. Post-incubation, culture medium was extracted, followed by dual phosphate-buffered saline rinses, and subsequent fixation utilizing 4% paraformaldehyde for 20 min. Cotton swabs gently cleared non-mobile cells from the upper chamber’s interior membrane surface, succeeded by 0.1% crystal violet staining for 30 min. A DM2000LED microscope facilitated cell observation and image capture. The invasiveness assessment incorporated Matrigel pre-coating on the upper chamber’s polycarbonate membrane, maintaining identical subsequent protocols as the motility assessment. Quantification of cellular motility or invasiveness relied on membrane-traversing cell counts. The analysis encompassed five arbitrarily chosen microscopic fields per membrane, calculating mean cell numbers. Each experimental condition underwent triple replication. 2.9 Correlation and prognostic analysis The X-tile software (version 3.6.1, Yale University, USA) was utilized to define cutoff values for DAPP1 protein expression, ranging from 0–42.06 and 44.13–100, based on OS, thereby stratifying BC patients into high- and low-expression cohorts. Survival curves examining DAPP1 expression’s prognostic impact on OS were generated utilizing the Kaplan–Meier approach. The associations between DAPP1 level and clinicopathological characteristics were examined through Spearman’s rank correlation tests, whereas hazard ratios along with 95% confidence intervals were calculated using univariate and multivariate Cox proportional hazards regression analyses. Furthermore, Pearson’s correlation tests assessed the connections between the DAPP1 level and both TIICs and immune checkpoint molecules. Statistical evaluations were executed with IBM SPSS Statistics (v.27.0), GraphPad Prism 10.0, and the R programming platform. The quantitative data were denoted as mean ± standard deviation (mean ± SD), and comparisons among groups were assessed via two-tailed t-tests or one-way analysis of variance (ANOVA). Statistical significance was defined as P 0.2. 3. Results 3.1 DAPP1 level is elevated in BC tissues and associated with poor prognosis in patients Through bioinformatics analysis, DAPP1 mRNA expression was found to be markedly elevated in BC tissues versus adjacent non-tumor tissues ( P < 0.001, Fig. 1 A). Moreover, Kaplan–Meier survival analysis demonstrated that individuals with elevated DAPP1 levels displayed markedly reduced OS relative to those with lower expression levels ( P < 0.001, Fig. 2 B). Given the partial concordance between mRNA and protein expression [ 13 ] , mIHC analysis confirmed that DAPP1 protein levels were also markedly elevated in BC tissues versus paired normal counterparts ( P < 0.0001, Fig. 1 C). Consistent with the transcriptomic findings, Kaplan–Meier curves based on protein expression revealed a poorer prognosis among patients in the high DAPP1 level group ( P = 0.0026, Fig. 1 D). Collectively, these results suggest that the DAPP1 level holds prognostic value in BC patients. 3.2 Correlation between DAPP1 protein expression and clinicopathological characteristics of BC patients To explore the association between DAPP1 protein expression and the clinical characteristics of individuals with BC, samples were stratified into high- and low-expression groups per their expression levels, and correlations with variables such as age, TNM stage, and pathological grade were assessed using pretreatment clinicopathological data. Based on clinical information obtained prior to therapeutic intervention, the analysis revealed that elevated DAPP1 protein expression within tumor regions was markedly associated with T ( P = 0.044, Table 1 ), N ( P = 0.045, Table 1 ), and TNM stage ( P = 0.048, Table 1 ). In stromal regions, high DAPP1 levels exhibited significant correlations with N ( P = 0.006, Table 1 ), M ( P = 0.047, Table 1 ), and TNM stage ( P = 0.007, Table 1 ). These observations indicate that DAPP1 might function as a prospective indicator for monitoring BC advancement. Table 1 Correlation between DAPP1 protein expression in breast tissue and clinicopathological characteristics Characteristic n DAPP1 expression in tumor Pearson x² P DAPP1 expression in stroma Pearson x² P Low or no High Low or no High Total 242 120(49.6) 122(50.4) 124(51.2) 118(48.8) Age 0.006 0.94 0.051 0.821 ≤ 60 180 89(49.4) 91(50.6) 93(51.7) 87(48.3) >60 62 31(50.0) 31(50.0) 31(50.0) 31(50.0) ER 0.082 0.775 0.534 0.465 Negative 80 41(51.3) 39(48.8) 39(48.8) 41(51.3) Positive 126 62(49.2) 64(50.8) 68(54.0) 58(46.0) Unknow 36 17 19 17 19 PR 0.586 0.444 0.088 0.767 Negative 91 48(52.7) 43(47.3) 46(50.5) 45(49.5) Positive 114 54(47.4) 60(52.6) 60(52.6) 54(47.4) Unknow 37 18 19 18 19 Her-2 7.367 0.007* 0.161 0.689 Negative 112 66(58.9) 46(41.1) 57(50.9) 55(49.1) Positive 95 38(40.0) 57(60.0) 51(53.7) 44(46.3) Unknow 35 16 19 16 19 T Stage 4.063 0.044* 0.647 0.421 T1-2 221 114(51.6) 107(48.4) 115(52.0) 106(48.0) T3-4 21 6(28.6) 15(71.4) 9(42.9) 12(57.1) N Stage 4.014 0.045* 7.461 0.006* N0-1 162 73(45.1) 89(54.9) 93(57.4) 69(42.6) N2-3 80 47(58.8) 33(41.3) 31(38.8) 49(61.3) M Stage 0.131 0.718 3.94 0.047* M0 235 117(49.8) 118(50.2) 123(52.3) 112(47.7) M1 7 3(42.9) 4(47.1) 1(14.3) 6(85.7) TNM Stage 3.904 0.048* 7.315 0.007* Ⅰ-Ⅱ 156 70(44.9) 86(55.1) 90(57.7) 66(42.3) Ⅲ-Ⅳ 86 50(58.1) 36(41.9) 34(39.5) 52(60.5) ER: estrogen receptor PR༚progesterone receptor HER-2:human epidermalgrowth factor receptor2 * P <0.05;T: Tumor size; N: Lymph node metastasis; M: Distant metastasis 3.3 DAPP1 potentially functions as a prognostic risk factor for BC patients Subsequent univariate and multivariate Cox regression analyses were executed to evaluate the prognostic significance of DAPP1 as a potential risk factor. In the univariate model, differential expression of DAPP1 in both tumor and stromal cells was detected to be statistically significant (all P < 0.01, Table 2 ). Moreover, significant associations with survival outcomes were observed for progesterone receptor (PR, P = 0.013, Table 2 ), T classification ( P = 0.024, Table 2 ), N classification ( P < 0.001, Table 2 ), M classification ( P = 0.003, Table 2 ), and TNM staging ( P < 0.001, Table 2 ). In the multivariate model, DAPP1 level in both tumor and stromal compartments remained markedly linked to survival (all P < 0.05, Table 2 ), along with PR ( P = 0.048, Table 2 ) and TNM stage ( P < 0.001, Table 2 ). Collectively, these observations indicate that DAPP1 protein expression may serve as an independent prognostic indicator in BC patients. Table 2 Univariate and multivariate prognostic factor analysis of overall survival in breast cancer patients Univariate analysis Multivariate analysis HR(95%CI) P>|z| HR(95%CI) P>|z| DAPP1 expression in tumor 2.260 0.003* 2.264 0.010* Low vs High (1.311–3.894) (1.215–4.217) DAPP1 expression in stroma 4.545 60 (0.709–2.199) ER 0.736 0.281 Negative vs Positive (0.422–1.285) PR 0.488 0.013* 0.560 0.048* Negative vs Positive (0.277–0.859) (0.315–0.995) Her-2 0.923 0.779 Negative vs Positive (0.525–1.620) T Stage 2.266 0.024* T1&T2 vs T3&T4 (1.111–4.619) N Stage 3.246 <0.001* N0&NI vs N2&N3 (1.929–5.461) M Stage 4.108 0.003* M0 vs M1 (1.635–10.319) TNM Stage 3.614 <0.001* 3.487 < 0.001* I&II vs III&IV (2.125–6.146) (1.897–6.411) ER: estrogen receptor PR༚progesterone receptor HER-2:human epidermalgrowth factor receptor2 * P <0.05;T: Tumor size; N: Lymph node metastasis; M: Distant metastasis 3.4 Association between DAPP1 level and TIICs To examine the link between DAPP1 level and the immune microenvironment in BC, mIHC was conducted. The analysis demonstrated a notable association between DAPP1 level and CD68⁺CD86⁺ macrophage infiltration within the tumor compartment (r = 0.210, P = 0.001, Fig. 2 A) as well as within the stromal compartment (r = 0.226, P < 0.001, Fig. 2 B). These observations imply that DAPP1 may contribute to BC progression through modulation of immune cell infiltration in the TME. Elevated DAPP1 level may facilitate tumor advancement and immune escape by promoting the recruitment of these immune cell subsets. 3.5 Association between DAPP1 level and immune checkpoints The connection between the DAPP1 level and essential immune checkpoint molecules, encompassing CTLA-4, PD-1, and PD-L1, underwent examination through mIHC analysis. A positive link was identified between CTLA-4 expression and DAPP1 levels within tumor regions of BC (r = 0.217, P < 0.001, Fig. 4 A), and a similar link was detected in the stromal regions (r = 0.224, P < 0.001, Fig. 4 B). These immune checkpoint molecules are known to participate in BC immune evasion mechanisms, and the observed association with the DAPP1 level suggests a potential role in modulating the tumor immune microenvironment via immunosuppressive signaling pathways. 3.6 Expression of DAPP1 in BC cells and construction of stably transfected cell lines WB analysis suggested that the DAPP1 level was markedly elevated in BC cell lines MDA-MB-468 and SKBR-3, whereas relatively low expression levels were observed in MDA-MB-231, MCF-7, and BT474 cells (Fig. 5 A). Based on these expression profiles, MDA-MB-468 and SKBR-3 were selected for the generation of stable DAPP1 knockdown cell lines, while MDA-MB-231 and MCF-7 were utilized to establish stable OE-DAPP1 cell lines. Following lentiviral transduction in MDA-MB-468 and SKBR-3, WB analysis confirmed a substantial decrease in DAPP1 protein levels in both shDAPP1#1 and shDAPP1#2 groups versus the shNC control ( P < 0.05, Fig. 5 B–C), indicating the successful establishment of knockdown cell models. Similarly, after overexpression lentiviral transduction of MDA-MB-231 and MCF-7 cells, upregulation of DAPP1 and Flag-tagged proteins was validated in OE-DAPP1 cell lines via WB analysis ( P < 0.05, Fig. 5 D–E). 3.7 DAPP1 knockdown inhibited proliferation, migration, and invasion of BC cells MDA-MB-468 and SKBR-3 CCK-8 proliferation assays and colony formation assays suggested that, in MDA-MB-468 and SKBR-3 cells, both cell viability (Fig. 5 A–B) and colony numbers (Fig. 5 C) were markedly decreased in the shDAPP1#1 and shDAPP1#2 groups versus the shNC group ( P < 0.05), indicating that silencing of DAPP1 suppressed BC cell proliferation and clonogenic capacity. In addition, results from the Transwell invasion assay demonstrated that, in the same cell lines, the number of migrating cells (Fig. 5 D) and invading cells within the chambers (Fig. 5 E) was markedly reduced following DAPP1 knockdown ( P < 0.001), suggesting that DAPP1 silencing impairs the migratory and invasive potential of BC cells. 3.8 OE-DAPP1 promotes the proliferation, migration, and invasion of BC cells MDA-MB-231 and MCF-7 To comprehensively assess the potential of DAPP1 to influence the tumor biology of BC cells, multiple in vitro experiments utilized MDA-MB-231 and MCF-7 cell lines. The observations from CCK-8 and colony formation assays indicated that DAPP1 overexpression markedly enhanced BC cell viability and clonogenic growth (Fig. 5 F–H). Transwell assays further demonstrated that increased DAPP1 levels markedly promoted the migratory and invasive abilities of MDA-MB-231 and MCF-7 cells (Fig. 5 I–J). Collectively, these findings suggest that elevated DAPP1 level facilitates the proliferation, migration, and invasion of BC cells under in vitro conditions. Discussion As the most prevalent malignancy impacting women’s health, BC presents substantial challenges for personalized therapeutic strategies owing to its pronounced heterogeneity [ 14 ] . Although continuous progress has been achieved in multidisciplinary treatment modalities, considerable variation in patient outcomes remains [ 15 ][ 16 ] , highlighting the urgent necessity to identify robust prognostic biomarkers and actionable therapeutic targets [ 17 ][ 18 ] . In this study, the function of the B-cell adaptor protein DAPP1 in BC was systematically characterized through an integrative approach combining bioinformatics analysis, clinical sample validation, and molecular experimentation. Analysis of clinical datasets demonstrated that both mRNA and DAPP1 levels were markedly upregulated in BC tissues versus adjacent non-tumorous counterparts, with elevated expression markedly correlated with advanced TNM stage and unfavorable prognosis. Importantly, multivariate Cox regression analysis suggested that DAPP1 functions as an independent risk factor influencing BC patient survival, thereby underscoring its potential utility in clinical prognostic evaluation. TME, recognized as a complex ecosystem essential for tumor cell survival and progression, has been confirmed to exhibit an immunosuppressive state that critically contributes to BC development [ 19 ][ 20 ] . Within this highly heterogeneous milieu, reciprocal interactions among immune cells, extracellular matrix components, and diverse cytokines collectively form defensive barriers enabling tumor cells to evade immune surveillance [ 21 ][ 22 ][ 23 ] . Based on mIHC assessment, a notable positive link was detected between elevated DAPP1 level and increased infiltration of CD68⁺CD86⁺ macrophages in both tumor areas (r = 0.210, P = 0.001) and stromal compartments (r = 0.226, P < 0.001). CD68⁺ and CD86⁺ markers are typically indicative of immunocompetent macrophages [ 24 ][ 25 ][ 26 ] . Despite CD86⁺ being characteristic of M1-type cells, it is hypothesized that inflammatory stimuli within the triple-negative BC environment may drive CD68⁺CD86⁺ macrophages toward a phenotype with immunosuppressive features resembling M2 functionality [ 27 ] . M2-like macrophages are known to facilitate angiogenesis by releasing immunoinhibitory mediators such as IL-10 and TGF-β [ 28 ][ 29 ] , thereby supplying metabolic and metastatic routes to tumor cells. Moreover, they are capable of directly suppressing cytotoxic immune cells, encompassing effector T cells and NK cells, thereby enabling tumor cells to circumvent immune eradication [ 30 ][ 31 ] . The observed elevation in CD68⁺CD86⁺ macrophage infiltration therefore suggests that DAPP1 may participate in fostering an immunosuppressive microenvironment. Concurrently, a marked positive link was also detected between DAPP1 level and CTLA-4 levels in both tumors (r = 0.217, P < 0.001) and stromal regions (r = 0.224, P < 0.001). As a principal negative regulator of T cell activation, CTLA-4 competitively binds B7 ligands on antigen-presenting cells, thereby inhibiting T cell proliferation and activation while enhancing the differentiation and function of regulatory T cells (Treg) [ 32 ] . Treg cells, which are pivotal in tumor-mediated immune evasion, exert immunosuppressive effects in TME through cytokine secretion and direct contact-mediated inhibition of effector T cells [ 33 ] . The co-upregulation of DAPP1 and CTLA-4 implies that DAPP1 may contribute to impaired antitumor immunity by promoting the expansion of immunosuppressive subsets such as Treg or by activating immune checkpoint cascades, thereby facilitating malignant progression. Although no significant association between DAPP1 and the PD-1/PD-L1 axis was identified in this study, CTLA-4, being a central inhibitory checkpoint of T cell activation [ 34 ] , may synergistically participate in immune escape pathways through its simultaneous upregulation with DAPP1. DAPP1, recognized as a pivotal adaptor protein in the BCR signaling cascade [ 35 ][ 36 ] , facilitates phosphotyrosine- and phosphoinositol-mediated signal transmission via its SH2 and PH domains, thereby modulating lymphocyte activation processes [ 37 ][ 38 ] . The present study revealed that aberrant upregulation of DAPP1 level in BC may directly contribute to the advancement of malignant phenotypes. In vitro functional assays demonstrated that silencing DAPP1 in MDA-MB-468 and SKBR-3 cell lines markedly suppressed cellular proliferation, clonogenic potential, and migratory as well as invasive behavior. In contrast, enforced DAPP1 levels in MDA-MB-231 and MCF-7 cells markedly augmented these oncogenic traits. Collectively, these findings suggest that DAPP1 serves a direct function in tumor progression by orchestrating the proliferative and metastatic properties of BC cells. Conclusion In conclusion, this investigation suggested that DAPP1 exhibits elevated expression levels in BC and is markedly linked to unfavorable clinical outcomes. Functional assays revealed its facilitative role in promoting the proliferation, migratory capacity, and invasive potential of tumor cells. Moreover, preliminary mechanistic insights were provided, indicating its oncogenic influence may be mediated through alterations in the immune microenvironment. Based on these discoveries, DAPP1 exhibits significant potential as an innovative prognostic indicator in BC and emerges as a prospective molecular candidate for developing targeted treatments. Nonetheless, certain constraints exist: the patient cohort analyzed was modest in scale and procured from a single medical center; the detailed mechanisms by which DAPP1 regulates immune microenvironmental dynamics and downstream signaling pathways remain insufficiently defined; and in vivo validation using animal models was not conducted. Future investigations should incorporate gene-edited animal models to further clarify the function of DAPP1 in tumorigenic processes in vivo . Abbreviations The following abbreviations are used in this manuscript: BC Breast cancer DAPP1 Dual Adaptor of Phosphotyrosine and 3-Phosphoinositides TME Tumor Microenvironment TAM Tumor-associated macrophages mIHC Multivariate immunohistochemistry TIICs Tumor immune infiltrating cells OS Overall survival Declarations Author Contributions: All authors contributed to the study conception and design. Material preparation were performed by Zhengyi Lian、 Tang Liu and Lei Yang, data collection were performed by Bingyi Liu、 Kefan Wu and Xuan Li， data analysis were performed by Qichao Ni and Jun Fang. The first draft of the manuscript was written by Jiali Shan and Qiming Gu. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding: This work was supported by [Postgraduate Research & Practice Innovation Program of Jiangsu Province] (Grant numbers [SJCX24_2053] ) Institutional Review Board Statement : The Human Research Ethics Committee of the Affiliated Hospital of Nantong University approved the research method (2018-K020) Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Acknowledgments : We thank all study participants who contributed to this study. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Footnotes : Qiming Gu, Jiali Shan and Yan Li contributed equally to this work. Qichao Ni and Jun Fang are both corresponding authors of this article. References FILHO A M, LAVERSANNE M, FERLAY J, et al. 2025. The GLOBOCAN 2022 cancer estimates: Data sources, methods, and a snapshot of the cancer burden worldwide. Int J Cancer [J], 156: 1336-1346. TARI D U 2024. Breast Cancer: A Multi-Disciplinary Approach from Imaging to Therapy. Curr Oncol [J], 31: 598-602. AKINPELU A, AKINSIPE T, AVILA L A, et al. 2024. The impact of tumor microenvironment: unraveling the role of physical cues in breast cancer progression. Cancer Metastasis Rev [J], 43: 823-844. HINSHAW D C, SHEVDE L A 2019. The Tumor Microenvironment Innately Modulates Cancer Progression. Cancer Res [J], 79: 4557-4566. LI C, XU X, WEI S, et al. 2021. Tumor-associated macrophages: potential therapeutic strategies and future prospects in cancer. J Immunother Cancer [J], 9. GALBO P M, JR., ZANG X, ZHENG D 2021. Molecular Features of Cancer-associated Fibroblast Subtypes and their Implication on Cancer Pathogenesis, Prognosis, and Immunotherapy Resistance. Clin Cancer Res [J], 27: 2636-2647. JAVED S A, NAJMI A, AHSAN W, et al. 2024. Targeting PD-1/PD-L-1 immune checkpoint inhibition for cancer immunotherapy: success and challenges. Front Immunol [J], 15: 1383456. KUMARI N, CHOI S H 2022. Tumor-associated macrophages in cancer: recent advancements in cancer nanoimmunotherapies. J Exp Clin Cancer Res [J], 41: 68. ZHOU Z Q, ZHAO J J, PAN Q Z, et al. 2019. PD-L1 expression is a predictive biomarker for CIK cell-based immunotherapy in postoperative patients with breast cancer. J Immunother Cancer [J], 7: 228. HAN A, SAIJO K, MECKLENBRäUKER I, et al. 2003. Bam32 links the B cell receptor to ERK and JNK and mediates B cell proliferation but not survival. Immunity [J], 19: 621-632. HAO L, MARSHALL A J, LIU L 2021. Suppressive Role of Bam32/DAPP1 in Chemokine-Induced Neutrophil Recruitment. Int J Mol Sci [J], 22. ZHANG X, MAITY T, KASHYAP M K, et al. 2017. Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response. Mol Cell Proteomics [J], 16: 891-910. BUCCITELLI C, SELBACH M 2020. mRNAs, proteins and the emerging principles of gene expression control. Nat Rev Genet [J], 21: 630-644. LAMBALLE F, AHMAD F, VINIK Y, et al. 2021. Modeling Heterogeneity of Triple-Negative Breast Cancer Uncovers a Novel Combinatorial Treatment Overcoming Primary Drug Resistance. Adv Sci (Weinh) [J], 8: 2003049. SARHANGI N, HAJJARI S, HEYDARI S F, et al. 2022. Breast cancer in the era of precision medicine. Mol Biol Rep [J], 49: 10023-10037. WAKS A G, WINER E P 2019. Breast Cancer Treatment: A Review. Jama [J], 321: 288-300. PASSARO A, AL BAKIR M, HAMILTON E G, et al. 2024. Cancer biomarkers: Emerging trends and clinical implications for personalized treatment. Cell [J], 187: 1617-1635. COCCO S, PIEZZO M, CALABRESE A, et al. 2020. Biomarkers in Triple-Negative Breast Cancer: State-of-the-Art and Future Perspectives. Int J Mol Sci [J], 21. KANG Z, WANG J, LIU J, et al. 2025. Epigenetic modifications in breast cancer: from immune escape mechanisms to therapeutic target discovery. Front Immunol [J], 16: 1584087. YIN J, GU T, CHAUDHRY N, et al. 2023. Epigenetic modulation of antitumor immunity and immunotherapy response in breast cancer: biological mechanisms and clinical implications. Front Immunol [J], 14: 1325615. AKINSIPE T, MOHAMEDELHASSAN R, AKINPELU A, et al. 2024. Cellular interactions in tumor microenvironment during breast cancer progression: new frontiers and implications for novel therapeutics. Front Immunol [J], 15: 1302587. DUSHYANTHEN S, BEAVIS P A, SAVAS P, et al. 2015. Relevance of tumor-infiltrating lymphocytes in breast cancer. BMC Med [J], 13: 202. BADR N M, BERDITCHEVSKI F, SHAABAN A M 2020. The Immune Microenvironment in Breast Carcinoma: Predictive and Prognostic Role in the Neoadjuvant Setting. Pathobiology [J], 87: 61-74. NI C, YANG L, XU Q, et al. 2019. CD68- and CD163-positive tumor infiltrating macrophages in non-metastatic breast cancer: a retrospective study and meta-analysis. J Cancer [J], 10: 4463-4472. MEHTA A K, KADEL S, TOWNSEND M G, et al. 2021. Macrophage Biology and Mechanisms of Immune Suppression in Breast Cancer. Front Immunol [J], 12: 643771. CHEN R, YANG D, SHEN L, et al. 2022. Overexpression of CD86 enhances the ability of THP-1 macrophages to defend against Talaromyces marneffei. Immun Inflamm Dis [J], 10: e740. TALAAT I M, ELEMAM N M, ABDULLAH H W 2022. CD68, CD86 and CD163 Expression Profile in Breast Cancer Molecular Subtypes. The FASEB Journal [J], 36. ALIAZIS K, CHRISTOFIDES A, SHAH R, et al. 2025. The tumor microenvironment's role in the response to immune checkpoint blockade. Nat Cancer [J]. NOY R, POLLARD J W 2014. Tumor-associated macrophages: from mechanisms to therapy. Immunity [J], 41: 49-61. KRNETA T, GILLGRASS A, POZNANSKI S, et al. 2017. M2-polarized and tumor-associated macrophages alter NK cell phenotype and function in a contact-dependent manner. J Leukoc Biol [J], 101: 285-295. ZHENG N, WANG T, LUO Q, et al. 2023. M2 macrophage-derived exosomes suppress tumor intrinsic immunogenicity to confer immunotherapy resistance. Oncoimmunology [J], 12: 2210959. SOBHANI N, TARDIEL-CYRIL D R, DAVTYAN A, et al. 2021. CTLA-4 in Regulatory T Cells for Cancer Immunotherapy. Cancers (Basel) [J], 13. HUANG T, LI F, WANG Y, et al. 2024. Tumor-infiltrating regulatory T cell: A promising therapeutic target in tumor microenvironment. Chin Med J (Engl) [J], 137: 2996-3009. LINSLEY P S, GREENE J L, BRADY W, et al. 1994. Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities but distinct kinetics to CD28 and CTLA-4 receptors. Immunity [J], 1: 793-801. SOMMERS C L, GURSON J M, SURANA R, et al. 2008. Bam32: a novel mediator of Erk activation in T cells. Int Immunol [J], 20: 811-818. ROUQUETTE-JAZDANIAN A K, SOMMERS C L, KORTUM R L, et al. 2012. LAT-independent Erk activation via Bam32-PLC-γ1-Pak1 complexes: GTPase-independent Pak1 activation. Mol Cell [J], 48: 298-312. FOURNIER E, ISAKOFF S J, KO K, et al. 2003. The B cell SH2/PH domain-containing adaptor Bam32/DAPP1 is required for T cell-independent II antigen responses. Curr Biol [J], 13: 1858-1866. MARSHALL A J, NIIRO H, LERNER C G, et al. 2000. A novel B lymphocyte-associated adaptor protein, Bam32, regulates antigen receptor signaling downstream of phosphatidylinositol 3-kinase. J Exp Med [J], 191: 1319-1332. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-7220359","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512551728,"identity":"796f6920-c93f-4ebc-b73a-541c4cb6450a","order_by":0,"name":"Qiming Gu","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University \u0026 Medical School of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Qiming","middleName":"","lastName":"Gu","suffix":""},{"id":512551732,"identity":"45ab41e1-2a7c-4523-9719-229404b1a241","order_by":1,"name":"Jiali Shan","email":"","orcid":"","institution":"Affiliated 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University","correspondingAuthor":false,"prefix":"","firstName":"Kefan","middleName":"","lastName":"Wu","suffix":""},{"id":512551742,"identity":"3a9f6a3f-0969-4c3a-af7c-d95b29235339","order_by":8,"name":"Xuan Li","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University \u0026 Medical School of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Li","suffix":""},{"id":512551743,"identity":"5487141a-067b-4734-882a-54c0708fad67","order_by":9,"name":"Qichao Ni","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University \u0026 Medical School of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Qichao","middleName":"","lastName":"Ni","suffix":""},{"id":512551745,"identity":"4de6ad14-6b0f-4a23-bd18-0cdd79f093ee","order_by":10,"name":"Jun Fang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBAC9oYDDAwJBRIM8uzNBx8kVNQQ1sJzAKTFQILBsOdYssGDM8eI0QIiDYD4Ro6a5MMWZiK0MJ59JvHAwCKPsSGHrSKxgY2Bv707Ab8WhuNmEkCHFbMznD12I3GHDIPEmbMb8GqxZzjGBtKS2NjYl3Yj8Qwbg4FELn4tPDAtDYd5zAoS25hJ0XKMx4yBWC3MFiAtG3vYkiUSzhzjIegXHoljjDd/VNQlzpd/fPDjj4oaOf72XvxaGCQOoFtLEPA3EFYzCkbBKBgFIxwAAHkUSJ630D39AAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Nantong University \u0026 Medical School of Nantong University","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Fang","suffix":""}],"badges":[],"createdAt":"2025-07-26 10:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7220359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7220359/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91505755,"identity":"1b47d025-acfe-4241-b7dc-a06b79298b53","added_by":"auto","created_at":"2025-09-17 08:21:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":649275,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of DAPP1 in different cancer tissues. (A) Analysis of DAPP1 mRNA expression in breast cancer patients and normal breast tissues using the TCGA database. (B) Kaplan-Meier analysis of overall survival in breast cancer patients with high and low DAPP1 expression; (C) Expression of DAPP1 protein in breast cancer tissues and normal tissues. (D) Prognostic analysis of breast cancer patients with high DAPP1 expression versus the low-expression group in Kaplan-Meier survival curves. (E-F) mIHC shows the expression of DAPP1 protein in (E) normal breast tissues and (F) breast cancer tissues. CK: Cytokeratin. Scale bar, 20 μm. ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7220359/v1/86f61220b5baa8d6e0e03d94.png"},{"id":91505759,"identity":"7d5f9a9f-b417-414d-871e-bb6eb55d01ca","added_by":"auto","created_at":"2025-09-17 08:21:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1831075,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between DAPP1 and tumor immune infiltrating cells. (A-B) The cloud plots show that the expression of DAPP1 protein is positively correlated with CD68+CD86+ macrophages in both the tumor area (A) and the stromal area (B), and there is no significant correlation with other immune cells. (C) CD68+CD86+ macrophages are significantly enriched in the tumor area (a) and the stromal area (b). Panel C is a multispectral composite image of DAPP1, CD68+, CD86+, CK, and DAPI. Scale bar, 20 μm. CK: Cytokeratin.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7220359/v1/59e60af3bbaedd3185f666fc.png"},{"id":91506972,"identity":"71b52089-b824-484a-b881-7ad7ad7aee27","added_by":"auto","created_at":"2025-09-17 08:29:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1805285,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between DAPP1 and immune checkpoints. (A-B) In breast cancer tissues, the expression level of DAPP1 protein is associated with the infiltration levels of immune checkpoints PD1, PD-L1, and CTLA-4 in the tumor area (A) and stromal area (B), among which CTLA-4 is enriched in the tumor area (c) and stromal area (d) with high DAPP1 protein expression. (C) Representative mIHC images. Scale bar, 20 μm. CK: Cytokeratin.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7220359/v1/45d0223c1b6be7b69e0229f1.png"},{"id":91510743,"identity":"8831c385-bd8f-4ca4-8d40-df47fa5a4e98","added_by":"auto","created_at":"2025-09-17 08:45:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":324274,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of DAPP1 in breast cancer cell lines and identification of stably transfected cell lines. (A) Western blotting was used to detect the expression of DAPP1 protein in breast cancer cell lines; (B-C) Western blotting was used to detect the knockdown effect of DAPP1 in MDA-MB-468 cells and SKBR-3 cells; (D-E) Western blotting was used to detect the overexpression effect of DAPP1 in MDA-MB-231 cells and MCF-7 cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7220359/v1/0e2451f548d6085bf107a479.png"},{"id":91505757,"identity":"a0e948d9-8c03-4ff4-ad9a-bb48f5218730","added_by":"auto","created_at":"2025-09-17 08:21:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":927779,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of DAPP1 knockdown on proliferation, migration and invasion of MDA-MB-468 and SKBR-3 cells. (A-B): CCK-8 assay was used to detect the viability of MDA-MB-468 and SKBR-3 cells; (C): Colony formation assay showed the proliferation and clonogenic ability of MDA-MB-468 and SKBR-3 cells; (D-E): Results of Transwell invasion assay showed the migration and invasion abilities of MDA-MB-468 and SKBR-3 cells. Cell morphology was observed under an optical microscope, scale bar: 100 μm. NC: Control group; shDAPP1#1 and shDAPP1#2: DAPP1 knockdown groups. Overexpression of DAPP1 promotes proliferation, migration and invasion of breast cancer cells in vitro. (F-H): CCK-8 and colony formation assays showed the viability and clonogenic growth ability of breast cancer cells MDA-MB-231 and MCF-7; (I-J): Transwell assay showed the migration and invasion abilities of MDA-MB-231 and MCF-7 cells. Cell morphology was observed under an optical microscope, scale bar: 100 μm. In all results, Vector: Control group; OE-DAPP1: DAPP1 overexpression group. All results are presented as mean ± SD from three independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7220359/v1/abcbe64465095f817f136de5.png"},{"id":92987662,"identity":"f596db06-05e6-4ffb-b7f5-d561b1fd90b0","added_by":"auto","created_at":"2025-10-07 23:46:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7043749,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7220359/v1/2921e3fb-27e5-43ef-99ff-28f43593cfbe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"DAPP1 Elevated Expression Influences Macrophages and Predicts Poor Prognosis in Breast Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast cancer (BC) has emerged as the most prevalent malignant neoplasm among women globally, representing a significant threat to female life and health \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Alongside advancements in contemporary medical technologies and an increasingly profound comprehension of BC pathogenesis, treatment approaches have transitioned from a singular surgical focus to an integrated multidisciplinary therapeutic framework. Despite notable progress achieved in diagnostic and therapeutic techniques, a considerable proportion of patients continue to suffer from tumor recurrence and distant metastasis, while the prognosis for those at advanced stages remains unsatisfactory \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Consequently, establishing more effective therapeutic approaches remains an urgent priority to enhance patient outcomes.\u003c/p\u003e\u003cp\u003eThe progression and dissemination of BC are influenced not only by the intrinsic malignancy of cancer cells but also by regulatory cues originating from the tumor microenvironment (TME) \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The TME comprises immune cells, stromal components, extracellular matrix structures, and soluble mediators \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Among these constituents, tumor-associated macrophages (TAMs) contribute to tumor neovascularization and the invasion\u0026ndash;metastasis cascade through the secretion of cytokines, including vascular endothelial growth factor and matrix metalloproteinases \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Cancer-associated fibroblasts facilitate the migratory potential of tumor cells by modulating the rigidity of the extracellular matrix \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, whereas the dysregulated expression of immune checkpoint proteins like programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) enables immune evasion by impairing T cell activation \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. A strong correlation has been observed between elevated TAM infiltration and lymph node metastasis, chemoresistance, and poor clinical outcomes in BC patients \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, while individuals expressing PD-L1 positivity tend to exhibit enhanced responsiveness to immunotherapy \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, indicating that TME components hold promise as predictive biomarkers of therapeutic efficacy. Nevertheless, the upstream molecular pathways governing the TME remain incompletely characterized, and the identification of pivotal regulatory molecules has emerged as a critical objective in the pursuit of precision BC therapy.\u003c/p\u003e\u003cp\u003eDual adaptor of phosphotyrosine and 3-phosphoinositides 1 (DAPP1, also referred to as BAM32 or B lymphocyte adaptor protein) is an adaptor molecule that becomes translocated to the plasma membrane in a phosphoinositide 3-kinase-dependent fashion upon B cell receptor (BCR) crosslinking \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, and exhibits significant involvement in modulating cellular proliferation, apoptosis, and immune responses through the recruitment of downstream signaling proteins \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Previous investigations have indicated that DAPP1 might be involved in modulating tumor cell growth and dissemination, with alterations in its expression levels potentially correlating with tumor aggressiveness and clinical prognosis. For instance, silencing of DAPP1 has been shown to markedly suppress the proliferation of epidermal growth factor receptor-mutant lung adenocarcinoma cells \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. While current findings suggest a possible role for DAPP1 in tumor development and advancement, its specific function in BC remains incompletely understood.\u003c/p\u003e\u003cp\u003eTherefore, this investigation sought to examine the immune-associated relevance of DAPP1 within the BC TME and to assess its clinical prognostic implications. Throughout the investigation, a comprehensive approach incorporating bioinformatics analyses and multivariate immunohistochemistry (mIHC) was adopted to systematically characterize DAPP1 protein expression in BC tissue samples, as well as its associations with tumor-infiltrating immune cells (TIICs) and immune checkpoint markers. By integrating clinicopathological parameters, Cox regression modeling was applied to rigorously evaluate the link between DAPP1 expression patterns and the clinicopathological features of individuals with BC. Moreover, \u003cem\u003ein vitro\u003c/em\u003e functional assays involving cell migration, invasion, and proliferation were conducted in conjunction with DAPP1 overexpression regulatory interventions, with the aim of further elucidating the intrinsic linkages between DAPP1, the BC TME, and malignant cellular phenotypes. The findings indicated that the DAPP1 level was markedly elevated in BC patients and exhibited a significant association with aggressive tumor features, thereby offering novel insights and a theoretical framework for advancing precision therapeutic strategies in BC.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Bioinformatics analysis\u003c/h2\u003e\u003cp\u003eExpression profiling data for the ENSG00000070190 (DAPP1) gene were procured from the UCSC Xena platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://xenabrowser.net/\u003c/span\u003e\u003cspan address=\"https://xenabrowser.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), encompassing 1,092 BC samples and 292 adjacent non-tumor tissue specimens. RNA-Seq datasets derived from The Cancer Genome Atlas for BC patients were also accessed, comprising 2,032 tumor tissue entries, with 1,012 classified as high-expression cases and 1,020 as low-expression cases. The comparative examination of the DAPP1 level between cancerous and corresponding normal specimens was executed through the \u0026ldquo;limma\u0026rdquo; package in the R programming environment. The significance criteria were developed as log\u003csub\u003e2\u003c/sub\u003e(fold change)\u0026thinsp;\u0026gt;\u0026thinsp;1.5 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Associations between DAPP1 mRNA expression levels and overall survival (OS) in individuals with BC were evaluated by Kaplan\u0026ndash;Meier survival curve analysis, while group-based survival distinctions were analyzed utilizing the log-rank test, with \u003cem\u003eP\u003c/em\u003e-values below 0.05 interpreted as statistically significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Clinical sample and data collection\u003c/h2\u003e\u003cp\u003eTumor specimens from 242 BC patients pathologically confirmed between 2010 and 2017, along with 113 adjacent normal tissues, were harvested and subsequently paraffin-embedded to generate tissue microarrays (TMA). The TMA was constructed by organizing the specimens into 2 mm cores. Concurrently, comprehensive clinicopathological information was obtained and documented, encompassing variables such as age, sex, molecular subtype, tumor size, lymph node metastasis status, tumor-node-metastasis (TNM) classification, and follow-up outcomes. Samples from individuals who had undergone preoperative neoadjuvant therapy were excluded from the analysis. This study was reviewed and sanctioned by the institutional ethics board (2018-K020), and informed consent was procured from all enrolled participants.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Multiplex immunofluorescence staining\u003c/h2\u003e\u003cp\u003eBC TMA sections were subjected to baking, deparaffinization, and rehydration. Antigen retrieval was executed utilizing a 10 mM sodium citrate buffer (pH 6.0), succeeded by overnight incubation with a validated and specific anti-DAPP1 primary antibody. The staining procedure was conducted with a commercially available multiplex immunofluorescence kit (PANOVUE). Each antibody pairing was processed through repeated staining cycles, and nuclear counterstaining was subsequently performed using DAPI (Sigma, USA). Multispectral images were acquired through the high-resolution Vectra 3.0 system (PerkinElmer, USA), and quantitative evaluation of DAPP1-positive cell proportions was performed within histologically verified tumor regions and stromal compartments using the integrated image analysis software InForm.\u003c/p\u003e\u003cp\u003eThe following primary antibodies were utilized in this investigation: anti-DAPP1 (1:400, 14722-1-AP, Proteintech, USA); anti-cytokeratin (1:4000, orb69073, Biorbyt, UK); anti-CD3 (1:800, 85061s, CST, USA); anti-CD4 (1:400, ab133616, Novus, USA); anti-CD8 (1:800, 85336s, CST, USA); anti-CD20 (1:400, ab78237, Abcam, USA); anti-CD66b (1:400, arg66287, Arigo, China); anti-CD68 (1:400, 76437s, CST, USA); anti-CD86 (1:400, orb388891, Biorbyt, UK); anti-CD163 (1:200, 93498s, CST, USA); anti-LAMP3 (1:400, orb1433531, Biorbyt, USA); anti-PD-1 (1:200, 13684T, CST, USA); anti-PD-L1 (1:200, 18616s, CST, USA); and anti-CTLA-4 (1:200, orb527271, Biorbyt, UK).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 BC cell lines and cell culture\u003c/h2\u003e\u003cp\u003eHuman BC cell lines MDA-MB-468, MDA-MB-231, SKBR-3, MCF-7, and BT474 were procured from ATCC (Nanjing, China). MDA-MB-468, MDA-MB-231, and MCF-7 cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) comprising 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. SKBR-3 cells received cultivation in a 5A medium comprising 10% FBS and 1% penicillin-streptomycin. BT474 cells underwent incubation in DMEM comprising 10% FBS, 1% penicillin-streptomycin, and 0.2% 474Advance (Cobioer, Nanjing). All cell lines received cultivation in humidified conditions at 37\u0026deg;C with 5% CO₂.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Western blotting (WB) analysis\u003c/h2\u003e\u003cp\u003eTotal proteins were procured from MDA-MB-468, MDA-MB-231, SKBR-3, MCF-7, and BT474 cells using a protease inhibitor (NCM Biotech, Suzhou, China) combined with pre-chilled radioimmunoprecipitation assay buffer (NCM Biotech, Suzhou, China) at a dilution ratio of 1:100. The protein extracts underwent denaturation through heat treatment for 10 min and were separated via 12.5% sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis (Ya-Enzyme, Shanghai, China), before transferring to 0.45 \u0026micro;m polyvinylidene fluoride membranes (Millipore, USA). The membranes underwent blocking in Tris-buffered saline containing Tween-20 (TBST) with 5% non-fat milk for 2 h, followed by overnight incubation with anti-DAPP1 (1:3000) at 4\u0026deg;C, triple washing with TBST, and subsequent exposure to secondary antibody for 1 h at ambient conditions. Detection of DAPP1 protein expression in BC cell lines was accomplished using an enhanced chemiluminescence kit (Biosharp, Shanghai, China) and documented via Bio-Rad system (USA). β-actin (1:10000, 66009-1-IG, Proteintech, USA) functioned as the loading control. The secondary antibody used was goat anti-rabbit (1:5000, LF102, EpiZyme, Shanghai, China). Relative DAPP1s were quantified using ImageJ software based on the gray value ratio (DAPP1/β-Actin), and all experiments were independently conducted in triplicate or more.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Cell transfection\u003c/h2\u003e\u003cp\u003ePer the supplier\u0026rsquo;s protocol (OBiO Technology, Shanghai), cells were placed into 6-well plates at 2 \u0026times; 10⁵ cells per well 24 h before transfection. The next day, the growth medium was exchanged with serum- and antibiotic-free medium, and the cells were incubated for 8 h prior to viral transduction, which was facilitated using polybrene plus. The constructs shDAPP1#1 (TargetSeq: GATGAGTGGATCAAGATATTA), shDAPP1#2(TargetSeq:AGCTGTACAATTCGATTATTC), and a non-coding shRNA control (shNC, TargetSeq: CCTAAGGTTAAGTCGCCCTCG) were introduced into MDA-MB-468 and SKBR-3 cells, while DAPP1 overexpression (OE-DAPP1) plasmids were delivered into MDA-MB-231 and MCF-7 cells. For generating stable expression lines, cells with successful transduction were isolated utilizing 2.0 \u0026micro;g/mL puromycin (Solarbio) selection 48 h after infection. The efficiency of viral infection was validated through WB analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Detection of cell proliferation activity by Cell Counting Kit-8 (CCK-8) assay\u003c/h2\u003e\u003cp\u003eThe transfected cells underwent placement in 96-well plates, with each well containing 5 \u0026times; 10\u0026sup3; cells. During specific time points (6/24/48/72 h), the addition of CCK-8 reagent solution occurred, succeeded by a 2-hour incubation period at 37\u0026deg;C in an environment with 5% CO₂. A Multiskan Go microplate reader (Thermo Fisher Scientific) measured the absorbance at 450 nm wavelength, which indicated the number of viable cells and their metabolic functions. Each group contained five technical replicates, and the entire experiment was independently performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Transwell assay\u003c/h2\u003e\u003cp\u003eThe evaluation of cellular motility and invasiveness utilized the Transwell chamber methodology. For motility examination, the experimental setup involved placing cells in a medium containing 3% bovine serum albumin, with 1 \u0026times; 10⁵ cells introduced into the Transwell insert\u0026rsquo;s upper portion in 200 \u0026micro;l total volume, while the bottom compartment received 600 \u0026micro;l of medium enriched with 10% FBS. The experimental plate underwent incubation at 37\u0026deg;C in moisture-controlled conditions with 5% CO₂ for 24 h. Post-incubation, culture medium was extracted, followed by dual phosphate-buffered saline rinses, and subsequent fixation utilizing 4% paraformaldehyde for 20 min. Cotton swabs gently cleared non-mobile cells from the upper chamber\u0026rsquo;s interior membrane surface, succeeded by 0.1% crystal violet staining for 30 min. A DM2000LED microscope facilitated cell observation and image capture. The invasiveness assessment incorporated Matrigel pre-coating on the upper chamber\u0026rsquo;s polycarbonate membrane, maintaining identical subsequent protocols as the motility assessment. Quantification of cellular motility or invasiveness relied on membrane-traversing cell counts. The analysis encompassed five arbitrarily chosen microscopic fields per membrane, calculating mean cell numbers. Each experimental condition underwent triple replication.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Correlation and prognostic analysis\u003c/h2\u003e\u003cp\u003eThe X-tile software (version 3.6.1, Yale University, USA) was utilized to define cutoff values for DAPP1 protein expression, ranging from 0\u0026ndash;42.06 and 44.13\u0026ndash;100, based on OS, thereby stratifying BC patients into high- and low-expression cohorts. Survival curves examining DAPP1 expression\u0026rsquo;s prognostic impact on OS were generated utilizing the Kaplan\u0026ndash;Meier approach. The associations between DAPP1 level and clinicopathological characteristics were examined through Spearman\u0026rsquo;s rank correlation tests, whereas hazard ratios along with 95% confidence intervals were calculated using univariate and multivariate Cox proportional hazards regression analyses. Furthermore, Pearson\u0026rsquo;s correlation tests assessed the connections between the DAPP1 level and both TIICs and immune checkpoint molecules. Statistical evaluations were executed with IBM SPSS Statistics (v.27.0), GraphPad Prism 10.0, and the R programming platform. The quantitative data were denoted as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), and comparisons among groups were assessed via two-tailed t-tests or one-way analysis of variance (ANOVA). Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 with r\u0026thinsp;\u0026gt;\u0026thinsp;0.2.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1 DAPP1 level is elevated in BC tissues and associated with poor prognosis in patients\u003c/h2\u003e\u003cp\u003eThrough bioinformatics analysis, DAPP1 mRNA expression was found to be markedly elevated in BC tissues versus adjacent non-tumor tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Moreover, Kaplan\u0026ndash;Meier survival analysis demonstrated that individuals with elevated DAPP1 levels displayed markedly reduced OS relative to those with lower expression levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Given the partial concordance between mRNA and protein expression \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, mIHC analysis confirmed that DAPP1 protein levels were also markedly elevated in BC tissues versus paired normal counterparts (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Consistent with the transcriptomic findings, Kaplan\u0026ndash;Meier curves based on protein expression revealed a poorer prognosis among patients in the high DAPP1 level group (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0026, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Collectively, these results suggest that the DAPP1 level holds prognostic value in BC patients.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Correlation between DAPP1 protein expression and clinicopathological characteristics of BC patients\u003c/h2\u003e\u003cp\u003eTo explore the association between DAPP1 protein expression and the clinical characteristics of individuals with BC, samples were stratified into high- and low-expression groups per their expression levels, and correlations with variables such as age, TNM stage, and pathological grade were assessed using pretreatment clinicopathological data. Based on clinical information obtained prior to therapeutic intervention, the analysis revealed that elevated DAPP1 protein expression within tumor regions was markedly associated with T (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.044, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and TNM stage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In stromal regions, high DAPP1 levels exhibited significant correlations with N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), M (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and TNM stage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These observations indicate that DAPP1 might function as a prospective indicator for monitoring BC advancement.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCorrelation between DAPP1 protein expression in breast tissue and clinicopathological characteristics\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCharacteristic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eDAPP1 expression in tumor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePearson x\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003eDAPP1 expression in stroma\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePearson x\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLow or no\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLow or no\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e242\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120(49.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e122(50.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e124(51.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e118(48.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.051\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.821\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e180\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e89(49.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e91(50.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e93(51.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e87(48.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026gt;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31(50.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31(50.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e31(50.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e31(50.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eER\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.082\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.775\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.534\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.465\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e41(51.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39(48.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e39(48.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e41(51.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e126\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62(49.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64(50.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e68(54.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e58(46.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUnknow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.586\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.444\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.088\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.767\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48(52.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43(47.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e46(50.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e45(49.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e114\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54(47.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60(52.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e60(52.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e54(47.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUnknow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHer-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.367\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.007*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.161\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.689\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e112\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66(58.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46(41.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e57(50.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e55(49.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e38(40.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e57(60.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e51(53.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e44(46.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUnknow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.063\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.044*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.647\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.421\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT1-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e221\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e114(51.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e107(48.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e115(52.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e106(48.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT3-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6(28.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15(71.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e9(42.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e12(57.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.045*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e7.461\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.006*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN0-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e162\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e73(45.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e89(54.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e93(57.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e69(42.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN2-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47(58.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33(41.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e31(38.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e49(61.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.718\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e3.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.047*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e235\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e117(49.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e118(50.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e123(52.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e112(47.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3(42.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4(47.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1(14.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6(85.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNM Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.904\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.048*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e7.315\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.007*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅠ-Ⅱ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e156\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70(44.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e86(55.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e90(57.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e66(42.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅢ-Ⅳ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50(58.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36(41.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e34(39.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e52(60.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003eER: estrogen receptor PR༚progesterone receptor\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003eHER-2:human epidermalgrowth factor receptor2\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003e*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05;T: Tumor size; N: Lymph node metastasis; M: Distant metastasis\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 DAPP1 potentially functions as a prognostic risk factor for BC patients\u003c/h2\u003e\u003cp\u003eSubsequent univariate and multivariate Cox regression analyses were executed to evaluate the prognostic significance of DAPP1 as a potential risk factor. In the univariate model, differential expression of DAPP1 in both tumor and stromal cells was detected to be statistically significant (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, significant associations with survival outcomes were observed for progesterone receptor (PR, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), T classification (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), N classification (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), M classification (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and TNM staging (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the multivariate model, DAPP1 level in both tumor and stromal compartments remained markedly linked to survival (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), along with PR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and TNM stage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Collectively, these observations indicate that DAPP1 protein expression may serve as an independent prognostic indicator in BC patients.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eUnivariate and multivariate prognostic factor analysis of overall survival in breast cancer patients\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eUnivariate analysis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eMultivariate analysis\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHR(95%CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP\u0026gt;|z|\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHR(95%CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP\u0026gt;|z|\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDAPP1 expression in tumor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.260\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.003*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.264\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.010*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow vs High\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(1.311\u0026ndash;3.894)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e(1.215\u0026ndash;4.217)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDAPP1 expression in stroma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.545\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.722\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.004*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow vs High\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(2.450\u0026ndash;8.431)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e(1.365\u0026ndash;5.428)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.249\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.441\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;60 vs\u0026thinsp;\u0026gt;\u0026thinsp;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(0.709\u0026ndash;2.199)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eER\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.736\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.281\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative vs Positive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(0.422\u0026ndash;1.285)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.488\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.013*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.560\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.048*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative vs Positive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(0.277\u0026ndash;0.859)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e(0.315\u0026ndash;0.995)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHer-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.923\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.779\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative vs Positive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(0.525\u0026ndash;1.620)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.266\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.024*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT1\u0026amp;T2 vs T3\u0026amp;T4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(1.111\u0026ndash;4.619)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN0\u0026amp;NI vs N2\u0026amp;N3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(1.929\u0026ndash;5.461)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.003*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM0 vs M1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(1.635\u0026ndash;10.319)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNM Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.614\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.487\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI\u0026amp;II vs III\u0026amp;IV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e(2.125\u0026ndash;6.146)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e(1.897\u0026ndash;6.411)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eER: estrogen receptor PR༚progesterone receptor\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eHER-2:human epidermalgrowth factor receptor2\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05;T: Tumor size; N: Lymph node metastasis; M: Distant metastasis\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Association between DAPP1 level and TIICs\u003c/h2\u003e\u003cp\u003eTo examine the link between DAPP1 level and the immune microenvironment in BC, mIHC was conducted. The analysis demonstrated a notable association between DAPP1 level and CD68⁺CD86⁺ macrophage infiltration within the tumor compartment (r\u0026thinsp;=\u0026thinsp;0.210, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) as well as within the stromal compartment (r\u0026thinsp;=\u0026thinsp;0.226, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These observations imply that DAPP1 may contribute to BC progression through modulation of immune cell infiltration in the TME. Elevated DAPP1 level may facilitate tumor advancement and immune escape by promoting the recruitment of these immune cell subsets.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Association between DAPP1 level and immune checkpoints\u003c/h2\u003e\u003cp\u003eThe connection between the DAPP1 level and essential immune checkpoint molecules, encompassing CTLA-4, PD-1, and PD-L1, underwent examination through mIHC analysis. A positive link was identified between CTLA-4 expression and DAPP1 levels within tumor regions of BC (r\u0026thinsp;=\u0026thinsp;0.217, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and a similar link was detected in the stromal regions (r\u0026thinsp;=\u0026thinsp;0.224, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These immune checkpoint molecules are known to participate in BC immune evasion mechanisms, and the observed association with the DAPP1 level suggests a potential role in modulating the tumor immune microenvironment via immunosuppressive signaling pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Expression of DAPP1 in BC cells and construction of stably transfected cell lines\u003c/h2\u003e\u003cp\u003eWB analysis suggested that the DAPP1 level was markedly elevated in BC cell lines MDA-MB-468 and SKBR-3, whereas relatively low expression levels were observed in MDA-MB-231, MCF-7, and BT474 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Based on these expression profiles, MDA-MB-468 and SKBR-3 were selected for the generation of stable DAPP1 knockdown cell lines, while MDA-MB-231 and MCF-7 were utilized to establish stable OE-DAPP1 cell lines. Following lentiviral transduction in MDA-MB-468 and SKBR-3, WB analysis confirmed a substantial decrease in DAPP1 protein levels in both shDAPP1#1 and shDAPP1#2 groups versus the shNC control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;C), indicating the successful establishment of knockdown cell models. Similarly, after overexpression lentiviral transduction of MDA-MB-231 and MCF-7 cells, upregulation of DAPP1 and Flag-tagged proteins was validated in OE-DAPP1 cell lines via WB analysis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.7 DAPP1 knockdown inhibited proliferation, migration, and invasion of BC cells MDA-MB-468 and SKBR-3\u003c/h2\u003e\u003cp\u003eCCK-8 proliferation assays and colony formation assays suggested that, in MDA-MB-468 and SKBR-3 cells, both cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B) and colony numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) were markedly decreased in the shDAPP1#1 and shDAPP1#2 groups versus the shNC group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that silencing of DAPP1 suppressed BC cell proliferation and clonogenic capacity. In addition, results from the Transwell invasion assay demonstrated that, in the same cell lines, the number of migrating cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and invading cells within the chambers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) was markedly reduced following DAPP1 knockdown (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting that DAPP1 silencing impairs the migratory and invasive potential of BC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.8 OE-DAPP1 promotes the proliferation, migration, and invasion of BC cells MDA-MB-231 and MCF-7\u003c/h2\u003e\u003cp\u003eTo comprehensively assess the potential of DAPP1 to influence the tumor biology of BC cells, multiple \u003cem\u003ein vitro\u003c/em\u003e experiments utilized MDA-MB-231 and MCF-7 cell lines. The observations from CCK-8 and colony formation assays indicated that DAPP1 overexpression markedly enhanced BC cell viability and clonogenic growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;H). Transwell assays further demonstrated that increased DAPP1 levels markedly promoted the migratory and invasive abilities of MDA-MB-231 and MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI\u0026ndash;J). Collectively, these findings suggest that elevated DAPP1 level facilitates the proliferation, migration, and invasion of BC cells under \u003cem\u003ein vitro\u003c/em\u003e conditions.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs the most prevalent malignancy impacting women\u0026rsquo;s health, BC presents substantial challenges for personalized therapeutic strategies owing to its pronounced heterogeneity \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Although continuous progress has been achieved in multidisciplinary treatment modalities, considerable variation in patient outcomes remains \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, highlighting the urgent necessity to identify robust prognostic biomarkers and actionable therapeutic targets \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e][\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. In this study, the function of the B-cell adaptor protein DAPP1 in BC was systematically characterized through an integrative approach combining bioinformatics analysis, clinical sample validation, and molecular experimentation. Analysis of clinical datasets demonstrated that both mRNA and DAPP1 levels were markedly upregulated in BC tissues versus adjacent non-tumorous counterparts, with elevated expression markedly correlated with advanced TNM stage and unfavorable prognosis. Importantly, multivariate Cox regression analysis suggested that DAPP1 functions as an independent risk factor influencing BC patient survival, thereby underscoring its potential utility in clinical prognostic evaluation.\u003c/p\u003e\u003cp\u003eTME, recognized as a complex ecosystem essential for tumor cell survival and progression, has been confirmed to exhibit an immunosuppressive state that critically contributes to BC development \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e][\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Within this highly heterogeneous milieu, reciprocal interactions among immune cells, extracellular matrix components, and diverse cytokines collectively form defensive barriers enabling tumor cells to evade immune surveillance \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e][\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e][\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Based on mIHC assessment, a notable positive link was detected between elevated DAPP1 level and increased infiltration of CD68⁺CD86⁺ macrophages in both tumor areas (r\u0026thinsp;=\u0026thinsp;0.210, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and stromal compartments (r\u0026thinsp;=\u0026thinsp;0.226, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). CD68⁺ and CD86⁺ markers are typically indicative of immunocompetent macrophages \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e][\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e][\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Despite CD86⁺ being characteristic of M1-type cells, it is hypothesized that inflammatory stimuli within the triple-negative BC environment may drive CD68⁺CD86⁺ macrophages toward a phenotype with immunosuppressive features resembling M2 functionality \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. M2-like macrophages are known to facilitate angiogenesis by releasing immunoinhibitory mediators such as IL-10 and TGF-β \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e][\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, thereby supplying metabolic and metastatic routes to tumor cells. Moreover, they are capable of directly suppressing cytotoxic immune cells, encompassing effector T cells and NK cells, thereby enabling tumor cells to circumvent immune eradication \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e][\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The observed elevation in CD68⁺CD86⁺ macrophage infiltration therefore suggests that DAPP1 may participate in fostering an immunosuppressive microenvironment. Concurrently, a marked positive link was also detected between DAPP1 level and CTLA-4 levels in both tumors (r\u0026thinsp;=\u0026thinsp;0.217, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and stromal regions (r\u0026thinsp;=\u0026thinsp;0.224, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). As a principal negative regulator of T cell activation, CTLA-4 competitively binds B7 ligands on antigen-presenting cells, thereby inhibiting T cell proliferation and activation while enhancing the differentiation and function of regulatory T cells (Treg) \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Treg cells, which are pivotal in tumor-mediated immune evasion, exert immunosuppressive effects in TME through cytokine secretion and direct contact-mediated inhibition of effector T cells \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. The co-upregulation of DAPP1 and CTLA-4 implies that DAPP1 may contribute to impaired antitumor immunity by promoting the expansion of immunosuppressive subsets such as Treg or by activating immune checkpoint cascades, thereby facilitating malignant progression. Although no significant association between DAPP1 and the PD-1/PD-L1 axis was identified in this study, CTLA-4, being a central inhibitory checkpoint of T cell activation \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, may synergistically participate in immune escape pathways through its simultaneous upregulation with DAPP1.\u003c/p\u003e\u003cp\u003eDAPP1, recognized as a pivotal adaptor protein in the BCR signaling cascade \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e][\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, facilitates phosphotyrosine- and phosphoinositol-mediated signal transmission via its SH2 and PH domains, thereby modulating lymphocyte activation processes \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e][\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. The present study revealed that aberrant upregulation of DAPP1 level in BC may directly contribute to the advancement of malignant phenotypes. \u003cem\u003eIn vitro\u003c/em\u003e functional assays demonstrated that silencing DAPP1 in MDA-MB-468 and SKBR-3 cell lines markedly suppressed cellular proliferation, clonogenic potential, and migratory as well as invasive behavior. In contrast, enforced DAPP1 levels in MDA-MB-231 and MCF-7 cells markedly augmented these oncogenic traits. Collectively, these findings suggest that DAPP1 serves a direct function in tumor progression by orchestrating the proliferative and metastatic properties of BC cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this investigation suggested that DAPP1 exhibits elevated expression levels in BC and is markedly linked to unfavorable clinical outcomes. Functional assays revealed its facilitative role in promoting the proliferation, migratory capacity, and invasive potential of tumor cells. Moreover, preliminary mechanistic insights were provided, indicating its oncogenic influence may be mediated through alterations in the immune microenvironment. Based on these discoveries, DAPP1 exhibits significant potential as an innovative prognostic indicator in BC and emerges as a prospective molecular candidate for developing targeted treatments. Nonetheless, certain constraints exist: the patient cohort analyzed was modest in scale and procured from a single medical center; the detailed mechanisms by which DAPP1 regulates immune microenvironmental dynamics and downstream signaling pathways remain insufficiently defined; and \u003cem\u003ein vivo\u003c/em\u003e validation using animal models was not conducted. Future investigations should incorporate gene-edited animal models to further clarify the function of DAPP1 in tumorigenic processes \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used in this manuscript:\u003c/p\u003e\n\u003cp\u003eBC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Breast cancer\u003c/p\u003e\n\u003cp\u003eDAPP1 \u0026nbsp; \u0026nbsp; Dual Adaptor of Phosphotyrosine and 3-Phosphoinositides\u003c/p\u003e\n\u003cp\u003eTME \u0026nbsp; \u0026nbsp; \u0026nbsp; Tumor Microenvironment\u003c/p\u003e\n\u003cp\u003eTAM \u0026nbsp; \u0026nbsp; \u0026nbsp; Tumor-associated macrophages\u003c/p\u003e\n\u003cp\u003emIHC \u0026nbsp; \u0026nbsp; \u0026nbsp;Multivariate immunohistochemistry\u003c/p\u003e\n\u003cp\u003eTIICs \u0026nbsp; \u0026nbsp; \u0026nbsp; Tumor immune infiltrating cells\u003c/p\u003e\n\u003cp\u003eOS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Overall survival\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003eAll authors contributed to the study conception and design. Material preparation were performed by Zhengyi Lian、\u0026nbsp;Tang Liu and Lei Yang, data collection were performed by Bingyi Liu、\u0026nbsp;Kefan Wu and Xuan Li，\u0026nbsp;data analysis were performed by Qichao Ni and Jun Fang. The first draft of the manuscript was written by Jiali Shan and Qiming Gu. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by [Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province] (Grant numbers [SJCX24_2053] )\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e The Human Research Ethics Committee of the Affiliated Hospital of Nantong University approved the research method (2018-K020)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Informed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e We thank all study participants who contributed to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFootnotes\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eQiming Gu, Jiali Shan and Yan Li contributed equally to this work.\u0026nbsp; Qichao Ni and Jun Fang are both corresponding authors of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col class=\"decimal_type\"\u003e\n\u003cli\u003eFILHO A M, LAVERSANNE M, FERLAY J, et al. 2025. 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Macrophage Biology and Mechanisms of Immune Suppression in Breast Cancer. Front Immunol [J], 12: 643771.\u003c/li\u003e\n\u003cli\u003eCHEN R, YANG D, SHEN L, et al. 2022. Overexpression of CD86 enhances the ability of THP-1 macrophages to defend against Talaromyces marneffei. Immun Inflamm Dis [J], 10: e740.\u003c/li\u003e\n\u003cli\u003eTALAAT I M, ELEMAM N M, ABDULLAH H W 2022. CD68, CD86 and CD163 Expression Profile in Breast Cancer Molecular Subtypes. The FASEB Journal [J], 36.\u003c/li\u003e\n\u003cli\u003eALIAZIS K, CHRISTOFIDES A, SHAH R, et al. 2025. The tumor microenvironment\u0026apos;s role in the response to immune checkpoint blockade. Nat Cancer [J].\u003c/li\u003e\n\u003cli\u003eNOY R, POLLARD J W 2014. Tumor-associated macrophages: from mechanisms to therapy. Immunity [J], 41: 49-61.\u003c/li\u003e\n\u003cli\u003eKRNETA T, GILLGRASS A, POZNANSKI S, et al. 2017. M2-polarized and tumor-associated macrophages alter NK cell phenotype and function in a contact-dependent manner. J Leukoc Biol [J], 101: 285-295.\u003c/li\u003e\n\u003cli\u003eZHENG N, WANG T, LUO Q, et al. 2023. M2 macrophage-derived exosomes suppress tumor intrinsic immunogenicity to confer immunotherapy resistance. Oncoimmunology [J], 12: 2210959.\u003c/li\u003e\n\u003cli\u003eSOBHANI N, TARDIEL-CYRIL D R, DAVTYAN A, et al. 2021. CTLA-4 in Regulatory T Cells for Cancer Immunotherapy. Cancers (Basel) [J], 13.\u003c/li\u003e\n\u003cli\u003eHUANG T, LI F, WANG Y, et al. 2024. Tumor-infiltrating regulatory T cell: A promising therapeutic target in tumor microenvironment. Chin Med J (Engl) [J], 137: 2996-3009.\u003c/li\u003e\n\u003cli\u003eLINSLEY P S, GREENE J L, BRADY W, et al. 1994. Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities but distinct kinetics to CD28 and CTLA-4 receptors. Immunity [J], 1: 793-801.\u003c/li\u003e\n\u003cli\u003eSOMMERS C L, GURSON J M, SURANA R, et al. 2008. Bam32: a novel mediator of Erk activation in T cells. Int Immunol [J], 20: 811-818.\u003c/li\u003e\n\u003cli\u003eROUQUETTE-JAZDANIAN A K, SOMMERS C L, KORTUM R L, et al. 2012. LAT-independent Erk activation via Bam32-PLC-\u0026gamma;1-Pak1 complexes: GTPase-independent Pak1 activation. Mol Cell [J], 48: 298-312.\u003c/li\u003e\n\u003cli\u003eFOURNIER E, ISAKOFF S J, KO K, et al. 2003. The B cell SH2/PH domain-containing adaptor Bam32/DAPP1 is required for T cell-independent II antigen responses. Curr Biol [J], 13: 1858-1866.\u003c/li\u003e\n\u003cli\u003eMARSHALL A J, NIIRO H, LERNER C G, et al. 2000. A novel B lymphocyte-associated adaptor protein, Bam32, regulates antigen receptor signaling downstream of phosphatidylinositol 3-kinase. J Exp Med [J], 191: 1319-1332.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, DAPP1, prognosis, tumor-infiltrating immune cells","lastPublishedDoi":"10.21203/rs.3.rs-7220359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7220359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eThe main purpose of this study is to clarify the intricate regulatory pathways in the breast cancer tumor microenvironment, with a specific focus on revealing the immunological relevance and prognostic significance of the adaptor protein DAPP1 (dual adaptor of phosphotyrosine and 3-phosphoinositides 1) in the breast cancer microenvironment. The core research question is to determine how DAPP1 influences breast cancer progression, particularly its association with immune-related factors and patient outcomes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThis study adopted an integrated research approach, including bioinformatics analysis, clinical tissue microarray verification, and molecular experiments. Multiplex immunohistochemistry was used to quantitatively assess the expression level of DAPP1 and analyze its correlation with tumor-infiltrating immune cells (such as specific macrophage subsets) and immune checkpoints. Western blotting was employed to identify the protein expression of DAPP1 in cell lines, and stable knockdown and overexpression models of DAPP1 were constructed. Functional validation was performed using Cell Counting Kit-8 assay to detect cell proliferation ability and Transwell assay to detect migration and invasion abilities.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe study found that DAPP1 was significantly highly expressed in breast cancer tissues and independently associated with advanced clinical stages and poor overall survival. In addition, the expression level of DAPP1 was positively correlated with the infiltration of CD68⁺CD86⁺ macrophages and the expression of CTLA4. Functional analyses further confirmed that DAPP1 could enhance the proliferation, migration, and invasion abilities of breast cancer cells.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese findings indicate that DAPP1 promotes the development of breast cancer by enhancing the malignant phenotypes of tumor cells and creating an immunosuppressive microenvironment. Therefore, DAPP1 can serve as a valuable prognostic indicator and a potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"DAPP1 Elevated Expression Influences Macrophages and Predicts Poor Prognosis in Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 08:21:08","doi":"10.21203/rs.3.rs-7220359/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"baa4cda3-fba8-48bd-8c33-369406eecb55","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-07T23:38:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 08:21:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7220359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7220359","identity":"rs-7220359","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}