SPDL1 Promotes Lung Metastasis of Triple-Negative Breast Cancer by Targeting the TGF-β/Smad Pathway to Regulate M2 Macrophage Polarization

SPDL1 Promotes Lung Metastasis of Triple-Negative Breast Cancer by Targeting the TGF-β/Smad Pathway to Regulate M2 Macrophage Polarization | 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 SPDL1 Promotes Lung Metastasis of Triple-Negative Breast Cancer by Targeting the TGF-β/Smad Pathway to Regulate M2 Macrophage Polarization Zeng you Xiao, Dongxiao Shen, Han Cai, Chang Lu, Zean Yang, Xiaotong Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7913702/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 7 You are reading this latest preprint version Abstract Breast cancer is a malignant tumor originating from breast epithelial tissue. Globally, there are approximately 2.3 million new breast cancer cases annually, accounting for 11.7% of all new cancer cases. Based on molecular subtype classification, triple-negative breast cancer (TNBC) has a high propensity for lung metastasis and is a major cause of death in breast cancer patients. As key innate immune cells in the tumor microenvironment (TME), tumor-associated macrophages (TAMs) have a functional status closely related to tumor progression. Recent studies have shown that macrophages in the TME play complex and critical regulatory roles in tumor evolution. Moreover, during the process of TNBC lung metastasis, TAMs are regulated by multiple signaling pathways, thereby driving metastasis. Previous studies have found that the TGF-β/Smad signaling pathway can induce TAM polarization toward the M2 type, which synergistically promotes TNBC lung metastasis. but its mechanism remains unclear. Spindle apparatus coiled-coil protein 1 (SPDL1) plays an important role in regulating cell mitosis and accurate chromosome segregation, and is involved in the occurrence, development, and prognosis evaluation of breast tumors. However, the impact of SPDL1 on the functional status of TAMs and whether the interaction between them is involved in regulating TNBC lung metastasis have not been clearly reported yet. Therefore, this study aims to explore whether SPDL1 affects the process of TNBC lung metastasis by regulating M2 macrophage polarization and clarify its potential molecular mechanism, so as to provide new ideas for the development of novel immune drugs based on SPDL1-macrophages. Triple-negative breast cancer SPDL1 M2 Macrophage TGF-β/Smad Pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Distant metastasis of breast cancer is the leading cause of death in female patients[ 1 ]. Although current clinical treatments can effectively control the primary tumor, the rapid deterioration of the disease often occurs after lung metastasis, making it a major challenge in clinical treatment[ 2 ]. Among these cases, the incidence of lung metastasis in patients with triple-negative breast cancer (TNBC) is as high as 21%-32%, and the lung is one of the most common target organs for distant metastasis[ 3 ]. Studies have shown that the progression of breast cancer lung metastasis is closely associated with driver gene mutations, such as BRCA1/2 deletion, HER2 amplification, and TP53 inactivation[ 4 ]. These mutations further exacerbate disease progression and lead to poor prognosis in patients by mechanisms including enhancing the invasive and metastatic potential of tumor cells, remodeling the pre-metastatic niche in the lung, inducing immune suppression, and promoting therapeutic resistance[ 5 ]. Notably, the molecular regulatory network underlying the directional metastatic propensity of breast cancer cells to lung tissue ("lung tropism") remains largely unclear, and its potential mechanism urgently requires in-depth investigation. The human SPDL1 gene is located in the 5q35.1 region of the chromosome, and its encoded product, Spindly protein, can maintain genomic stability. It also acts synergistically with the RZZ complex (Rod/ZW10/Zwilch) in the centromere region and is involved in regulating the signaling network related to cell migration[ 6 ] Previous studies by our research group found that SPDL1 not only participates in core events of the cell cycle but also exhibits oncogenic properties in various malignant tumors such as lung cancer and oral cancer[ 7 ]. Notably, SPDL1 has been identified as a novel independent prognostic marker for TNBC[ 8 ]. However, the mechanism of action of SPDL1 in breast cancer lung metastasis remains an unknown field, especially whether SPDL1 can affect the progression of breast cancer by regulating the tumor microenvironment has not been reported yet. Therefore, systematic analysis of the multi-dimensional regulatory network of SPDL1 in breast cancer lung metastasis will provide a key theoretical basis for the development of targeted intervention strategies. TAMs are the most abundant population of tumor-infiltrating immune cells in the TME. During tumor progression, the number of M2-like TAMs increases sharply, becoming the dominant cell population of TAMs in the TME, and at the same time secrete anti-inflammatory factors such as IL-10 and TGF-β[ 9 ]. They exhibit pro-tumor activities by promoting the formation of the pre-metastatic niche (PMN), regulating the immunosuppressive microenvironment, promoting angiogenesis, and adjusting energy metabolism[ 10 ]. Studies have found that exosomes derived from breast cancer carry molecules such as miR-138-5p, which induce M2 macrophage polarization by inhibiting KDM6B, thereby affecting the process of tumor metastasis. This reveals the crosstalk between macrophages and tumor cells in the progression of breast cancer[ 11 ]. In breast cancer, the abnormal activation of the TGF-β/Smad pathway is closely related to disease progression, drug resistance, and poor prognosis[ 12 ]. In the early stage of breast cancer, TGF-β activates the Smad2/3-Smad4 complex, inhibits the expression of cyclins, and induces cell apoptosis, thereby exerting a tumor-suppressive effect[ 13 ]. However, as the TME changes, cancer cells escape via gene mutations or epigenetic modifications and exert an oncogenic effect. Studies have found that inactivating mutations in TGF-β receptor I and II or Smad4 lead to off-target effects, which in turn promote epithelial-mesenchymal transition (EMT), angiogenesis, and immune escape, accelerating breast cancer metastasis[ 14 ]. GLI2 is a direct downstream target of the TGF-β pathway and is independent of the Hedgehog signaling axis[ 15 ]. Studies have found that GLI2 plays a direct functional role in solid tumors; GLI2 knockout can inhibit the proliferation of hepatocellular carcinoma cells and reduce the invasiveness of melanoma cells by downregulating E-cadherin[ 16 , 17 ]. In conclusion, the key role of GLI2 downstream of TGF-β signaling in promoting cancer progression toward metastasis has been documented in different cancer types. Interestingly, GLI2 also mediates the inflammatory regulation of the TME through cytokines such as IL-6 and TGF-β [ 18 , 19 ]. Both IL-6 and TGF-β can regulate the polarization of M2-type TAMs in the TME, thereby promoting cancer progression[ 20 , 21 ]. It has also been reported that IL-6 and TGF-β are directly related to the poor prognosis of TNBC[ 22 , 23 ]. Therefore, this study intends to clarify that SPDL1 mediates M2 macrophage polarization by regulating the TGF-β/Smad pathway, thereby promoting the process of TNBC lung metastasis. This finding may provide a novel molecular intervention strategy and theoretical basis for the targeted treatment of metastatic breast cancer. Materials and Methods Data Collection The pan-cancer gene expression, prognostic, and immune analysis data used in this study were all obtained from The Cancer Genome Atlas (TCGA) pan-cancer database ( https://portal.gdc.cancer.gov/ ); the immunohistochemical protein expression analysis data for SPDL1, CD163, and CD206 were retrieved from the Human Protein Atlas (HPA) web platform ( https://www.proteinatlas.org/ ); for the Mendelian randomization study, expression quantitative trait locus (eQTL) data of human breast tissue genes were downloaded from the GTEx Portal website ( https://www.gtexportal.org/home/ ), and breast tumor disease data were screened from the FinnGen database ( https://www.finngen.fi/en/access_results ). These two types of data were combined for subsequent analysis. Cell Culture Human triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were purchased from ATCC (USA), and the human monocytic leukemia cell line THP-1 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 and MDA-MB-468 cells were cultured in L15 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). THP-1 cells and their derived macrophages were cultured in RPMI-1640 medium containing 1% penicillin-streptomycin and 20% FBS. All cells were maintained in an incubator at 37°C with 5% CO₂. Collection of Conditioned Medium For the collection of conditioned medium from triple-negative breast cancer (TNBC) cells, we seeded MDA-MB-231 or MDA-MB-468 cells in 6-well plates at a density of 1×10⁶ cells/mL. After allowing the cells to adhere for 24 hours, the medium in each well was replaced according to the experimental conditions: either 2 mL of serum-free RPMI 1640 medium, or serum-free medium supplemented with 10 µmol/L of the TGF-β receptor inhibitor LY2109761. Following an additional 24-hour starvation culture, the conditioned medium was collected according to the experimental groups. Establishment of the Co-Culture System For the co-culture system of macrophages and TNBC cells, Transwell chambers were used for indirect co-culture. TNBC cells were seeded in the lower chamber of 24-well plates (3×10⁵ cells/well), and THP-1-derived M2 macrophages were seeded in the upper chamber of 24-well plates (3×10⁵ cells/well), with complete L15 medium added to both chambers. Control and experimental groups were set as follows: Control group: TNBC cells cultured alone; Experimental Group 1: SPDL1-overexpressing TNBC cells cultured alone; Experimental Group 2: TNBC cells co-cultured with M2 macrophages; Experimental Group 3: SPDL1-overexpressing TNBC cells co-cultured with M2 macrophages. After 48 hours of culture, TNBC cells were collected for Transwell and Western blot assays to detect the invasive and migratory abilities of TNBC cells. Cell Scratch Healing Assay MDA-MB-231 and MDA-MB-468 cells in the logarithmic growth phase were digested with 0.25% trypsin and seeded in 6-well plates at a density of 5×10⁵ cells/well. After 24 hours of culture, a horizontal line was drawn on the back of the culture plate using a 200 µL pipette tip, and then the cells were washed three times with PBS. Three random areas were selected for imaging observation, and another three random areas were selected for imaging observation 24 hours later to track the cell migration process in real time. Cell Migration and Invasion Assay After digestion and resuspension, the cells were resuspended again with serum-free L15 medium and counted to 3×10⁵ cells/mL using a cell counting chamber. 700 µL of medium containing 20% FBS was added to the lower chamber of a 24-well Transwell plate (Corning, New York, USA), and 300 µL of the cell suspension was added to the upper chamber of the Transwell. After incubation at 37°C for 48 hours, crystal violet staining was performed. The cells were washed and fixed with a fixative for 15 minutes, and the cells on the inner wall of the chamber were gently wiped off with a cotton swab. Then, the number of cells on the outer wall of the chamber was counted. In the invasion assay, 200 mg/mL Matrigel TM (BD, Sparks, MD, USA) was added to the bottom of the upper chamber of the Transwell, and the other operation steps were the same as above. Each experiment was repeated three times, and the average value was taken. Western Blotting After washing the cells with PBS, cell lysis buffer was added, and the cells were incubated on ice for 15 minutes. The cells were thoroughly scraped off with a cell scraper, and the lysate was collected into EP tubes. The samples were ultrasonically disrupted and then centrifuged to determine the protein concentration. After denaturation of the protein in a metal bath, Western blot experiments were performed, including sample loading, electrophoresis, and membrane transfer. After membrane transfer, the membrane was blocked with a rapid blocking solution for 10 minutes and incubated with primary antibodies at 4°C overnight. The next day, the corresponding secondary antibodies were added, and after incubation, the membrane was developed with a developing solution. The TGF-β antibody, Smad2 antibody, P-Smad2 antibody, and Vimentin antibody used were purchased from CST Company; TGF-βRII antibody, TGF-βRI antibody, and MMP9 antibody were purchased from Wanlei Company; GLI2 antibody was purchased from Sanying Company; N-cadherin antibody and SPDL1 antibody were purchased from Abcam Company; secondary antibodies and GAPDH antibody were purchased from Aibotai Company. Flow Cytometry After macrophage polarization, cells from each group were collected and resuspended in PBS for macrophage detection: 1×10⁶ cells were taken, and CD11b-phycoerythrin (PE) and CD206-fluorescein isothiocyanate (FITC) antibodies (BD Pharmingen Company) were added, followed by incubation at 4°C in the dark for 30 minutes. A flow cytometer was used for detection, and the sample results were analyzed. Quantitative RT-PCR Cell samples were collected, and total RNA was extracted using an RNA extraction kit from Accurate Biotechnology (Hunan) Co., Ltd. The concentration and purity of RNA were determined; cDNA synthesis was performed using a reverse transcription kit from the same company. The primer sequences used are as follows: H-TGF-β:5′-GGGACTATCCACCTGCAAGA-3′,5′-CCTCCTTGGCGTAGTAGTCG-3′;H-IL-10:5′-TGCCTTCAGCAGAGTGAAGA-3′,5′-GGTCTTGGTTCTCAGCTTGG-3′;H-SPDL1:5′-CCCCTAACTCTCCCAGGTCA-3′,5′-CATGCAGCGAGAGAGGAGAG-3′;H-GAPDH:5′-TGTGGGCATCAATGGATTTGG-3′,5′-ACACCATGTATTCCGGGTCAAT-3′. ELISA Animal serum or cell culture supernatant was collected, and the secretion levels of IL-6 and TGF-β were detected according to the instructions of the corresponding ELISA kit from Boster Biological Engineering Co., Ltd. (Wuhan, China). Animal Models MDA-MB-231 and 231SPDL1-OE cells were injected into the tail vein of 5-week-old female Balb/c mice (purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd.) at a concentration of 1×10⁷ cells/mL. Each mouse was injected with 200 µL of cell suspension to establish a breast cancer lung metastasis model. Seven days after inoculation, the mice were divided into 4 groups: Control group: intragastric administration of 200 µL normal saline once a day; Oxy treatment group: intragastric administration of 120 mg/kg oxymatrine once a day; SPDL1 overexpression group: intragastric administration of 200 µL normal saline once a day; SPDL1 overexpression combined with Oxy treatment group: intragastric administration of 120 mg/kg oxymatrine once a day. From day 7, the mice were weighed every other day. Two weeks after administration, all animals were sacrificed, and the number of lung metastatic tumors and the weight of the lungs were counted. Some tissues were fixed with formalin, and some were frozen in liquid nitrogen for subsequent experiments. Immunohistochemistry (IHC) Tissues dissected from animals were fixed with 10% formalin and then embedded in paraffin for sectioning. The sections were dewaxed with xylene and ethanol, and antigen retrieval was performed with 0.01 M citrate buffer at 98°C. After blocking with bovine serum albumin (BSA), the sections were incubated with primary antibodies at 4°C overnight. After washing with PBS, the sections were incubated with secondary antibodies at room temperature for 1 hour. After washing again, the sections were incubated with streptavidin-horseradish peroxidase (SABC) for 30 minutes. Finally, 3,3'-diaminobenzidine (DAB) was used for color development, and hematoxylin was used for counterstaining. Hematoxylin and Eosin (HE) Staining To examine various tissues of the mice, hematoxylin-eosin (HE) staining was used. Mouse tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and then sectioned at a thickness of 5 µm to observe the overall morphology. The samples were then hydrated through gradient concentrations of alcohol and dewaxed with xylene. The sections were first stained with hematoxylin for five minutes and then with eosin for one minute. Subsequently, the tissues were dehydrated with gradient concentrations of alcohol and then treated with xylene. Finally, each section was sealed and observed under a ×200 optical microscope (Olympus, Tokyo, Japan). Statistical Analysis GraphPad Prism 8 software was used for statistical analysis, and the experimental data were expressed as "mean ± standard deviation (mean ± SD)". Unpaired t-test was used for comparison between two groups, and one-way analysis of variance (one-way ANOVA) was used for comparison among multiple groups. P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 were considered to indicate statistically significant differences. Results SPDL1 is Highly Expressed in Breast Cancer and Associated with Poor Prognosis To clarify the expression characteristics and clinical significance of SPDL1 in cancer, a pan-cancer analysis was first conducted: samples of 33 cancer types were collected from the TCGA database for analysis. The results showed that there were significant differences in the expression of SPDL1 among different cancer types (Supplementary Fig. 1A-B), and it was highly expressed in breast cancer (Supplementary Fig. 1C, E). A Mendelian randomization study found that the expression level of SPDL1 was positively correlated with the risk of breast tumor, i.e., the higher the expression of SPDL1, the higher the risk of onset (Supplementary Fig. 1D). Prognostic analysis results confirmed that high expression of SPDL1 was positively correlated with poor prognosis of breast cancer patients within a certain period (Supplementary Fig. 1F). In conclusion, SPDL1 may play an oncogenic role in triple-negative breast cancer (TNBC) and participate in the disease progression of TNBC. SPDL1 Promotes the Invasive and Migratory Capacities of TNBC Cells To explore the effect of SPDL1 on the biological functions of TNBC cells, an SPDL1-overexpressing lentivirus was first constructed and transfected into TNBC cells. Western blot (WB) and qRT-PCR experiments were used to verify the transfection efficiency. The results showed that the protein and mRNA expression levels of SPDL1 in the transfected cells were significantly increased (Fig. 1 A-B). Wound healing assay and Transwell assay were used to detect cell migration and invasion abilities: compared with the control group, the wound healing rate of TNBC cells in the SPDL1 overexpression group was significantly accelerated (Fig. 1 C), and the number of transmembrane cells in the Transwell chamber was significantly increased (Fig. 1 D). The above results indicate that SPDL1 can significantly enhance the invasive and migratory capacities of TNBC cells. SPDL1 Expression is Positively Correlated with M2 Macrophage Infiltration M2 macrophages play a key regulatory role in the progression of TNBC. Based on this, immunotherapy targeting macrophages has potential application value in the treatment of TNBC. To explore the association between SPDL1 and M2 macrophages, bioinformatics analysis (including gene expression correlation analysis and immune cell infiltration scoring) showed that the expression level of SPDL1 was positively correlated with the infiltration degree of macrophages (Supplementary Fig. 2A-B). Moreover, analysis using the Human Protein Atlas found that SPDL1 was significantly positively correlated with M2 macrophages (Supplementary Fig. 2C), suggesting that there may be a functional synergistic effect between them. SPDL1 Drives M2 Macrophage Polarization by Promoting the Secretion of IL-6 and TGF-β from TNBC Cells Tumor cells in the tumor microenvironment (TME) can regulate macrophage polarization by secreting cytokines. To clarify the effect of SPDL1 on M2 macrophage polarization, the supernatant of TNBC cells was collected according to the experimental groups to prepare conditioned medium (CM), which was then co-cultured with M0 macrophages induced and differentiated from THP-1 cells for detection: Morphological observation showed that compared with the control group (CM from untreated TNBC cells), the CM from TNBC cells in the SPDL1 overexpression group could induce more M0 macrophages to exhibit the typical elongated spindle shape of M2 macrophages (Fig. 2 A); qRT-PCR detection results showed that after co-culture with TNBC cells in the SPDL1 overexpression group, the mRNA expression levels of M2 markers (IL-10, TGF-β) in macrophages were significantly increased (Fig. 2 B); flow cytometry verification showed that the proportion of CD11b⁺CD206⁺ cells (M2 phenotypic markers) on the surface of macrophages in the above co-culture system was significantly higher than that in the control group (Fig. 2 C).Given that IL-6 and TGF-β are known key regulatory factors for M2 macrophage polarization, the secretory function of TNBC cells was detected by ELISA: compared with the control group, the concentrations of IL-6 and TGF-β secreted by TNBC cells in the SPDL1 overexpression group were significantly increased (Fig. 2 D). In conclusion, SPDL1 may induce macrophage polarization toward the M2 type by promoting the secretion of IL-6 and TGF-β from TNBC cells. M2 Macrophages Synergistically Enhance the Invasive and Migratory Capacities of TNBC Cells Previous studies have confirmed that M2 macrophages can promote the malignant progression of TNBC[ 24 ]. To verify this effect, M2 macrophages induced by co-culture with TNBC cells were collected, and an indirect co-culture system was established using Transwell chambers for co-culture with TNBC cells. Detection results showed: Transwell assay showed that compared with TNBC cells not exposed to M2 macrophages, the number of transmembrane TNBC cells co-cultured with M2 macrophages was significantly increased (Fig. 3 A-B); Western blot detection showed that the expression levels of epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, MMP9, Vimentin) in TNBC cells in the above co-culture group were significantly increased (Fig. 3 C). The above results indicate that M2 macrophages can synergistically enhance the invasive and migratory capacities of TNBC cells. SPDL1 Activates the TGF-β/Smad Signaling Pathway in TNBC Cells The TGF-β signaling pathway plays a core regulatory role in the progression of TNBC. To clarify the association between SPDL1 and this pathway, transcriptome sequencing was performed on MDA-MB-231 cells (a TNBC cell line). GO functional annotation and KEGG pathway enrichment analysis results showed that the TGF-β signaling pathway was significantly enriched in the SPDL1 overexpression group (Fig. 4 A-B). The GSEA plot showed that the TGF-β signaling pathway was upregulated in the experimental group (Fig. 4 C). Previous studies have shown that GLI2, a downstream factor of the TGF-β/Smad signaling axis, can target and regulate the production of IL-6 and TGF-β. Based on this, it is hypothesized that SPDL1 may promote the expression of GLI2 by activating the TGF-β/Smad pathway, thereby increasing the secretion of IL-6 and TGF-β to promote M2 macrophage polarization and TNBC progression. To verify this mechanism: Western blot detection was performed, and the results showed that the protein expression levels of key molecules in the TGF-β/Smad pathway (TGF-β, Smad2, phosphorylated Smad2 (P-Smad2), TGF-β receptor I (TGF-βRI), TGF-β receptor II (TGF-βRII)) and the downstream factor GLI2 in TNBC cells in the SPDL1 overexpression group were significantly increased (Fig. 4 D); immunofluorescence experiment showed that the fluorescence intensity of TGF-β and GLI2 in cells in the SPDL1 overexpression group was significantly stronger than that in the control group (Fig. 4 E). In conclusion, SPDL1 can promote the secretion of IL-6 and TGF-β by activating the TGF-β/Smad signaling pathway in TNBC cells, thereby promoting the polarization of M2-type tumor-associated macrophages (TAMs) in the TME and the invasion and metastasis of TNBC cells. Inhibition of the TGF-β/Smad Signaling Pathway Attenuates SPDL1-Induced Promotion of M2 Macrophage Polarization LY2109761 is a dual inhibitor of TGF-βRI and TGF-βRII, which can inhibit the activity of the TGF-β/Smad signaling pathway by downregulating the phosphorylation level of Smad2. To evaluate the effect of inhibiting the TGF-β/Smad pathway on SPDL1-regulated M2 macrophage polarization, TNBC cells were divided into 4 groups: Control group, LY2109761 treatment group (LY group), SPDL1 overexpression group, and SPDL1 overexpression + LY2109761 combined treatment group (SPDL1 + LY group). The CM from TNBC cells from the above groups were collected and added to M0 macrophages. Morphological observation showed that the number of macrophages showing M2 morphology in the LY group and SPDL1 + LY group was significantly less than that in the control group and SPDL1 overexpression group (Fig. 5 A). Flow cytometry showed that the proportion of CD11b⁺CD206⁺ cells (M2 markers) in macrophages in the LY group and SPDL1 + LY group was significantly lower than that in the corresponding control groups (Fig. 5 B). The above results indicate that inhibition of the TGF-β/Smad pathway can attenuate the promoting effect of SPDL1 on M2 macrophage polarization. Inhibition of the TGF-β/Smad Signaling Pathway Reduces the Synergistic Promoting Effect of M2 Macrophages on TNBC Cell Invasion and Migration To evaluate the effect of inhibiting the TGF-β/Smad pathway on the synergistic function of M2 macrophages, Western blot was first used to verify the inhibition efficiency of the TGF-β/Smad pathway after adding LY2109761. The Western blot results showed that the expression levels of TGF-β/Smad pathway-related proteins in TNBC cells in the LY group and SPDL1 + LY group were lower than those in the control group and SPDL1 overexpression group (Fig. 6 A). Subsequently, M2 macrophages obtained previously were co-cultured with TNBC cells treated with LY2109761 in an indirect co-culture system established using Transwell chambers. The regulation of M2 macrophages on the invasive and migratory abilities of TNBC cells was detected by Transwell and Western blot assays. Transwell assay showed that compared with the control group and SPDL1 overexpression group, the number of transmembrane TNBC cells in the LY group and SPDL1 + LY group was significantly reduced (Fig. 6 B); Western blot results showed that the expression levels of N-cadherin, MMP9, and Vimentin proteins in TNBC cells in the LY group and SPDL1 + LY group were lower than those in the control group and SPDL1 overexpression group (Fig. 6 C). In conclusion, inhibition of the TGF-β/Smad pathway can reduce the synergistic promoting effect of M2 macrophages on TNBC cell invasion and migration. In Vivo Experiments Demonstrated That SPDL1 Promotes TNBC Progression by Inducing IL-6 and TGF-β Secretion to Drive M2 Macrophage Polarization To verify the function of SPDL1 in vivo, a mouse model of breast cancer lung metastasis via tail vein injection was established. The mice were divided into 4 groups: Control group, oxymatrine treatment group (Oxy group, a drug that inhibits the TGF-β pathway), SPDL1 overexpression group, and SPDL1 overexpression + Oxy combined treatment group (SPDL1 + Oxy group). The detection results were as follows:Lung metastasis: Compared with the control group, the number of lung metastatic foci in the Oxy group was reduced; compared with the SPDL1 overexpression group, the number of lung metastatic foci in the SPDL1 + Oxy group was also reduced (Fig. 7 A-C); Cytokine levels: ELISA detection showed that the concentrations of IL-6 and TGF-β in the serum of mice in the SPDL1 overexpression group were significantly higher than those in the control group, while Oxy treatment could significantly reduce the concentrations of the above cytokines (Fig. 7 D); Macrophage polarization: Immunohistochemistry (IHC) staining showed that the number of positive cells for M2 macrophage markers (CD163, CD206) in the tumor tissues of mice in the SPDL1 overexpression group was significantly higher than that in the control group, while Oxy treatment could significantly reduce the number of positive cells for the above markers (Fig. 7 E); Organ safety: HE staining results showed that Oxy and SPDL1 overexpression had no obvious pathological damage to the liver, kidney, and spleen of mice (Fig. 7 F). The above results indicate that SPDL1 can promote TNBC progression in vivo and drive M2 macrophage polarization by inducing the secretion of IL-6 and TGF-β, and Oxy can inhibit this effect without obvious organ toxicity. In Vivo Experiments Demonstrated That SPDL1 Promotes TNBC Invasion and Metastasis via Activating the TGF-β/Smad Signaling Pathway To clarify the molecular mechanism by which SPDL1 regulates TNBC invasion and metastasis in vivo, Western blot was used to detect the expression of key molecules in the TGF-β/Smad pathway and EMT-related proteins in the tumor tissues of mice in each group:The results showed that compared with the control group, the expression levels of TGF-β, Smad2, P-Smad2, TGF-βRI, TGF-βRII, GLI2, and EMT-related proteins (N-cadherin, MMP9, Vimentin) in the tumor tissues of the SPDL1 overexpression group were increased; compared with the SPDL1 overexpression group, the expression levels of the above proteins in the SPDL1 + Oxy group were significantly reduced (Fig. 8 A-B). In conclusion, in the in vivo environment, SPDL1 can promote M2 macrophage polarization by activating the TGF-β/Smad signaling pathway, thereby accelerating the invasion and metastasis process of TNBC. Discussion As a malignant tumor threatening the health of women worldwide, breast cancer involves complex interactions of multiple factors in its pathological mechanisms, including gene mutations, abnormal epigenetic regulation, chronic inflammatory microenvironment, immune escape mechanisms, metabolic reprogramming, and other intricate biological processes[ 25 – 28 ]. Although innovative therapies such as targeted therapy and immune checkpoint inhibitors have significantly improved patients' survival and prognosis in recent years, the high recurrence rate and metastatic risk of advanced breast cancer remain core challenges in clinical treatment. Notably, breast cancer exhibits significant subtype heterogeneity, and the aforementioned clinical dilemmas are particularly prominent in triple-negative breast cancer (TNBC). Compared with other breast cancer subtypes, TNBC not only has higher metastatic potential and recurrence risk but also shows limited response to conventional targeted therapies and immune checkpoint inhibitors. In current clinical practice, there is still an urgent need for therapeutic approaches that can effectively address the issues of high metastasis and high recurrence in this refractory subtype[ 29 ]. As a spindle pole-associated protein, spindle and centriole-associated protein 1 (SPDL1) not only participates in mitosis and cell division processes but also exhibits pro-carcinogenic activity[ 7 ]. Overexpression of SPDL1 has been shown to be associated with poor prognosis and higher metastatic potential in breast cancer patients[ 8 ]. Its potential mechanisms include regulating microtubule stability, interfering with the invasion and migration of tumor cells, and thereby promoting tumor cells to break through blood vessel walls and form distant metastatic foci. In addition, SPDL1 may also help tumor cells grow in the microenvironment of metastatic sites by regulating immune escape[ 30 ]. Therefore, SPDL1 is expected to be a potential therapeutic target for distant metastasis of breast cancer, and further research on its mechanism of action is of great significance for the early diagnosis and targeted therapy of breast cancer. For this purpose, we first conducted a pan-cancer analysis of SPDL1 using online databases and found that SPDL1 is highly expressed in breast cancer tissues and associated with poor prognosis. Meanwhile, Mendelian randomization analysis was used to explore the association between the SPDL1 gene and breast cancer risk, and the results showed a positive correlation between the SPDL1 gene and breast cancer risk. Furthermore, a TNBC cell line with stable overexpression of SPDL1 was constructed in in vitro experiments, which confirmed that SPDL1 has the function of promoting the invasion and metastasis of TNBC. Breast cancer lung metastasis is a metastatic cascade event. During this process, TNBC cells release cytokines, proteases, exosomes, and other substances to interact and crosstalk with immune cells in the lung microenvironment, forming a TME conducive to the colonization and growth of metastatic tumors. The heterogeneity of TAMs and their interaction with the TME are key factors affecting the progression of metastasis[ 31 ]. During this cancer progression process, cancer cells escape immune surveillance and activate proliferation. At this time, TAMs in lung metastatic foci interact with cancer cells and gradually show significant M2 polarization characteristics[ 32 ], and promote the invasion and metastasis of cancer cells by secreting factors that promote epithelial-mesenchymal transition[ 33 ]. In addition, it is worth noting that specific subsets of macrophages (such as TREM2 + Mregs) are enriched in the pre-metastatic niche, providing suitable conditions for metastasis by remodeling the extracellular matrix and mediating the immunosuppressive microenvironment[ 34 ]. Therefore, intervention measures targeting TAMs show therapeutic potential. To explore whether SPDL1 can regulate TAMs in breast cancer, we found through bioinformatics analysis that SPDL1 is positively correlated with M2-TAM markers CD163 and CD206. Later, in vitro co-culture systems confirmed that SPDL1 promotes M2 macrophage polarization, and the expression of M2 surface markers increases. Studies have shown that autocrine TGF-β signaling is one of the key factors for the survival of breast cancer cells[ 35 ]. To further explore the mechanism by which SPDL1 promotes M2 macrophage polarization in TNBC cells, we performed RNA-seq and found that the TGF-β signaling pathway is enriched therein. It was also found that the expression of IL-6 and TGF-β factors was higher in the supernatant of SPDL1-overexpressing TNBC cells. TGF-β activates downstream target genes through the classic Smad2/3-Smad4 complex, and at the same time upregulates the expression of GLI2, a key transcription factor of the Hedgehog signaling pathway, through a Smad-dependent pathway, promoting its nuclear translocation. As a core effector molecule, GLI2 can directly bind to the IL-6 promoter region to promote its expression. IL-6 further upregulates the expression of TGF-β by activating the STAT3 signal, thereby forming a positive feedback loop, and at the same time induces M2 macrophage polarization to form an immunosuppressive microenvironment[ 36 ]. In addition, IL-6 and TGF-β can synergistically promote EMT of cancer cells and enhance metastatic potential. Therefore, we confirmed in vitro experiments that SPDL1 can activate the TGF-β/Smad signaling pathway and found that M2 macrophages can synergistically promote TNBC cell invasion and metastasis. Subsequently, we used the TGF-β receptor inhibitor LY2109761 to perform a rescue experiment. The study found that after inhibiting the TGF-β/Smad signaling pathway, the polarization of M2 macrophages and the oncogenic activity promoted by SPDL1 were downregulated. To verify this finding, we also established a breast cancer lung metastasis model in mice. The experimental results showed that SPDL1 can promote M2 macrophage polarization and breast cancer lung metastasis in breast cancer through the TGF-β/Smad signaling pathway, and this result was inhibited after adding the in vivo TGF-β receptor inhibitor oxymatrine. In conclusion, our research results reveal a new mechanism by which SPDL1 drives breast cancer metastasis by regulating the phenotypic transformation of immune cells in the tumor microenvironment. Specifically, SPDL1 can induce the phenotypic transformation of M2-type TAMs and form a pro-metastatic positive feedback regulatory network by activating the TGF-β/Smad signaling axis of tumor cells and its downstream GLI2-dependent IL-6 and TGF-β autocrine loops, ultimately mediating the formation of distant organ metastatic foci. This finding clarifies the molecular biological significance of the SPDL1-TGFβ/Smad/GLI2-IL-6/TGF-β signal cascade from the perspective of tumor-immune interaction and provides key molecular targets for the development of therapeutic strategies targeting the tumor microenvironment. Declarations Conflict of interest The authors declare no competing interests. Ethical approval All animal experiments were conducted in accordance with guidelines and protocol approved by the institutional animal care and use committee of Putuo Hospital, Shanghai University of Traditional Chinese Medicine, China. (DWEC-A-2024-18-1-88). Consent for publication All authors have approved submission of the manuscript to this journal. Funding: This work was supported by National Natural Science Foundation of China (81973625); Key Medical Discipline Project of Shanghai Municipal Health Bureau(2024ZDXK0046); Science, Technology Innovation Project of Putuo District Health System (No. ptkwws202307); The One Hundred Talents Project of Putuo Hospital, Shanghai University of Traditional Chinese Medicine (2022-RCLH-03)and the Shen Hongquan’s Academic Experience Research Studio of Shanghai Famous Veteran TCM Expert (SHGZS-202224). Author Contribution Jie Wang, Wei Li and Fenfen Xiang con-ceived the original idea and designed the study. 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Cancer Sci 114:2429–2444. https://doi.org/10.1111/cas.15791 Zhao B, Yu X, Shi J et al (2024) A stepwise mode of TGFβ-SMAD signaling and DNA methylation regulates naïve-to-primed pluripotency and differentiation. Nat Commun 15:10123. https://doi.org/10.1038/s41467-024-54433-5 Additional Declarations No competing interests reported. Supplementary Files Onlinefloatimage1.png Fig.S1 SPDL1 is highly expressed in breast cancer and associated with poor prognosis. (A,B) The expression differences of SPDL1 in paired and unpaired samples across various cancers. (C) The expression differences of SPDL1 in paired and unpaired breast cancer samples. (D) Forest plot and scatter plot of the Mendelian randomization analysis of SPDL1 and breast tumors. (E) Expression Difference Radar Chart of SPDL1 in Various Cancers. (F) Prognostic analysis of SPDL1 in cases of high expression and low expression. Results are expressed as mean ± SD. *P < 0.05, **P < 0.01, **P < 0.001 Onlinefloatimage3.png Fig.S2 SPDL1 expression was positively correlated with M2 macrophage infiltration. (A) The correlation heatmap between SPDL1 and immune cells. (B) The correlation between SPDL1 and six common types of immune cells. (C) The correlation between SPDL1 and M2 macrophage markers (CD163, CD206) was analyzed in both normal and tumor tissues. Results are expressed as mean ± SD. P < 0.05 Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 04 Dec, 2025 Reviews received at journal 04 Nov, 2025 Reviewers agreed at journal 03 Nov, 2025 Reviewers invited by journal 24 Oct, 2025 Editor assigned by journal 23 Oct, 2025 Submission checks completed at journal 23 Oct, 2025 First submitted to journal 21 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7913702","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":539556171,"identity":"ab47b852-96b3-4ef5-933d-2e71d36f877e","order_by":0,"name":"Zeng you Xiao","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zeng","middleName":"you","lastName":"Xiao","suffix":""},{"id":539556173,"identity":"515239b4-202d-4249-97eb-321ede7929b7","order_by":1,"name":"Dongxiao Shen","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese 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1","display":"","copyAsset":false,"role":"figure","size":4119952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSPDL1 can promote the invasion and migration abilities of TNBC cells. (A,B) The transfection efficiency of the SPDL1 virus was measured using Western blot and qRT-PCR. (C) The effect of SPDL1 on TNBC cell migration was examined using a wound healing assay. (D) The migratory and invasive capacities of TNBC cells were examined using a Transwell assay following SPDL1 treatment. Results are expressed as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, **P \u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/3479c8dbd9507e7eb086b474.png"},{"id":95313327,"identity":"b6fa2ed8-6175-46ad-b116-79630b16606b","added_by":"auto","created_at":"2025-11-06 15:51:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2850714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSPDL1 drives M2 macrophage polarization by promoting the secretion of IL-6 and TGF-β from TNBC cells. (A) Representative microscopic images of macrophages cultured in conditioned medium from TNBC cells. Scale bar: 20 μm. (B) The mRNA expression levels of IL-10 and TGF-β in TNBC cells from different groups were detected by qRT-PCR. (C) The frequency of M2 markers (CD11b and CD206) in macrophages cultured with conditioned medium (CM) from different groups was analyzed by flow cytometry. (D) The concentrations of IL-6 and TGF-β in the supernatant of TNBC cells from different groups were measured by ELISA. Results are expressed as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/6860b4f0ba7dce6be6c71f79.png"},{"id":95260387,"identity":"cd034a03-ff41-4acf-8cc3-f168b5ba4739","added_by":"auto","created_at":"2025-11-06 04:17:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4804698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eM2 macrophages synergistically enhanced the invasive and migratory capacities of TNBC cells. (A,B) The invasive and migratory capacities of TNBC cells from different groups were detected using the Transwell assay. (C) The protein expression levels of N-cadherin, MMP9, and Vimentin in TNBC cells from different groups were detected by Western blot analysis. Results are expressed as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ***P \u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/df2249430c9ed41cf761fe31.png"},{"id":95260382,"identity":"26997bff-a0df-4453-838e-3c6971cb61c6","added_by":"auto","created_at":"2025-11-06 04:17:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3690087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSPDL1 activates the TGF-β/Smad signaling pathway in TNBC cells. (A,B) Functional enrichment plots of GO and KEGG analyses were generated from the transcriptome sequencing data of MDA-MB-231 cells. (C) GSEA plot of TGF-β signaling pathway from transcriptome sequencing in MDA-MB-231 cells. (D) Expression levels of TGF-β/Smad pathway proteins were measured using Western blot in various TNBC cell groups. (E) Immunofluorescence detection of TGF-β and GLI2 expression in the TGF-β/Smad pathway across TNBC cell groups. Results are expressed as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, **P \u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/5274dc749bd2d0af179a1dcf.png"},{"id":95260390,"identity":"f4fee02a-9db1-4752-bc6d-f79d8b018cea","added_by":"auto","created_at":"2025-11-06 04:17:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5164592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of the TGF-β/Smad signaling pathway attenuates SPDL1-induced promotion of M2 macrophage polarization. (A) Representative images of macrophages cultured in conditioned medium from inhibitor-treated TNBC cells. Scale bar: 20 μm. (B) Flow cytometric analysis of CD11b and CD206 expression in M2 macrophages cultured in conditioned medium from inhibitor-treated TNBC cells. Results are expressed as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, **P \u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/d44a873bc5e695656ae74c32.png"},{"id":95313365,"identity":"400d6bd5-7e36-4d77-bde8-490b16c6d545","added_by":"auto","created_at":"2025-11-06 15:51:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5189796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of the TGF-β/Smad signaling pathway reduces the synergistic promoting effect of M2 macrophages on TNBC cell invasion and migration. (A) The expression levels of TGF-β/Smad pathway-related proteins were analyzed by Western blot in inhibitor-treated TNBC cells from different groups. (B) The number of transmembrane cells in TNBC invasion and migration was quantified by Transwell assay across different groups. (C) The expression levels of N-cadherin, MMP9, and Vimentin proteins were detected by Western blot in TNBC cells from different groups. Results are expressed as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, **P \u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/f25b442ec5429b0722d4a2f9.png"},{"id":95260388,"identity":"549ddedf-21a5-4e6e-b82b-f7406ec2eb9d","added_by":"auto","created_at":"2025-11-06 04:17:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":667351,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo experiments demonstrated that SPDL1 promoted TNBC progression by inducing IL-6 and TGF-β secretion to drive M2 macrophage polarization. (A, B) [1] Macroscopic morphology of in vivo pulmonary metastatic foci. (C) Lung Weight and Number of Pulmonary Metastatic Nodules in Different Groups. (D) The concentrations of IL-6 and TGF-β were measured by ELISA in different groups of mice. (E) Expression levels of SPDL1 and M2 macrophage markers (CD163, CD206) were detected by IHC in different groups of mice. (F) Assessment of toxicity on the liver, kidney, and spleen was performed by HE staining in different groups of mice treated with SPDL1 and Oxy. Results are expressed as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, **P \u0026lt; 0.001\u003c/p\u003e\n\u003cp\u003eB图可以再放大一点\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/0c7275d3433078dfee0b38f8.png"},{"id":95260385,"identity":"07ab626c-ca3c-4b77-8f22-b84e2a3e49fb","added_by":"auto","created_at":"2025-11-06 04:17:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":207082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo experiments demonstrated that SPDL1 promotes TNBC invasion and metastasis via activating the TGF-β/Smad signaling pathway. (A) Expression levels of TGF-β/Smad pathway-related proteins were detected by Western blot in vivo in different groups of mice. (B) Expression levels of invasion and metastasis-related proteins were examined by Western blot in vivo in different groups of mice. Results are expressed as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ***P \u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/c048868a08eed210e823eeb1.png"},{"id":95315643,"identity":"620a48d7-aab1-4b31-8833-fe68e96ad3e3","added_by":"auto","created_at":"2025-11-06 15:56:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7196704,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/3621f189-6ff7-418e-9689-742683566fdb.pdf"},{"id":95260381,"identity":"58fc5dd8-2676-4564-865a-f605080a955e","added_by":"auto","created_at":"2025-11-06 04:17:37","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1404008,"visible":true,"origin":"","legend":"\u003cp\u003eFig.S1\u003cstrong\u003e SPDL1 is highly expressed in breast cancer and associated with poor prognosis. (A,B) The expression differences of SPDL1 in paired and unpaired samples across various cancers. (C) The expression differences of SPDL1 in paired and unpaired breast cancer samples. (D) Forest plot and scatter plot of the Mendelian randomization analysis of SPDL1 and breast tumors. (E) Expression Difference Radar Chart of SPDL1 in Various Cancers. (F) Prognostic analysis of SPDL1 in cases of high expression and low expression. Results are expressed as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, **P \u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/b096909846c2ff8e82a29f18.png"},{"id":95260386,"identity":"24af51c0-8b65-4005-888d-1ae281dea096","added_by":"auto","created_at":"2025-11-06 04:17:37","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4912396,"visible":true,"origin":"","legend":"\u003cp\u003eFig.S2\u003cstrong\u003e SPDL1 expression was positively correlated with M2 macrophage infiltration. (A) The correlation heatmap between SPDL1 and immune cells. (B) The correlation between SPDL1 and six common types of immune cells. (C) The correlation between SPDL1 and M2 macrophage markers (CD163, CD206) was analyzed in both normal and tumor tissues. Results are expressed as mean ± SD. P \u0026lt; 0.05\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7913702/v1/ef2f1a2a28ec8f0ba6eab50a.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"SPDL1 Promotes Lung Metastasis of Triple-Negative Breast Cancer by Targeting the TGF-β/Smad Pathway to Regulate M2 Macrophage Polarization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDistant metastasis of breast cancer is the leading cause of death in female patients[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although current clinical treatments can effectively control the primary tumor, the rapid deterioration of the disease often occurs after lung metastasis, making it a major challenge in clinical treatment[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among these cases, the incidence of lung metastasis in patients with triple-negative breast cancer (TNBC) is as high as 21%-32%, and the lung is one of the most common target organs for distant metastasis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Studies have shown that the progression of breast cancer lung metastasis is closely associated with driver gene mutations, such as BRCA1/2 deletion, HER2 amplification, and TP53 inactivation[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These mutations further exacerbate disease progression and lead to poor prognosis in patients by mechanisms including enhancing the invasive and metastatic potential of tumor cells, remodeling the pre-metastatic niche in the lung, inducing immune suppression, and promoting therapeutic resistance[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notably, the molecular regulatory network underlying the directional metastatic propensity of breast cancer cells to lung tissue (\"lung tropism\") remains largely unclear, and its potential mechanism urgently requires in-depth investigation.\u003c/p\u003e\u003cp\u003eThe human SPDL1 gene is located in the 5q35.1 region of the chromosome, and its encoded product, Spindly protein, can maintain genomic stability. It also acts synergistically with the RZZ complex (Rod/ZW10/Zwilch) in the centromere region and is involved in regulating the signaling network related to cell migration[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Previous studies by our research group found that SPDL1 not only participates in core events of the cell cycle but also exhibits oncogenic properties in various malignant tumors such as lung cancer and oral cancer[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Notably, SPDL1 has been identified as a novel independent prognostic marker for TNBC[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the mechanism of action of SPDL1 in breast cancer lung metastasis remains an unknown field, especially whether SPDL1 can affect the progression of breast cancer by regulating the tumor microenvironment has not been reported yet. Therefore, systematic analysis of the multi-dimensional regulatory network of SPDL1 in breast cancer lung metastasis will provide a key theoretical basis for the development of targeted intervention strategies.\u003c/p\u003e\u003cp\u003eTAMs are the most abundant population of tumor-infiltrating immune cells in the TME. During tumor progression, the number of M2-like TAMs increases sharply, becoming the dominant cell population of TAMs in the TME, and at the same time secrete anti-inflammatory factors such as IL-10 and TGF-β[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. They exhibit pro-tumor activities by promoting the formation of the pre-metastatic niche (PMN), regulating the immunosuppressive microenvironment, promoting angiogenesis, and adjusting energy metabolism[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Studies have found that exosomes derived from breast cancer carry molecules such as miR-138-5p, which induce M2 macrophage polarization by inhibiting KDM6B, thereby affecting the process of tumor metastasis. This reveals the crosstalk between macrophages and tumor cells in the progression of breast cancer[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn breast cancer, the abnormal activation of the TGF-β/Smad pathway is closely related to disease progression, drug resistance, and poor prognosis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the early stage of breast cancer, TGF-β activates the Smad2/3-Smad4 complex, inhibits the expression of cyclins, and induces cell apoptosis, thereby exerting a tumor-suppressive effect[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, as the TME changes, cancer cells escape via gene mutations or epigenetic modifications and exert an oncogenic effect. Studies have found that inactivating mutations in TGF-β receptor I and II or Smad4 lead to off-target effects, which in turn promote epithelial-mesenchymal transition (EMT), angiogenesis, and immune escape, accelerating breast cancer metastasis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. GLI2 is a direct downstream target of the TGF-β pathway and is independent of the Hedgehog signaling axis[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Studies have found that GLI2 plays a direct functional role in solid tumors; GLI2 knockout can inhibit the proliferation of hepatocellular carcinoma cells and reduce the invasiveness of melanoma cells by downregulating E-cadherin[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In conclusion, the key role of GLI2 downstream of TGF-β signaling in promoting cancer progression toward metastasis has been documented in different cancer types. Interestingly, GLI2 also mediates the inflammatory regulation of the TME through cytokines such as IL-6 and TGF-β [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Both IL-6 and TGF-β can regulate the polarization of M2-type TAMs in the TME, thereby promoting cancer progression[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It has also been reported that IL-6 and TGF-β are directly related to the poor prognosis of TNBC[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTherefore, this study intends to clarify that SPDL1 mediates M2 macrophage polarization by regulating the TGF-β/Smad pathway, thereby promoting the process of TNBC lung metastasis. This finding may provide a novel molecular intervention strategy and theoretical basis for the targeted treatment of metastatic breast cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData Collection\u003c/h2\u003e\u003cp\u003eThe pan-cancer gene expression, prognostic, and immune analysis data used in this study were all obtained from The Cancer Genome Atlas (TCGA) pan-cancer database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e); the immunohistochemical protein expression analysis data for SPDL1, CD163, and CD206 were retrieved from the Human Protein Atlas (HPA) web platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org/\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e); for the Mendelian randomization study, expression quantitative trait locus (eQTL) data of human breast tissue genes were downloaded from the GTEx Portal website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gtexportal.org/home/\u003c/span\u003e\u003cspan address=\"https://www.gtexportal.org/home/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and breast tumor disease data were screened from the FinnGen database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.finngen.fi/en/access_results\u003c/span\u003e\u003cspan address=\"https://www.finngen.fi/en/access_results\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These two types of data were combined for subsequent analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eHuman triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were purchased from ATCC (USA), and the human monocytic leukemia cell line THP-1 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 and MDA-MB-468 cells were cultured in L15 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). THP-1 cells and their derived macrophages were cultured in RPMI-1640 medium containing 1% penicillin-streptomycin and 20% FBS. All cells were maintained in an incubator at 37\u0026deg;C with 5% CO₂.\u003c/p\u003e\n\u003ch3\u003eCollection of Conditioned Medium\u003c/h3\u003e\n\u003cp\u003eFor the collection of conditioned medium from triple-negative breast cancer (TNBC) cells, we seeded MDA-MB-231 or MDA-MB-468 cells in 6-well plates at a density of 1\u0026times;10⁶ cells/mL. After allowing the cells to adhere for 24 hours, the medium in each well was replaced according to the experimental conditions: either 2 mL of serum-free RPMI 1640 medium, or serum-free medium supplemented with 10 \u0026micro;mol/L of the TGF-β receptor inhibitor LY2109761. Following an additional 24-hour starvation culture, the conditioned medium was collected according to the experimental groups.\u003c/p\u003e\n\u003ch3\u003eEstablishment of the Co-Culture System\u003c/h3\u003e\n\u003cp\u003eFor the co-culture system of macrophages and TNBC cells, Transwell chambers were used for indirect co-culture. TNBC cells were seeded in the lower chamber of 24-well plates (3\u0026times;10⁵ cells/well), and THP-1-derived M2 macrophages were seeded in the upper chamber of 24-well plates (3\u0026times;10⁵ cells/well), with complete L15 medium added to both chambers. Control and experimental groups were set as follows: Control group: TNBC cells cultured alone; Experimental Group 1: SPDL1-overexpressing TNBC cells cultured alone; Experimental Group 2: TNBC cells co-cultured with M2 macrophages; Experimental Group 3: SPDL1-overexpressing TNBC cells co-cultured with M2 macrophages. After 48 hours of culture, TNBC cells were collected for Transwell and Western blot assays to detect the invasive and migratory abilities of TNBC cells.\u003c/p\u003e\n\u003ch3\u003eCell Scratch Healing Assay\u003c/h3\u003e\n\u003cp\u003eMDA-MB-231 and MDA-MB-468 cells in the logarithmic growth phase were digested with 0.25% trypsin and seeded in 6-well plates at a density of 5\u0026times;10⁵ cells/well. After 24 hours of culture, a horizontal line was drawn on the back of the culture plate using a 200 \u0026micro;L pipette tip, and then the cells were washed three times with PBS. Three random areas were selected for imaging observation, and another three random areas were selected for imaging observation 24 hours later to track the cell migration process in real time.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell Migration and Invasion Assay\u003c/h2\u003e\u003cp\u003eAfter digestion and resuspension, the cells were resuspended again with serum-free L15 medium and counted to 3\u0026times;10⁵ cells/mL using a cell counting chamber. 700 \u0026micro;L of medium containing 20% FBS was added to the lower chamber of a 24-well Transwell plate (Corning, New York, USA), and 300 \u0026micro;L of the cell suspension was added to the upper chamber of the Transwell. After incubation at 37\u0026deg;C for 48 hours, crystal violet staining was performed. The cells were washed and fixed with a fixative for 15 minutes, and the cells on the inner wall of the chamber were gently wiped off with a cotton swab. Then, the number of cells on the outer wall of the chamber was counted. In the invasion assay, 200 mg/mL Matrigel TM (BD, Sparks, MD, USA) was added to the bottom of the upper chamber of the Transwell, and the other operation steps were the same as above. Each experiment was repeated three times, and the average value was taken.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cp\u003eAfter washing the cells with PBS, cell lysis buffer was added, and the cells were incubated on ice for 15 minutes. The cells were thoroughly scraped off with a cell scraper, and the lysate was collected into EP tubes. The samples were ultrasonically disrupted and then centrifuged to determine the protein concentration. After denaturation of the protein in a metal bath, Western blot experiments were performed, including sample loading, electrophoresis, and membrane transfer. After membrane transfer, the membrane was blocked with a rapid blocking solution for 10 minutes and incubated with primary antibodies at 4\u0026deg;C overnight. The next day, the corresponding secondary antibodies were added, and after incubation, the membrane was developed with a developing solution. The TGF-β antibody, Smad2 antibody, P-Smad2 antibody, and Vimentin antibody used were purchased from CST Company; TGF-βRII antibody, TGF-βRI antibody, and MMP9 antibody were purchased from Wanlei Company; GLI2 antibody was purchased from Sanying Company; N-cadherin antibody and SPDL1 antibody were purchased from Abcam Company; secondary antibodies and GAPDH antibody were purchased from Aibotai Company.\u003c/p\u003e\n\u003ch3\u003eFlow Cytometry\u003c/h3\u003e\n\u003cp\u003eAfter macrophage polarization, cells from each group were collected and resuspended in PBS for macrophage detection: 1\u0026times;10⁶ cells were taken, and CD11b-phycoerythrin (PE) and CD206-fluorescein isothiocyanate (FITC) antibodies (BD Pharmingen Company) were added, followed by incubation at 4\u0026deg;C in the dark for 30 minutes. A flow cytometer was used for detection, and the sample results were analyzed.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative RT-PCR\u003c/h2\u003e\u003cp\u003eCell samples were collected, and total RNA was extracted using an RNA extraction kit from Accurate Biotechnology (Hunan) Co., Ltd. The concentration and purity of RNA were determined; cDNA synthesis was performed using a reverse transcription kit from the same company. The primer sequences used are as follows:\u003c/p\u003e\u003cp\u003eH-TGF-β:5\u0026prime;-GGGACTATCCACCTGCAAGA-3\u0026prime;,5\u0026prime;-CCTCCTTGGCGTAGTAGTCG-3\u0026prime;;H-IL-10:5\u0026prime;-TGCCTTCAGCAGAGTGAAGA-3\u0026prime;,5\u0026prime;-GGTCTTGGTTCTCAGCTTGG-3\u0026prime;;H-SPDL1:5\u0026prime;-CCCCTAACTCTCCCAGGTCA-3\u0026prime;,5\u0026prime;-CATGCAGCGAGAGAGGAGAG-3\u0026prime;;H-GAPDH:5\u0026prime;-TGTGGGCATCAATGGATTTGG-3\u0026prime;,5\u0026prime;-ACACCATGTATTCCGGGTCAAT-3\u0026prime;.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eELISA\u003c/h2\u003e\u003cp\u003e Animal serum or cell culture supernatant was collected, and the secretion levels of IL-6 and TGF-β were detected according to the instructions of the corresponding ELISA kit from Boster Biological Engineering Co., Ltd. (Wuhan, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAnimal Models\u003c/h2\u003e\u003cp\u003eMDA-MB-231 and 231SPDL1-OE cells were injected into the tail vein of 5-week-old female Balb/c mice (purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd.) at a concentration of 1\u0026times;10⁷ cells/mL. Each mouse was injected with 200 \u0026micro;L of cell suspension to establish a breast cancer lung metastasis model. Seven days after inoculation, the mice were divided into 4 groups: Control group: intragastric administration of 200 \u0026micro;L normal saline once a day; Oxy treatment group: intragastric administration of 120 mg/kg oxymatrine once a day; SPDL1 overexpression group: intragastric administration of 200 \u0026micro;L normal saline once a day; SPDL1 overexpression combined with Oxy treatment group: intragastric administration of 120 mg/kg oxymatrine once a day. From day 7, the mice were weighed every other day. Two weeks after administration, all animals were sacrificed, and the number of lung metastatic tumors and the weight of the lungs were counted. Some tissues were fixed with formalin, and some were frozen in liquid nitrogen for subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e\u003cp\u003eTissues dissected from animals were fixed with 10% formalin and then embedded in paraffin for sectioning. The sections were dewaxed with xylene and ethanol, and antigen retrieval was performed with 0.01 M citrate buffer at 98\u0026deg;C. After blocking with bovine serum albumin (BSA), the sections were incubated with primary antibodies at 4\u0026deg;C overnight. After washing with PBS, the sections were incubated with secondary antibodies at room temperature for 1 hour. After washing again, the sections were incubated with streptavidin-horseradish peroxidase (SABC) for 30 minutes. Finally, 3,3'-diaminobenzidine (DAB) was used for color development, and hematoxylin was used for counterstaining.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eHematoxylin and Eosin (HE) Staining\u003c/h2\u003e\u003cp\u003eTo examine various tissues of the mice, hematoxylin-eosin (HE) staining was used. Mouse tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and then sectioned at a thickness of 5 \u0026micro;m to observe the overall morphology. The samples were then hydrated through gradient concentrations of alcohol and dewaxed with xylene. The sections were first stained with hematoxylin for five minutes and then with eosin for one minute. Subsequently, the tissues were dehydrated with gradient concentrations of alcohol and then treated with xylene. Finally, each section was sealed and observed under a \u0026times;200 optical microscope (Olympus, Tokyo, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eGraphPad Prism 8 software was used for statistical analysis, and the experimental data were expressed as \"mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\". Unpaired t-test was used for comparison between two groups, and one-way analysis of variance (one-way ANOVA) was used for comparison among multiple groups. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 were considered to indicate statistically significant differences.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eSPDL1 is Highly Expressed in Breast Cancer and Associated with Poor Prognosis\u003c/h2\u003e\u003cp\u003eTo clarify the expression characteristics and clinical significance of SPDL1 in cancer, a pan-cancer analysis was first conducted: samples of 33 cancer types were collected from the TCGA database for analysis. The results showed that there were significant differences in the expression of SPDL1 among different cancer types (Supplementary Fig.\u0026nbsp;1A-B), and it was highly expressed in breast cancer (Supplementary Fig.\u0026nbsp;1C, E). A Mendelian randomization study found that the expression level of SPDL1 was positively correlated with the risk of breast tumor, i.e., the higher the expression of SPDL1, the higher the risk of onset (Supplementary Fig.\u0026nbsp;1D). Prognostic analysis results confirmed that high expression of SPDL1 was positively correlated with poor prognosis of breast cancer patients within a certain period (Supplementary Fig.\u0026nbsp;1F). In conclusion, SPDL1 may play an oncogenic role in triple-negative breast cancer (TNBC) and participate in the disease progression of TNBC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eSPDL1 Promotes the Invasive and Migratory Capacities of TNBC Cells\u003c/h2\u003e\u003cp\u003eTo explore the effect of SPDL1 on the biological functions of TNBC cells, an SPDL1-overexpressing lentivirus was first constructed and transfected into TNBC cells. Western blot (WB) and qRT-PCR experiments were used to verify the transfection efficiency. The results showed that the protein and mRNA expression levels of SPDL1 in the transfected cells were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B).\u003c/p\u003e\u003cp\u003eWound healing assay and Transwell assay were used to detect cell migration and invasion abilities: compared with the control group, the wound healing rate of TNBC cells in the SPDL1 overexpression group was significantly accelerated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and the number of transmembrane cells in the Transwell chamber was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The above results indicate that SPDL1 can significantly enhance the invasive and migratory capacities of TNBC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSPDL1 Expression is Positively Correlated with M2 Macrophage Infiltration\u003c/h2\u003e\u003cp\u003eM2 macrophages play a key regulatory role in the progression of TNBC. Based on this, immunotherapy targeting macrophages has potential application value in the treatment of TNBC. To explore the association between SPDL1 and M2 macrophages, bioinformatics analysis (including gene expression correlation analysis and immune cell infiltration scoring) showed that the expression level of SPDL1 was positively correlated with the infiltration degree of macrophages (Supplementary Fig.\u0026nbsp;2A-B). Moreover, analysis using the Human Protein Atlas found that SPDL1 was significantly positively correlated with M2 macrophages (Supplementary Fig.\u0026nbsp;2C), suggesting that there may be a functional synergistic effect between them.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSPDL1 Drives M2 Macrophage Polarization by Promoting the Secretion of IL-6 and TGF-β from TNBC Cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTumor cells in the tumor microenvironment (TME) can regulate macrophage polarization by secreting cytokines. To clarify the effect of SPDL1 on M2 macrophage polarization, the supernatant of TNBC cells was collected according to the experimental groups to prepare conditioned medium (CM), which was then co-cultured with M0 macrophages induced and differentiated from THP-1 cells for detection: Morphological observation showed that compared with the control group (CM from untreated TNBC cells), the CM from TNBC cells in the SPDL1 overexpression group could induce more M0 macrophages to exhibit the typical elongated spindle shape of M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA); qRT-PCR detection results showed that after co-culture with TNBC cells in the SPDL1 overexpression group, the mRNA expression levels of M2 markers (IL-10, TGF-β) in macrophages were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB); flow cytometry verification showed that the proportion of CD11b⁺CD206⁺ cells (M2 phenotypic markers) on the surface of macrophages in the above co-culture system was significantly higher than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).Given that IL-6 and TGF-β are known key regulatory factors for M2 macrophage polarization, the secretory function of TNBC cells was detected by ELISA: compared with the control group, the concentrations of IL-6 and TGF-β secreted by TNBC cells in the SPDL1 overexpression group were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In conclusion, SPDL1 may induce macrophage polarization toward the M2 type by promoting the secretion of IL-6 and TGF-β from TNBC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eM2 Macrophages Synergistically Enhance the Invasive and Migratory Capacities of TNBC Cells\u003c/h2\u003e\u003cp\u003ePrevious studies have confirmed that M2 macrophages can promote the malignant progression of TNBC[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To verify this effect, M2 macrophages induced by co-culture with TNBC cells were collected, and an indirect co-culture system was established using Transwell chambers for co-culture with TNBC cells. Detection results showed: Transwell assay showed that compared with TNBC cells not exposed to M2 macrophages, the number of transmembrane TNBC cells co-cultured with M2 macrophages was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B); Western blot detection showed that the expression levels of epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, MMP9, Vimentin) in TNBC cells in the above co-culture group were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The above results indicate that M2 macrophages can synergistically enhance the invasive and migratory capacities of TNBC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eSPDL1 Activates the TGF-β/Smad Signaling Pathway in TNBC Cells\u003c/h2\u003e\u003cp\u003eThe TGF-β signaling pathway plays a core regulatory role in the progression of TNBC. To clarify the association between SPDL1 and this pathway, transcriptome sequencing was performed on MDA-MB-231 cells (a TNBC cell line). GO functional annotation and KEGG pathway enrichment analysis results showed that the TGF-β signaling pathway was significantly enriched in the SPDL1 overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). The GSEA plot showed that the TGF-β signaling pathway was upregulated in the experimental group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Previous studies have shown that GLI2, a downstream factor of the TGF-β/Smad signaling axis, can target and regulate the production of IL-6 and TGF-β. Based on this, it is hypothesized that SPDL1 may promote the expression of GLI2 by activating the TGF-β/Smad pathway, thereby increasing the secretion of IL-6 and TGF-β to promote M2 macrophage polarization and TNBC progression. To verify this mechanism: Western blot detection was performed, and the results showed that the protein expression levels of key molecules in the TGF-β/Smad pathway (TGF-β, Smad2, phosphorylated Smad2 (P-Smad2), TGF-β receptor I (TGF-βRI), TGF-β receptor II (TGF-βRII)) and the downstream factor GLI2 in TNBC cells in the SPDL1 overexpression group were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD); immunofluorescence experiment showed that the fluorescence intensity of TGF-β and GLI2 in cells in the SPDL1 overexpression group was significantly stronger than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In conclusion, SPDL1 can promote the secretion of IL-6 and TGF-β by activating the TGF-β/Smad signaling pathway in TNBC cells, thereby promoting the polarization of M2-type tumor-associated macrophages (TAMs) in the TME and the invasion and metastasis of TNBC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eInhibition of the TGF-β/Smad Signaling Pathway Attenuates SPDL1-Induced Promotion of M2 Macrophage Polarization\u003c/h2\u003e\u003cp\u003eLY2109761 is a dual inhibitor of TGF-βRI and TGF-βRII, which can inhibit the activity of the TGF-β/Smad signaling pathway by downregulating the phosphorylation level of Smad2. To evaluate the effect of inhibiting the TGF-β/Smad pathway on SPDL1-regulated M2 macrophage polarization, TNBC cells were divided into 4 groups: Control group, LY2109761 treatment group (LY group), SPDL1 overexpression group, and SPDL1 overexpression\u0026thinsp;+\u0026thinsp;LY2109761 combined treatment group (SPDL1\u0026thinsp;+\u0026thinsp;LY group). The CM from TNBC cells from the above groups were collected and added to M0 macrophages. Morphological observation showed that the number of macrophages showing M2 morphology in the LY group and SPDL1\u0026thinsp;+\u0026thinsp;LY group was significantly less than that in the control group and SPDL1 overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Flow cytometry showed that the proportion of CD11b⁺CD206⁺ cells (M2 markers) in macrophages in the LY group and SPDL1\u0026thinsp;+\u0026thinsp;LY group was significantly lower than that in the corresponding control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The above results indicate that inhibition of the TGF-β/Smad pathway can attenuate the promoting effect of SPDL1 on M2 macrophage polarization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eInhibition of the TGF-β/Smad Signaling Pathway Reduces the Synergistic Promoting Effect of M2 Macrophages on TNBC Cell Invasion and Migration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the effect of inhibiting the TGF-β/Smad pathway on the synergistic function of M2 macrophages, Western blot was first used to verify the inhibition efficiency of the TGF-β/Smad pathway after adding LY2109761. The Western blot results showed that the expression levels of TGF-β/Smad pathway-related proteins in TNBC cells in the LY group and SPDL1\u0026thinsp;+\u0026thinsp;LY group were lower than those in the control group and SPDL1 overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequently, M2 macrophages obtained previously were co-cultured with TNBC cells treated with LY2109761 in an indirect co-culture system established using Transwell chambers. The regulation of M2 macrophages on the invasive and migratory abilities of TNBC cells was detected by Transwell and Western blot assays. Transwell assay showed that compared with the control group and SPDL1 overexpression group, the number of transmembrane TNBC cells in the LY group and SPDL1\u0026thinsp;+\u0026thinsp;LY group was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB); Western blot results showed that the expression levels of N-cadherin, MMP9, and Vimentin proteins in TNBC cells in the LY group and SPDL1\u0026thinsp;+\u0026thinsp;LY group were lower than those in the control group and SPDL1 overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In conclusion, inhibition of the TGF-β/Smad pathway can reduce the synergistic promoting effect of M2 macrophages on TNBC cell invasion and migration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vivo Experiments Demonstrated That SPDL1 Promotes TNBC Progression by Inducing IL-6 and TGF-β Secretion to Drive M2 Macrophage Polarization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify the function of SPDL1 in vivo, a mouse model of breast cancer lung metastasis via tail vein injection was established. The mice were divided into 4 groups: Control group, oxymatrine treatment group (Oxy group, a drug that inhibits the TGF-β pathway), SPDL1 overexpression group, and SPDL1 overexpression\u0026thinsp;+\u0026thinsp;Oxy combined treatment group (SPDL1\u0026thinsp;+\u0026thinsp;Oxy group). The detection results were as follows:Lung metastasis: Compared with the control group, the number of lung metastatic foci in the Oxy group was reduced; compared with the SPDL1 overexpression group, the number of lung metastatic foci in the SPDL1\u0026thinsp;+\u0026thinsp;Oxy group was also reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C); Cytokine levels: ELISA detection showed that the concentrations of IL-6 and TGF-β in the serum of mice in the SPDL1 overexpression group were significantly higher than those in the control group, while Oxy treatment could significantly reduce the concentrations of the above cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eD); Macrophage polarization: Immunohistochemistry (IHC) staining showed that the number of positive cells for M2 macrophage markers (CD163, CD206) in the tumor tissues of mice in the SPDL1 overexpression group was significantly higher than that in the control group, while Oxy treatment could significantly reduce the number of positive cells for the above markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eE); Organ safety: HE staining results showed that Oxy and SPDL1 overexpression had no obvious pathological damage to the liver, kidney, and spleen of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). The above results indicate that SPDL1 can promote TNBC progression in vivo and drive M2 macrophage polarization by inducing the secretion of IL-6 and TGF-β, and Oxy can inhibit this effect without obvious organ toxicity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vivo Experiments Demonstrated That SPDL1 Promotes TNBC Invasion and Metastasis via Activating the TGF-β/Smad Signaling Pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo clarify the molecular mechanism by which SPDL1 regulates TNBC invasion and metastasis in vivo, Western blot was used to detect the expression of key molecules in the TGF-β/Smad pathway and EMT-related proteins in the tumor tissues of mice in each group:The results showed that compared with the control group, the expression levels of TGF-β, Smad2, P-Smad2, TGF-βRI, TGF-βRII, GLI2, and EMT-related proteins (N-cadherin, MMP9, Vimentin) in the tumor tissues of the SPDL1 overexpression group were increased; compared with the SPDL1 overexpression group, the expression levels of the above proteins in the SPDL1\u0026thinsp;+\u0026thinsp;Oxy group were significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). In conclusion, in the in vivo environment, SPDL1 can promote M2 macrophage polarization by activating the TGF-β/Smad signaling pathway, thereby accelerating the invasion and metastasis process of TNBC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a malignant tumor threatening the health of women worldwide, breast cancer involves complex interactions of multiple factors in its pathological mechanisms, including gene mutations, abnormal epigenetic regulation, chronic inflammatory microenvironment, immune escape mechanisms, metabolic reprogramming, and other intricate biological processes[\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although innovative therapies such as targeted therapy and immune checkpoint inhibitors have significantly improved patients' survival and prognosis in recent years, the high recurrence rate and metastatic risk of advanced breast cancer remain core challenges in clinical treatment. Notably, breast cancer exhibits significant subtype heterogeneity, and the aforementioned clinical dilemmas are particularly prominent in triple-negative breast cancer (TNBC). Compared with other breast cancer subtypes, TNBC not only has higher metastatic potential and recurrence risk but also shows limited response to conventional targeted therapies and immune checkpoint inhibitors. In current clinical practice, there is still an urgent need for therapeutic approaches that can effectively address the issues of high metastasis and high recurrence in this refractory subtype[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs a spindle pole-associated protein, spindle and centriole-associated protein 1 (SPDL1) not only participates in mitosis and cell division processes but also exhibits pro-carcinogenic activity[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Overexpression of SPDL1 has been shown to be associated with poor prognosis and higher metastatic potential in breast cancer patients[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Its potential mechanisms include regulating microtubule stability, interfering with the invasion and migration of tumor cells, and thereby promoting tumor cells to break through blood vessel walls and form distant metastatic foci. In addition, SPDL1 may also help tumor cells grow in the microenvironment of metastatic sites by regulating immune escape[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, SPDL1 is expected to be a potential therapeutic target for distant metastasis of breast cancer, and further research on its mechanism of action is of great significance for the early diagnosis and targeted therapy of breast cancer. For this purpose, we first conducted a pan-cancer analysis of SPDL1 using online databases and found that SPDL1 is highly expressed in breast cancer tissues and associated with poor prognosis. Meanwhile, Mendelian randomization analysis was used to explore the association between the SPDL1 gene and breast cancer risk, and the results showed a positive correlation between the SPDL1 gene and breast cancer risk. Furthermore, a TNBC cell line with stable overexpression of SPDL1 was constructed in in vitro experiments, which confirmed that SPDL1 has the function of promoting the invasion and metastasis of TNBC.\u003c/p\u003e\u003cp\u003eBreast cancer lung metastasis is a metastatic cascade event. During this process, TNBC cells release cytokines, proteases, exosomes, and other substances to interact and crosstalk with immune cells in the lung microenvironment, forming a TME conducive to the colonization and growth of metastatic tumors. The heterogeneity of TAMs and their interaction with the TME are key factors affecting the progression of metastasis[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. During this cancer progression process, cancer cells escape immune surveillance and activate proliferation. At this time, TAMs in lung metastatic foci interact with cancer cells and gradually show significant M2 polarization characteristics[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and promote the invasion and metastasis of cancer cells by secreting factors that promote epithelial-mesenchymal transition[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, it is worth noting that specific subsets of macrophages (such as TREM2\u0026thinsp;+\u0026thinsp;Mregs) are enriched in the pre-metastatic niche, providing suitable conditions for metastasis by remodeling the extracellular matrix and mediating the immunosuppressive microenvironment[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, intervention measures targeting TAMs show therapeutic potential. To explore whether SPDL1 can regulate TAMs in breast cancer, we found through bioinformatics analysis that SPDL1 is positively correlated with M2-TAM markers CD163 and CD206. Later, in vitro co-culture systems confirmed that SPDL1 promotes M2 macrophage polarization, and the expression of M2 surface markers increases.\u003c/p\u003e\u003cp\u003eStudies have shown that autocrine TGF-β signaling is one of the key factors for the survival of breast cancer cells[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. To further explore the mechanism by which SPDL1 promotes M2 macrophage polarization in TNBC cells, we performed RNA-seq and found that the TGF-β signaling pathway is enriched therein. It was also found that the expression of IL-6 and TGF-β factors was higher in the supernatant of SPDL1-overexpressing TNBC cells. TGF-β activates downstream target genes through the classic Smad2/3-Smad4 complex, and at the same time upregulates the expression of GLI2, a key transcription factor of the Hedgehog signaling pathway, through a Smad-dependent pathway, promoting its nuclear translocation. As a core effector molecule, GLI2 can directly bind to the IL-6 promoter region to promote its expression. IL-6 further upregulates the expression of TGF-β by activating the STAT3 signal, thereby forming a positive feedback loop, and at the same time induces M2 macrophage polarization to form an immunosuppressive microenvironment[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In addition, IL-6 and TGF-β can synergistically promote EMT of cancer cells and enhance metastatic potential. Therefore, we confirmed in vitro experiments that SPDL1 can activate the TGF-β/Smad signaling pathway and found that M2 macrophages can synergistically promote TNBC cell invasion and metastasis. Subsequently, we used the TGF-β receptor inhibitor LY2109761 to perform a rescue experiment. The study found that after inhibiting the TGF-β/Smad signaling pathway, the polarization of M2 macrophages and the oncogenic activity promoted by SPDL1 were downregulated. To verify this finding, we also established a breast cancer lung metastasis model in mice. The experimental results showed that SPDL1 can promote M2 macrophage polarization and breast cancer lung metastasis in breast cancer through the TGF-β/Smad signaling pathway, and this result was inhibited after adding the in vivo TGF-β receptor inhibitor oxymatrine.\u003c/p\u003e\u003cp\u003eIn conclusion, our research results reveal a new mechanism by which SPDL1 drives breast cancer metastasis by regulating the phenotypic transformation of immune cells in the tumor microenvironment. Specifically, SPDL1 can induce the phenotypic transformation of M2-type TAMs and form a pro-metastatic positive feedback regulatory network by activating the TGF-β/Smad signaling axis of tumor cells and its downstream GLI2-dependent IL-6 and TGF-β autocrine loops, ultimately mediating the formation of distant organ metastatic foci. This finding clarifies the molecular biological significance of the SPDL1-TGFβ/Smad/GLI2-IL-6/TGF-β signal cascade from the perspective of tumor-immune interaction and provides key molecular targets for the development of therapeutic strategies targeting the tumor microenvironment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cb\u003eConflict of interest\u003c/b\u003e\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cb\u003eEthical approval\u003c/b\u003e\u003c/strong\u003e\u003cp\u003e All animal experiments were conducted in accordance with guidelines and protocol approved by the institutional animal care and use committee of Putuo Hospital, Shanghai University of Traditional Chinese Medicine, China. (DWEC-A-2024-18-1-88).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003e All authors have approved submission of the manuscript to this journal.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis work was supported by National Natural Science Foundation of China (81973625); Key Medical Discipline Project of Shanghai Municipal Health Bureau(2024ZDXK0046); Science, Technology Innovation Project of Putuo District Health System (No. ptkwws202307); The One Hundred Talents Project of Putuo Hospital, Shanghai University of Traditional Chinese Medicine (2022-RCLH-03)and the Shen Hongquan\u0026rsquo;s Academic Experience Research Studio of Shanghai Famous Veteran TCM Expert (SHGZS-202224).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJie Wang, Wei Li and Fenfen Xiang con-ceived the original idea and designed the study. 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Nat Commun 15:10123. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-024-54433-5\u003c/span\u003e\u003cspan address=\"10.1038/s41467-024-54433-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Triple-negative breast cancer, SPDL1, M2 Macrophage, TGF-β/Smad Pathway","lastPublishedDoi":"10.21203/rs.3.rs-7913702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7913702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreast cancer is a malignant tumor originating from breast epithelial tissue. Globally, there are approximately 2.3\u0026nbsp;million new breast cancer cases annually, accounting for 11.7% of all new cancer cases. Based on molecular subtype classification, triple-negative breast cancer (TNBC) has a high propensity for lung metastasis and is a major cause of death in breast cancer patients. As key innate immune cells in the tumor microenvironment (TME), tumor-associated macrophages (TAMs) have a functional status closely related to tumor progression. Recent studies have shown that macrophages in the TME play complex and critical regulatory roles in tumor evolution. Moreover, during the process of TNBC lung metastasis, TAMs are regulated by multiple signaling pathways, thereby driving metastasis. Previous studies have found that the TGF-β/Smad signaling pathway can induce TAM polarization toward the M2 type, which synergistically promotes TNBC lung metastasis. but its mechanism remains unclear. Spindle apparatus coiled-coil protein 1 (SPDL1) plays an important role in regulating cell mitosis and accurate chromosome segregation, and is involved in the occurrence, development, and prognosis evaluation of breast tumors. However, the impact of SPDL1 on the functional status of TAMs and whether the interaction between them is involved in regulating TNBC lung metastasis have not been clearly reported yet. Therefore, this study aims to explore whether SPDL1 affects the process of TNBC lung metastasis by regulating M2 macrophage polarization and clarify its potential molecular mechanism, so as to provide new ideas for the development of novel immune drugs based on SPDL1-macrophages.\u003c/p\u003e","manuscriptTitle":"SPDL1 Promotes Lung Metastasis of Triple-Negative Breast Cancer by Targeting the TGF-β/Smad Pathway to Regulate M2 Macrophage Polarization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 04:17:32","doi":"10.21203/rs.3.rs-7913702/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-04T10:54:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-04T05:16:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169780835803562001280466549277161240454","date":"2025-11-04T01:06:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-24T09:15:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-23T12:51:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-23T12:50:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-10-21T10:48:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6e78f6c3-1a27-4bd5-b8b4-fd02bc318984","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-12-04T11:08:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-06 04:17:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7913702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7913702","identity":"rs-7913702","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}