Extracellular Vesicles from Canine Mammary Tumor Cells Promote Macrophage M2 Polarization and Enhance Tumor Progression

Extracellular Vesicles from Canine Mammary Tumor Cells Promote Macrophage M2 Polarization and Enhance Tumor Progression | 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 Extracellular Vesicles from Canine Mammary Tumor Cells Promote Macrophage M2 Polarization and Enhance Tumor Progression Na-Kyoung Chi, Se-Hoon Kim, Ga-Hyun Lim, Ki-Hoon Song, Ju-Hyun An, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9043816/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Mar, 2026 Read the published version in Veterinary Research Communications → Version 1 posted 9 You are reading this latest preprint version Abstract Extracellular vesicles (EVs) derived from tumor cells play a crucial role in modulating and reprogramming the tumor microenvironment (TME), thereby promoting tumor progression. While previous studies have investigated the impact of tumor-derived EVs on macrophages in various cancers, such as bladder cancer and osteosarcoma, and in canines, particularly in melanoma, little is known about the effects of canine mammary tumors influence on macrophage polarization. This study aimed to elucidate the functional role of EVs derived from CIPp cells, a canine mammary tumor cell line, in macrophage polarization and tumor progression. CIPp-derived EVs were isolated and characterized, and their effects on macrophage phenotype were examined using the canine macrophage cell line DH82. Exposure to CIPp-EVs induced DH82 macrophages to adopt a tumor-associated macrophage (TAM)-like M2 phenotype, as confirmed by quantitative RT-PCR, immunofluorescence, and flow cytometry. CIPp-EV treatment significantly upregulated M2-associated markers (CD206, VEGF-A, IL-10, and COX2) while downregulating M1-associated markers (iNOS and IL-6). Furthermore, conditioned media (CM) from CIPp-EV–treated macrophages enhanced CIPp tumor cell migration and induced epithelial–mesenchymal transition (EMT), evidenced by the reduction of E-cadherin and increased expression of vimentin, fibronectin, and α-SMA. Together, these findings demonstrate that CIPp-derived EVs reprogram the TME by driving TAM-like M2 macrophage polarization, which in turn promotes tumor cell migration and EMT, thereby facilitating tumor progression and metastasis. Canine mammary tumor Epithelial-mesenchymal transition (EMT) Extracellular vesicle Tumor-associated macrophages (TAMs) Tumor microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In the evolving landscape of cancer biology, Tumor-derived extracellular vesicles (TDEs) have recently emerged as key regulators of tumor growth, metastasis, and immune evasion (Zhang and Yu 2019 ; Olejarz et al. 2020 ). TDEs are nanosized, lipid bilayer-enclosed vesicles that carry various bioactive molecules, including tumor-derived proteins, lipids, microRNAs, and mRNA (Whiteside 2016 ). These vesicles deliver molecular cargo to recipient cells within the tumor microenvironment (TME), reprogramming it into an immunosuppressive and pro-tumoral niche (Reale et al. 2022 ). The TME is a complex and dynamic ecosystem composed of immune cells, fibroblasts, and endothelial cells, which interact closely with tumor cells (Yang et al. 2020a ). Among the immune cells, macrophages play a central role in shaping the immune landscape under the influence of TDEs. Depending on external stimuli, macrophages can differentiate into either tumor-suppressive M1 or tumor-promoting M2 phenotypes (Boutilier and Elsawa 2021 ). In many solid tumors, TDEs have been shown to induce M2 polarization, leading to the formation of tumor-associated macrophages (TAMs) (Baig et al. 2020 ). TAMs secrete immunosuppressive cytokines (e.g., IL-10, TGF-β) and tumor-promoting factors (e.g., VEGF, MMPs), which suppress immune responses, promote angiogenesis, and induce epithelial–mesenchymal transition (EMT), thereby enhancing tumor cell motility and invasiveness (Noy and Pollard 2014 ; Chen et al. 2019 ). EMT is a key process in cancer metastasis and is strongly associated with poor prognosis (Ribatti et al. 2020 ) Through these mechanisms, TDEs create a tumor-favorable microenvironment via the TDE–TAM axis (Chen et al. 2024 ). This axis has been extensively studied in various human solid tumors (Guo et al. 2020 ; Chen et al. 2021 ), including breast cancer, lung cancer (Pritchard et al. 2020 ), and glioblastoma (Gabrusiewicz et al. 2018 ). In breast cancer, TDEs have been reported to not only promote M2 polarization (Hao et al. 2023 ) but also enhance cancer cell migration and lymph node metastasis (Piao et al. 2018 ). Consequently, inhibitors of extracellular vesicle (EV) secretion are currently being explored as potential anti-cancer therapies (Li et al. 2022b ; Zhang et al. 2020 ). Despite significant advances in human oncology, studies on EV–immune cell interactions remain limited in veterinary oncology. Canine mammary tumors (CMTs), one of the most common neoplasms in intact female dogs (Sleeckx et al. 2011 ), share clinical and molecular similarities with human breast cancer, making them valuable comparative oncology models (Abdelmegeed and Mohammed 2018). Previous studies have demonstrated that EVs derived from the CMT cell line CIPp may enhance proliferation and migration of CIPp cells themselves (Moccia et al. 2023). However, their effects on immune cells—particularly on macrophage polarization—remain unclear. To date, research on EV–macrophage interactions in dogs has been limited to a few melanoma models (Kim et al. 2022), in which EVs were shown to promote M2 polarization and reinforce immunosuppressive conditions. This study was designed to test the hypothesis that EVs derived from the canine mammary gland tumor cell line CIPp polarize canine macrophages toward a TAM-like M2 phenotype, thereby promoting tumor progression via enhanced migration and EMT. This study provides novel insights into the role of EVs in shaping the immune landscape of canine mammary tumors. Given the conserved nature of EV–TAM interactions across species, this work may inform the development of TAM-targeted therapies in both veterinary and human oncology. Materials and methods Cell culture and isolation of EV The canine malignant mammary gland tumor cell line CIPp, established from a primary tumor, was obtained from the Department of Veterinary Clinicopathology at Seoul National University. Cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA; #11875093) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific; #16000-044) and 1% penicillin-streptomycin (PS; Thermo Fisher Scientific; #15140122) at 37◦C in a humidified atmosphere containing 5% CO2. For conditioned media collection, CIPp cells were seeded at 5000 cells/cm² in 10-layer Culture Chambers (BioPioneer Inc., San Diego, CA, USA) and cultured in RPMI with 10% FBS until 80% confluence. The cells were washed three times with phosphate-buffered saline (PBS, Welgene, Gyeongsan, Gyeongbuk, Republic of Korea; #LB 004 − 01) to remove residual FBS and incubated for 24 hours in serum-free CD 293 medium (Thermo Fisher Scientific; #11913-019) supplemented with 1% GlutaMAX and 1% sodium pyruvate before harvesting the conditioned media (CM). EVs were isolated from the CM using the tangential flow filtration (TFF)-based method. The conditioned media was first filtered through a 0.22-µm polyethersulfone membrane filter (Thermo Fisher Scientific) to remove cell debris and non-EV particles. The filtrate was then concentrated using a 700 kDa hollow-fiber cartridge (Cytiva, Marlborough, MA, USA) and diafiltered with PBS. The EV suspension was aliquoted into polypropylene disposable tubes, and stored at − 80°C. Before use, frozen EVs were thawed completely at 4°C. Characterization and profiling of EVs were performed in accordance with the Minimal Information for Studies of Extracellular Vesicles 2018 (Théry et al. 2018 ) recommended by the International Society of Extracellular Vesicles. Characterization of EV derived from CIPp cells To verify that the isolated particles were EVs, western blotting was performed on CIPp cell lysates and EV preparations to assess the enrichment of EV-specific protein markers. Cells were lysed using a lysis buffer (Elpis Biotech, Daejeon, Republic of Korea; #EBA-1149) and kept on ice for 30 minutes. The lysates were then centrifuged at 12,000 rpm for 20 minutes at 4°C to remove cell debris. Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific; #23227). Equal amounts of protein were resolved by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Darmstadt, Germany; #IPVH00010). Membranes were blocked for 1 hour at 21°C in TBST buffer containing either 5% skim milk (LPS Solution, Daejeon, Republic of Korea; #SKI500) or 5% bovine serum albumin (BSA, GeorgiaChem, Hwaseong, Republic of Korea; #G.C-BS1005). After blocking, the membranes were incubated overnight at 4°C with primary antibodies (1:1,000). Specifically, antibodies against CD9 (Cell Signaling Technology, Danvers, MA, USA; #13174), CD81 (System Biosciences, Palo Alto, CA, USA; #EXOAB-CD81A-1) and Alix (Cell Signaling Technology; #2171) were used as EV-positive markers. Additionally, β-actin (Cell Signaling Technology; #4970) was employed as a negative marker to assess the depletion of cellular contaminants. Following TBST washes, the membranes were incubated for 1 hour at 21°C with HRP-conjugated secondary antibodies (1:2,000), including anti-rabbit and anti-mouse antibodies (ABclonal Technology; #AS014, #AS003). Protein bands were visualized using an enhanced chemiluminescence detection reagent (SmartGene, Seongnam, Republic of Korea; #SG-PR-HECL), and band images were acquired using the LAS-4000 imaging system (GE Healthcare Biosciences, Milwaukee, WI, USA). Nanoparticle tracking analysis (NTA) was performed to assess the size and concentration of EV samples, using a 642-nm laser NanoSight LM10 (Malvern Panalytical, Amesbury, UK). EV samples were diluted in PBS and analyzed using NTA 3.2 software (Malvern Panalytical Ltd., Worcestershire, UK). For each sample, at least five videos were recorded and averaged to obtain representative measurements. EV morphology was further evaluated by transmission electron microscopy (TEM). A 10 µl EV suspension was applied on a carbon-coated grid and allowed to absorb for 1 minute, followed by removal of excess liquid with filter paper. A 2% uranyl acetate solution was then applied for 20 seconds as a negative stain, and excess stain was gently blotted before imaging. Images were acquired using a JEM1010 transmission electron microscope (Jeol Ltd., Tokyo, Japan) operating at 80 kV. All procedures were performed in triplicate to ensure reproducibility. DH82 cell culture and EV treatment The canine macrophage cell line DH82 was purchased from ATCC (ATCC number: CRL-10389, Manassas, VA, USA). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Solbio, Suwon, Republic of Korea, #DME-001) supplemented with 10% FBS and 1% PS, maintained at 37°C in a 5% CO₂ incubator. DH82 cells were seeded for 12 hours, followed by a media change and treated with CIPp-EVs for an additional 36 hours. Cell viability assay DH82 cell viability following exposure to CIPp-EVs was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan, #CK04), following the manufacturer's protocol. DH82 cells were seeded in 96-well plates at a density of 1.5 × 10³ cells/well and cultured overnight to allow attachment. The medium was then replaced, and cells were treated with either fresh medium alone, 20% PBS, 10% EVs (19.5 µg/ml), or 20% EVs (39 µg/ml) for 36 hours. After adding 10 µl of CCK solution and incubating for 2 hours, cell viability was measured using Epoch Microplate Spectrophotometer (BioTek Korea Co., Ltd., Seoul, Republic of Korea). Morphological observation DH82 cells were seeded at a density of 1 × 10 5 cells/well in 6-well plates for 12 hours. Seeded DH82 cells were treated with CIPp-EVs (39 µg/ml) for 36 hours. Cell morphology was observed using a Leica DMil microscope (Leica Microsystems, Wetzlar, Germany) to assess EV-induced morphological changes. Quantitative real-time PCR (qRT-PCR) of DH82 cells DH82 cells were seeded at 2 × 10⁵ cells/well in 6-well plates and allowed to adhere for 12 hours before being treated with CIPp-EVs (39 µg/ml) for 36 hours. After treatment, total RNA was extracted using Easy-Blue Total RNA Extraction Kit (Intron Biotechnology, Daejeon, Korea, #17061) according to the manufacturer’s instructions, and RNA purity and concentration were measured using a spectrophotometer (NanoPhotometer NP80, Implen GmbH, Munich, Germany). Quantitative PCR was performed on a Qunatstudio 1 (Applied Biosystems, CA, USA) using SYBR Green qPCR Master Mix (SmartGene, #SG-SYBR-ROXH). Target gene expression levels were normalized to GAPDH, which served as the housekeeping control. Primer sequences utilized in this study are provided in Table S1. Triplicate reactions were carried out for all samples. Immunofluorescence assay To analyze M1/M2 surface marker expression in DH82 cells, an immunofluorescence assay was performed. DH82 cells were seeded at a density of 2 × 10⁴ cells per confocal dish (SPL Life Sciences, Pocheon, Gyeonggi-do, Republic of Korea, #101350) and incubated for 12 hours. Cells were then treated with CIPp-EVs (39 µg/ml) for 36 hours. As a positive control for M1 polarization, cells were exposed to 100 ng/ml Lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA, #L4391) for the same duration. Following treatment, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at 21°C. After additional PBS washes, cells were blocked with 5% BSA for 30 minutes at 21°C to prevent nonspecific binding. FITC-conjugated anti-mouse CD206 antibody (1:100 dilution, BioLegend, San Diego, CA, USA, #141703) and APC-conjugated anti-mouse CD86 antibody (1:10 dilution, Miltenyi Biotec, Bergisch Gladbach, Germany, #130-102-558) were applied to the cells, followed by incubation for 1 hour at 21°C. Cells were then washed three times with PBS, and coverslips were mounted with DAPI-containing mounting solution (Thermo Fisher Scientific, #00-4959-52). Fluorescence images were acquired using an EVOS FL imaging system (Thermo Fisher Scientific). Flow cytometry analysis Flow cytometric analysis was performed to evaluate M1 and M2 surface marker expression in DH82 cells. Cells were seeded at a density of 2 × 10⁵ cells per well in 6‑well plates and allowed to adhere for 12 hours. The cultures were then divided into three groups: untreated controls, LPS-treated cells (200 ng/mL, 36 hours) to induce M1 polarization, and CIPp-EV-treated cells (39 µg/mL, 36 hours) to assess EV-mediated modulation of macrophage phenotype. Following treatment, cells were collected, washed with PBS, and fixed in 4% paraformaldehyde for 15 min at 21°C. To reduce nonspecific binding, cells were blocked with 5% BSA for 30 minutes. Staining was performed using the same FITC-conjugated CD206 and APC-conjugated CD86 antibodies described in the immunofluorescence assay, using identical dilutions. After a 1 hour incubation at 21°C in the dark, the cells were washed with PBS and resuspended in 200 µL PBS to obtain a single-cell suspension. Samples were analyzed using a FACS Aria II flow cytometer (BD Biosciences, San Jose, CA, USA), and data were processed with FlowJo software (Tree Star, Ashland, OR, USA). Conditioned media preparation DH82 cells were seeded in 100 mm dishes at a density of 1 × 10 6 cells in complete DMEM medium for 12 hours. After attachment, the cultures were divided into three groups. The control group was maintained in fresh complete DMEM without EVs for an additional 36 hours. For EV treatment, cells were exposed to CIPp-EVs at 20% (v/v) for either 24 or 36 hours. Following the respective incubation periods, the cells were washed twice with PBS to completely remove any residual CIPp-EVs. Subsequently, the medium was replaced with fresh DMEM medium with 10% Exo-free FBS (Thermo Fisher Scientific, #A2720803) and 1% PS, and the cells were cultured for an additional 24 hours to produce CM. The CM was harvested, centrifuged at 800 x g for 3 minutes to remove residual cells, and the supernatant was stored at − 80°C until use. Wound healing assay CIPp cells were seeded in 12-well plates at a density of 1×10 5 cells/well and cultured until confluence. To inhibit proliferation, cells were treated with 2 µg/ml of mitomycin (Enzo Life Science, Farmingdale, NY, USA, #BML-GR311) for 2 hours. The cell monolayer was then scratched with a 1 ml pipette tip and cell debris was removed by washing twice with PBS. The remaining adherent cells were cultured in complete DMEM supplemented with 50% CM. Wound healing was monitored at 0, 3, and 6 hours under a microscope using a TCapture program (Tucsen Photonics, Fuzhou, Fujian, China). Wound width was quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and relative width closure (%) was calculated using the following formula: [(Relative width at 0 h – relative width at 3 h or 6 h)/relative width at 0 h] ×100. qRT-PCR of CIPp cells CIPp cells were seeded at a density of 2 × 10 5 cells/well in 6-well plates and cultured for 12 hours. Seeded CIPp cells were then treated with 50% complete DMEM and 50% CM for 48 hours. Following incubation, total RNA was extracted and EMT marker expression was analyzed by qRT-PCR using the same procedure described for DH82 cells. Statistical Analysis All data were analyzed using the GraphPad Prism software (version 10.1.2, GraphPad Software Inc., San Diego, CA, USA). For the CCK-8 cell viability assay, data were analyzed using one-way ANOVA with Tukey’s multiple comparison test to assess differences among groups. For experiments involving comparisons between two groups (e.g., qRT-PCR, flow cytometry and wound healing), unpaired two-tailed Student’s t-tests were used. Data are presented as the mean ± SD, and p < 0.05 was considered statistically significant. Results Characterization of CIPp-EVs EVs were isolated from CIPp cells using TFF and the overall isolation process was schematically illustrated (Fig. 1 A). TEM confirmed that the isolated EVs displayed a typical round morphology with an approximate diameter of 50 nm (Fig. 1 B). As expected from the distinct measurement principles, NTA showed that the size distribution of CIPp-EVs was centered around 140 nm (Fig. 1 C). Western blotting confirmed the enrichment of EV markers CD9 and CD81 in CIPp-EVs compared with CIPp cells (Fig. 1 D). Taken together, these findings verify that TFF-based isolation effectively yields structurally intact EVs with uniform characteristics, supporting their suitability for downstream applications. Impact of CIPp-EVs on Macrophage Viability and Morphology The effect of CIPp-EVs on the viability of DH82 cells was evaluated using a CCK-8 assay. Cells were exposed to two concentrations of CIPp-EVs (19.5 and 39 µg/ml) for 36 hours. No significant difference in cell viability was observed between the control and EV-treated groups (Fig. 2 A), indicating that CIPp-EVs did not significantly affect cell viability at either dose. Based on these results, the higher concentration (39 µg/ml) was selected for subsequent experiments. In addition to evaluating cell viability, morphological changes in DH82 cells were examined after 36 hours of treatment with or without CIPp-EVs. The EV-treated cells exhibited a more elongated shape compared to the control group, suggesting alterations in cell morphology and a shift toward an M2-like phenotype in response to EV exposure (Fig. 2 B). This observation is consistent with previous studies (McWhorter FY et al. 2013 ; Heinrich et al. 2017 ; Pe et al. 2022 ), which showed that M2-polarized macrophages characteristically adopt an elongated morphology, in contrast to the rounded form of M1 or unstimulated M0 macrophages. CIPp-EVs Promote M2 Macrophage Polarization To investigate the effects of CIPp-EVs on macrophage polarization, DH82 cells were co-cultured with CIPp-EVs (39 µg/ml) for 36 hours. The expression of both M1 and M2 macrophage markers was analyzed using qRT-PCR, immunofluorescence, and flow cytometry. qRT-PCR analysis showed significant upregulation of M2-associated genes (CD206, VEGF-A, IL-10, and COX2) and downregulation of M1-associated genes (iNOS and IL-6) in EV-treated macrophages compared with controls (Fig. 3 A). These transcriptional changes are consistent with a shift toward an M2 phenotype. Immunofluorescence staining further supported this finding. Control cells exhibited minimal expression of either marker, while LPS-treated cells (positive control) displayed bright CD86 (red) staining characteristic of M1 polarization. By contrast, CIPp-EV–treated macrophages showed a pronounced increase in CD206 (green) signal, indicating M2 polarization at the protein level (Fig. 3 B). Additionally, flow cytometry was performed to quantify the populations of M1 and M2 macrophages by measuring the surface expression of CD86 and CD206. The percentage of CD86 + /CD206 − (M1) macrophages did not differ significantly between control and CIPp-EV-treated groups. However, CIPp-EV-treated cells showed a significant increase in CD86 − /CD206 + (M2) macrophages ( p < 0.05), confirming the polarization towards the M2, TAM-like phenotype (Fig. 3 C). Taken together, these findings demonstrate that CIPp-EVs effectively induce TAM-like M2 macrophage polarization, as indicated by the upregulation of M2 markers at both the gene and protein levels, and the increase in M2 macrophage populations. This shift towards the M2 phenotype may contribute to the immunosuppressive and tumor promoting properties observed in the tumor microenvironment. TAMs Induced by CIPp-EVs Enhance CIPp Tumor Progression Having confirmed that CIPp-EVs induce TAM-like M2 macrophage polarization in DH82 cells (Fig. 3 ), the impact of these macrophages on CIPp tumor cell behavior was subsequently evaluated. Treatment of CIPp tumor cells with CM derived from DH82 macrophages exposed to CIPp-EVs for 24 hours (CIPp-EV–DH82-CM) significantly increased the migratory activity of CIPp cells compared to CM from untreated DH82 macrophages (Control DH82-CM) (Fig. 4 A). In contrast, CM collected from 36-hour EV-treated DH82 cells showed no significant effect (Fig. S1). These findings suggest that 24-hour CM contains a higher level of TAM-associated factors, and was therefore selected for all subsequent experiments. Representative images taken at 0, 3, and 6 hours illustrated a pronounced reduction in the wound gap in the CIPp-EV–DH82-CM group compared to the Control DH82-CM group. Quantitative analysis confirmed a significant increase in relative migration at 3 hours ( p < 0.01) and an even greater effect at 6 hours ( p < 0.0001) (Fig. 4 B). To further assess TAM-mediated effects on tumor progression, EMT-related markers were analyzed. The CIPp-EV–DH82-CM group exhibited marked downregulation of the epithelial marker E-cadherin ( p < 0.05) and upregulation of mesenchymal markers such as vimentin, fibronectin, and α-SMA ( p < 0.01 for vimentin and fibronectin; p < 0.05 for α-SMA) (Fig. 5 ). These results indicate that TAM-derived factors promote EMT and enhance tumor invasiveness. Collectively, the results demonstrate that CIPp-EVs induce TAM-like polarization in macrophages, which in turn promotes CIPp tumor cell migration and EMT, thereby contributing to a more aggressive tumor microenvironment. Discussions This study demonstrates that CIPp-EVs promote M2 polarization of canine macrophage cell line DH82, and that these M2-polarized macrophages, in turn, enhance EMT and migratory potential in tumor cells. Together, these findings suggest that interactions among EVs, macrophages, and tumor cells establish a self-reinforcing feedback loop within the TME, thereby driving tumor malignancy and progression (Fig. 6 ). TDEs are recognized as key modulators of the TME, notably by polarizing macrophages toward an M2 TAM phenotype (Baig et al. 2020 ; Wang et al. 2024a ). In both canine and human mammary carcinomas, studies show that infiltration of M2 TAMs is strongly associated with advanced clinical stage, shorter survival, and disrupted extracellular matrix organization (Monteiro et al. 2018 ; Jeong et al. 2019 ; Allison et al. 2022 ; Garcia et al. 2025 ). TAMs have been shown to promote tumor progression through multiple mechanisms, including the secretion of immunosuppressive cytokines, angiogenic factors, and extracellular matrix–remodeling enzymes, thereby fostering an invasive and immune-suppressive microenvironment (Yang et al. 2020b ; Bied et al. 2023 ). Beyond these correlative findings, mechanistic studies in human oncology have demonstrated that tumor-derived EVs act upstream of macrophage polarization toward the M2 phenotype, thereby promoting the establishment of a tumor-supportive microenvironment and enhancing tumor progression (Chen et al. 2020 ; Reed et al. 2021 ; Tian et al. 2023 ; Wang et al. 2024a ). However, whether a comparable EV–TAM axis exists in canine tumors remains largely unexplored. The present study addresses this gap by providing functional evidence for EV-driven TAM polarization and its downstream effects in a canine mammary tumor model. Previous studies have shown that the activation of the STAT3 signaling pathway reprograms macrophages toward a tumor-promoting M2-like phenotype across multiple cancers. (Takaishi et al. 2010 ; Fu et al. 2017 ; Ham et al. 2018 ; Mu et al. 2018 ; Irey et al. 2019 ; Mohammad et al. 2025 ). In our study, CIPp-EVs induced hallmark features of M2 polarization, including elongated morphology, increased CD206 expression, and elevated secretion of cytokines such as VEGF-A and IL-10 (McWhorter et al. 2013 ; Rőszer 2015 ; Wang et al. 2024b ). Given that IL-10 is a known upstream activator of STAT3 in macrophages (Murray 2006 ; Hutchins et al. 2013 ), it is hypothesized that CIPp-EVs may engage a positive feedback mechanism wherein EV-induced IL-10 secretion reinforces STAT3 signaling, thereby stabilizing the TAM-like phenotype. While the direct activation of this signaling pathway was not experimentally validated in the current study, the observed pattern of IL-10 upregulation provides a strong rationale for future investigations into STAT3-dependent mechanisms for conditioning and the establishment of a protumorigenic microenvironment. Importantly, M2-polarized macrophages are not only immunosuppressive but also potent inducers of EMT via secretion of IL-10, VEGF, and TGF-β (Feng et al. 2018 ; Li et al. 2022a ; Zhang et al. 2024 ; Zhao et al. 2024 ). These cytokines repress epithelial markers such as E-cadherin while inducing mesenchymal markers including vimentin, N-cadherin, and fibronectin, thereby enhancing tumor cell motility and invasiveness (Ribatti et al. 2020 ). Multiple studies have demonstrated that IL-10, VEGF, and TGF-β secreted by TAMs are critical mediators of EMT, and inhibition of each factor effectively attenuates TAM-induced EMT and tumor cell invasiveness (Liu et al. 2013 ; Feng et al. 2018 ; Cai et al. 2019 ). In this study, CM from CIPp-EV–treated macrophages downregulated E-cadherin and upregulated vimentin and fibronectin in CIPp tumor cells, accompanied by enhanced migratory capacity, confirming EMT induction. It should be noted that the term "TAM-like macrophages" used in this study is based on marker expression patterns and in vitro functional effects, rather than comprehensive in vivo functional validation. Nevertheless, the collective findings suggest that CIPp-EVs reprogram macrophages toward TAM-like phenotype, potentially via a STAT3–IL-10 pathway, and these reprogrammed macrophages, in turn, drive EMT through IL-10 and VEGF signaling—establishing a self-reinforcing EV–TAM–tumor axis that promotes tumor progression in canine mammary carcinoma. In this study, to ensure the high purity and integrity of EVs required for functional assays, tangential Flow Filtration (TFF) was utilized. This method allows for the scalable isolation of EVs with high yield and purity even from large-volume or diluted samples (Busatto et al. 2018 ; Liangsupree et al. 2021 ). Subsequent NTA analysis demonstrated that TFF-isolated EVs exhibited a narrow and uniform size distribution, supporting the suitability of this method for elucidating the biological roles of CIPp-EVs. Given the central role of the EV–TAM axis in establishing a tumor-supportive microenvironment, targeting this pathway has emerged as a promising therapeutic strategy in preclinical cancer models (Im et al. 2019 ; Jiang et al. 2021 ; Li et al. 2022b ; Peng et al. 2022 ). Our findings demonstrate that TDEs are key drivers of macrophage polarization. Therefore, strategies aimed at inhibiting EV biogenesis or secretion could effectively disrupt this pro-tumorigenic crosstalk. While specific inhibitors were not evaluated in this study, the potential to modulate the TME by severing the EV-mediated communication loop suggests that EV-targeted therapies warrant further investigation as a novel treatment approach for canine mammary carcinoma. This study has several limitations. As the experiments were conducted in vitro, these conditions cannot fully recapitulate the complexity of the in vivo tumor microenvironment. Notably, while EV dosing in this study was standardized by protein concentration (µg/mL), future in vivo investigations should adopt particle-based dosing strategies to ensure greater physiological relevance. Additionally, the use of single tumor (CIPp) and macrophage (DH82) cell lines limits generalizability. Thus, validation in primary cells or multiple tumor models is required. Finally, although the STAT3 axis was implicated as a potential mediator, the precise intracellular signaling cascades driving M2 polarization warrant further detailed molecular characterization. In conclusion, this study highlights the pivotal role of CIPp-EVs in remodeling the TME through the induction of a pro-tumorigenic feedback loop. These EVs promote the polarization of macrophages into TAMs, which in turn secrete factors that enhance tumor cell migration and EMT, thereby increasing invasive and metastatic potential. Collectively, the findings underscore the significance of the EV–TAM axis in the progression of canine mammary carcinoma. A deeper understanding of this EV–TAM–tumor cell axis may provide a conceptual framework for developing EV- or TAM-targeted therapeutic strategies in canine mammary tumors and other human cancer models that share similar microenvironmental mechanism. Declarations Competing interest The authors have no relevant financial or non-financial interests to disclose. Compliance with ethical standards Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. Funding This study was supported by the BK 21 Plus Program for Creative Veterinary Science Research, the Research Institute for Veterinary Science, and the College of Veterinary Medicine. Author Contribution Na-Kyoung Chi conceived and designed the study, conducted the experiments, and drafted the manuscript. Se-Hoon Kim, Ju-Hyun An and Ga-Hyun Lim contributed to the execution of the assays and data acquisition. Ki-Hoon Song and Min-Ok Ryu critically reviewed and edited the manuscript for intellectual content. Kyoung-Won Seo validated the original data, provided critical revisions, and supervised the overall study. All authors have read and approved the final version of the manuscript. Acknowledgement Sincere appreciation is extended to Dr. Sung Youl Kim at GNG CELL Co., Ltd. (R&D Center, 122 Unjung-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 13466, Republic of Korea) for generously providing the CIPp-derived extracellular vesicles used in the study. Data Availability The datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request. References Allison E, Edirimanne S, Matthews J, Fuller SJ (2022) Breast cancer survival outcomes and tumor-associated macrophage markers: a systematic review and meta-analysis. 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Supplementary Files CIPpEVsTAMPolarizationTableS1.xlsx CIPpEVsTAMPolarizationFigS1.docx Cite Share Download PDF Status: Published Journal Publication published 30 Mar, 2026 Read the published version in Veterinary Research Communications → Version 1 posted Editorial decision: Accepted 21 Mar, 2026 Reviews received at journal 21 Mar, 2026 Reviews received at journal 17 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers agreed at journal 14 Mar, 2026 Reviewers invited by journal 11 Mar, 2026 Editor assigned by journal 10 Mar, 2026 Submission checks completed at journal 10 Mar, 2026 First submitted to journal 05 Mar, 2026 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9043816","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":604483189,"identity":"c4078eb6-c781-4e6c-a586-245bc4008015","order_by":0,"name":"Na-Kyoung Chi","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Na-Kyoung","middleName":"","lastName":"Chi","suffix":""},{"id":604483190,"identity":"c59fa65a-ce0a-4648-8fb8-6d5ab0ec697e","order_by":1,"name":"Se-Hoon Kim","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Se-Hoon","middleName":"","lastName":"Kim","suffix":""},{"id":604483191,"identity":"755ef662-046f-48fe-83b3-ebb54cf74b01","order_by":2,"name":"Ga-Hyun Lim","email":"","orcid":"","institution":"VIP animal medical center","correspondingAuthor":false,"prefix":"","firstName":"Ga-Hyun","middleName":"","lastName":"Lim","suffix":""},{"id":604483197,"identity":"801f5c9f-1cf4-48db-ab64-76876755b1dc","order_by":3,"name":"Ki-Hoon Song","email":"","orcid":"","institution":"ViroCure Inc","correspondingAuthor":false,"prefix":"","firstName":"Ki-Hoon","middleName":"","lastName":"Song","suffix":""},{"id":604483199,"identity":"838e6bdd-82f4-4913-b27e-f96f559e88b2","order_by":4,"name":"Ju-Hyun An","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Ju-Hyun","middleName":"","lastName":"An","suffix":""},{"id":604483206,"identity":"1238c014-3945-4ea2-be45-193a9a88e782","order_by":5,"name":"Min-Ok Ryu","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Min-Ok","middleName":"","lastName":"Ryu","suffix":""},{"id":604483207,"identity":"ddc310b6-3345-4e44-a040-6485b472a13d","order_by":6,"name":"Kyoung-Won Seo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACCTBZAeMeIFrLGZK1MLaRokVyRvrDBx/n1eUZHGB++IHhzD3CWqQlcowNZ247XGxwgM1YguFGMWEtctI5bNK82w4kbjjAYMbA8CGBGC3pz3/zzqkDamH/RpwWaekEM2beBmagFh6gLTeI0CI5/42x5IxjhxNnHuYplkg4Q4QWiTPHH374UFOX2He8feOHD8eI0IIAzEBMkoZRMApGwSgYBbgBANYjOelu+aMUAAAAAElFTkSuQmCC","orcid":"","institution":"Seoul National University","correspondingAuthor":true,"prefix":"","firstName":"Kyoung-Won","middleName":"","lastName":"Seo","suffix":""}],"badges":[],"createdAt":"2026-03-05 19:53:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9043816/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9043816/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11259-026-11179-3","type":"published","date":"2026-03-30T15:59:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":104599156,"identity":"40309e7b-1c14-4ebd-bd21-b6336f71c1a5","added_by":"auto","created_at":"2026-03-13 19:40:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":193912,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of EVs derived from CIPp cells. \u003cstrong\u003e(A)\u003c/strong\u003eSchematic overview of EV isolation using TFF. \u003cstrong\u003e(B)\u003c/strong\u003e TEM image showing the typical round morphology of CIPp-EVs. Scale bar: 50 nm. \u003cstrong\u003e(C)\u003c/strong\u003e Representative NTA histogram demonstrating the size distribution of CIPp-EVs, with a mean diameter of approximately 140 nm. \u003cstrong\u003e(D)\u003c/strong\u003eWestern blot showing selective enrichment of EV-associated markers (CD9 and CD81) in CIPp-EVs relative to parental cell lysates.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9043816/v1/d3c4f4375ba617aeddafc4f1.jpg"},{"id":104599162,"identity":"8707a25a-8ff1-4bed-8fa0-b799d953dc33","added_by":"auto","created_at":"2026-03-13 19:40:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111776,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CIPp-EVs on Canine Macrophages. \u003cstrong\u003e(A)\u003c/strong\u003e Viability of DH82 cells after 36 hours of exposure to CIPp-EVs (19.5 and 39 μg/mL), assessed using the CCK-8 assay. No significant differences were detected compared with untreated controls. Data are presented as mean ± SD; statistical analysis was performed using one-way ANOVA. \u003cstrong\u003e(B)\u003c/strong\u003e Morphological changes in DH82 cells following 36 hours of treatment with or without CIPp-EVs. EV-treated cells exhibited a noticeably elongated, spindle-like morphology compared with control cells. Images were acquired at 100× magnification.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9043816/v1/72fb243a72c70a837178a46f.jpg"},{"id":104782071,"identity":"ceb46129-9111-4948-97a2-dd5a25426d96","added_by":"auto","created_at":"2026-03-17 07:56:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":160100,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CIPp-EVs on macrophage polarization. DH82 cells were incubated with CIPp-EVs (39 μg/mL) for 36 hours to evaluate EV-mediated polarization toward a tumor-associated macrophage (TAM)-like phenotype. \u003cstrong\u003e(A)\u003c/strong\u003e Relative mRNA expression of M1- and M2-associated cytokines was quantified by qRT-PCR. EV-treated cells showed increased expression of M2 markers (CD206, VEGF-A, IL-10, and COX2) and reduced expression of M1 markers (iNOS and IL-6) compared with untreated controls. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01 versus control. \u003cstrong\u003e(B)\u003c/strong\u003e Immunofluorescence staining of CD86⁺ (M1, red) and CD206⁺ (M2, green) cells. EV treatment increased the proportion of CD206⁺ cells. Scale bar: 100 μm. \u003cstrong\u003e(C)\u003c/strong\u003e Surface expression of CD86\u003csup\u003e+\u003c/sup\u003e/CD206\u003csup\u003e-\u003c/sup\u003e (M1) and CD86\u003csup\u003e-\u003c/sup\u003e/CD206\u003csup\u003e+\u003c/sup\u003e (M2) macrophages assessed by flow cytometry. Data are presented as mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 by unpaired t-test analysis.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9043816/v1/120a899dc89e1eaa214acb17.jpg"},{"id":104599161,"identity":"5c1c044c-f2d2-49a1-86de-30999407429b","added_by":"auto","created_at":"2026-03-13 19:40:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":205207,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of TAM-CM on the migratory activity of CIPp cells. CIPp cells were cultured in complete DMEM supplemented with 50% CM derived from DH82 macrophages. CM obtained from untreated DH82 macrophages is referred to as Control DH82-CM, whereas CM collected from DH82 macrophages treated with CIPp-EVs for 24 hours to induce a TAM-like phenotype is referred to as CIPp-EV–DH82-CM. All experiments were performed in triplicate and repeated as three independent biological experiments. (A) Representative images of wound closure at 0, 3, and 6 hours following treatment. Scale bar: 500 μm. (B)Quantification of relative migration rates. Data are presented as mean ± SD (n = 3). **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 versus control.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9043816/v1/10b2c83a899be2b97690488f.jpg"},{"id":104599158,"identity":"beec6c9f-f9f0-4c94-bf61-8fb18014e62b","added_by":"auto","created_at":"2026-03-13 19:40:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":58660,"visible":true,"origin":"","legend":"\u003cp\u003eTAM-derived factors promote EMT in CIPp tumor cells.\u003cstrong\u003e \u003c/strong\u003eCIPp cells were treated for 48 hours with CM derived from M2 polarized macrophages. qRT-PCR revealed downregulation of the epithelial marker (E-cadherin) and upregulation of mesenchymal markers (vimentin, fibronectin, and α-SMA), consistent with EMT induction. Data are presented as mean ± SD from three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus control group.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9043816/v1/a167ccc9e9c67bab49462041.jpg"},{"id":104599159,"identity":"a765f30b-190c-4f2d-94a1-f91f41b336c2","added_by":"auto","created_at":"2026-03-13 19:40:49","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":41012,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the role of CIPp-EVs in tumor microenvironment remodeling and metastasis promotion. CIPp-EVs activate macrophages into TAMs, which secrete M2-associated cytokines, including IL-10, COX2, and VEGF-A. These cytokines contribute to an immunosuppressive environment and enhance tumor cell behavior. TAMs also promote metastatic potential by inducing EMT, characterized by increased tumor cell migration and invasiveness. This feedback loop between tumor cells and TAMs facilitates tumor progression and metastasis.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9043816/v1/e4bee32fc378a258dad4989a.jpg"},{"id":106343581,"identity":"a09f2c4d-b2bf-4347-b3e5-d534fec444c6","added_by":"auto","created_at":"2026-04-07 16:06:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1586028,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9043816/v1/2bcc8d85-6252-4004-ab82-d926001fa1d9.pdf"},{"id":104599163,"identity":"d03ef5f7-b1db-41ef-8c29-84c2f714fe7e","added_by":"auto","created_at":"2026-03-13 19:40:51","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11910,"visible":true,"origin":"","legend":"","description":"","filename":"CIPpEVsTAMPolarizationTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9043816/v1/a3eeda984782812a8065d1ea.xlsx"},{"id":104599160,"identity":"3ced10ce-793b-40ba-b1e8-d9eeafbcfe91","added_by":"auto","created_at":"2026-03-13 19:40:49","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1141544,"visible":true,"origin":"","legend":"","description":"","filename":"CIPpEVsTAMPolarizationFigS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9043816/v1/68511cf135e03a9fd0af146d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extracellular Vesicles from Canine Mammary Tumor Cells Promote Macrophage M2 Polarization and Enhance Tumor Progression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the evolving landscape of cancer biology, Tumor-derived extracellular vesicles (TDEs) have recently emerged as key regulators of tumor growth, metastasis, and immune evasion (Zhang and Yu \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Olejarz et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). TDEs are nanosized, lipid bilayer-enclosed vesicles that carry various bioactive molecules, including tumor-derived proteins, lipids, microRNAs, and mRNA (Whiteside \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These vesicles deliver molecular cargo to recipient cells within the tumor microenvironment (TME), reprogramming it into an immunosuppressive and pro-tumoral niche (Reale et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe TME is a complex and dynamic ecosystem composed of immune cells, fibroblasts, and endothelial cells, which interact closely with tumor cells (Yang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Among the immune cells, macrophages play a central role in shaping the immune landscape under the influence of TDEs. Depending on external stimuli, macrophages can differentiate into either tumor-suppressive M1 or tumor-promoting M2 phenotypes (Boutilier and Elsawa \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In many solid tumors, TDEs have been shown to induce M2 polarization, leading to the formation of tumor-associated macrophages (TAMs) (Baig et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). TAMs secrete immunosuppressive cytokines (e.g., IL-10, TGF-β) and tumor-promoting factors (e.g., VEGF, MMPs), which suppress immune responses, promote angiogenesis, and induce epithelial\u0026ndash;mesenchymal transition (EMT), thereby enhancing tumor cell motility and invasiveness (Noy and Pollard \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). EMT is a key process in cancer metastasis and is strongly associated with poor prognosis (Ribatti et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThrough these mechanisms, TDEs create a tumor-favorable microenvironment via the TDE\u0026ndash;TAM axis (Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This axis has been extensively studied in various human solid tumors (Guo et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), including breast cancer, lung cancer (Pritchard et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and glioblastoma (Gabrusiewicz et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In breast cancer, TDEs have been reported to not only promote M2 polarization (Hao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) but also enhance cancer cell migration and lymph node metastasis (Piao et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Consequently, inhibitors of extracellular vesicle (EV) secretion are currently being explored as potential anti-cancer therapies (Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite significant advances in human oncology, studies on EV\u0026ndash;immune cell interactions remain limited in veterinary oncology. Canine mammary tumors (CMTs), one of the most common neoplasms in intact female dogs (Sleeckx et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), share clinical and molecular similarities with human breast cancer, making them valuable comparative oncology models (Abdelmegeed and Mohammed 2018). Previous studies have demonstrated that EVs derived from the CMT cell line CIPp may enhance proliferation and migration of CIPp cells themselves (Moccia et al. 2023). However, their effects on immune cells\u0026mdash;particularly on macrophage polarization\u0026mdash;remain unclear. To date, research on EV\u0026ndash;macrophage interactions in dogs has been limited to a few melanoma models (Kim et al. 2022), in which EVs were shown to promote M2 polarization and reinforce immunosuppressive conditions.\u003c/p\u003e \u003cp\u003eThis study was designed to test the hypothesis that EVs derived from the canine mammary gland tumor cell line CIPp polarize canine macrophages toward a TAM-like M2 phenotype, thereby promoting tumor progression via enhanced migration and EMT. This study provides novel insights into the role of EVs in shaping the immune landscape of canine mammary tumors. Given the conserved nature of EV\u0026ndash;TAM interactions across species, this work may inform the development of TAM-targeted therapies in both veterinary and human oncology.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and isolation of EV\u003c/h2\u003e \u003cp\u003eThe canine malignant mammary gland tumor cell line CIPp, established from a primary tumor, was obtained from the Department of Veterinary Clinicopathology at Seoul National University. Cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA; #11875093) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific; #16000-044) and 1% penicillin-streptomycin (PS; Thermo Fisher Scientific; #15140122) at 37◦C in a humidified atmosphere containing 5% CO2.\u003c/p\u003e \u003cp\u003eFor conditioned media collection, CIPp cells were seeded at 5000 cells/cm\u0026sup2; in 10-layer Culture Chambers (BioPioneer Inc., San Diego, CA, USA) and cultured in RPMI with 10% FBS until 80% confluence. The cells were washed three times with phosphate-buffered saline (PBS, Welgene, Gyeongsan, Gyeongbuk, Republic of Korea; #LB 004\u0026thinsp;\u0026minus;\u0026thinsp;01) to remove residual FBS and incubated for 24 hours in serum-free CD 293 medium (Thermo Fisher Scientific; #11913-019) supplemented with 1% GlutaMAX and 1% sodium pyruvate before harvesting the conditioned media (CM).\u003c/p\u003e \u003cp\u003eEVs were isolated from the CM using the tangential flow filtration (TFF)-based method. The conditioned media was first filtered through a 0.22-\u0026micro;m polyethersulfone membrane filter (Thermo Fisher Scientific) to remove cell debris and non-EV particles. The filtrate was then concentrated using a 700 kDa hollow-fiber cartridge (Cytiva, Marlborough, MA, USA) and diafiltered with PBS. The EV suspension was aliquoted into polypropylene disposable tubes, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Before use, frozen EVs were thawed completely at 4\u0026deg;C. Characterization and profiling of EVs were performed in accordance with the Minimal Information for Studies of Extracellular Vesicles 2018 (Th\u0026eacute;ry et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) recommended by the International Society of Extracellular Vesicles.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCharacterization of EV derived from CIPp cells\u003c/h3\u003e\n\u003cp\u003eTo verify that the isolated particles were EVs, western blotting was performed on CIPp cell lysates and EV preparations to assess the enrichment of EV-specific protein markers. Cells were lysed using a lysis buffer (Elpis Biotech, Daejeon, Republic of Korea; #EBA-1149) and kept on ice for 30 minutes. The lysates were then centrifuged at 12,000 rpm for 20 minutes at 4\u0026deg;C to remove cell debris. Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific; #23227). Equal amounts of protein were resolved by SDS\u0026ndash;polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Darmstadt, Germany; #IPVH00010).\u003c/p\u003e \u003cp\u003eMembranes were blocked for 1 hour at 21\u0026deg;C in TBST buffer containing either 5% skim milk (LPS Solution, Daejeon, Republic of Korea; #SKI500) or 5% bovine serum albumin (BSA, GeorgiaChem, Hwaseong, Republic of Korea; #G.C-BS1005). After blocking, the membranes were incubated overnight at 4\u0026deg;C with primary antibodies (1:1,000). Specifically, antibodies against CD9 (Cell Signaling Technology, Danvers, MA, USA; #13174), CD81 (System Biosciences, Palo Alto, CA, USA; #EXOAB-CD81A-1) and Alix (Cell Signaling Technology; #2171) were used as EV-positive markers. Additionally, β-actin (Cell Signaling Technology; #4970) was employed as a negative marker to assess the depletion of cellular contaminants.\u003c/p\u003e \u003cp\u003eFollowing TBST washes, the membranes were incubated for 1 hour at 21\u0026deg;C with HRP-conjugated secondary antibodies (1:2,000), including anti-rabbit and anti-mouse antibodies (ABclonal Technology; #AS014, #AS003). Protein bands were visualized using an enhanced chemiluminescence detection reagent (SmartGene, Seongnam, Republic of Korea; #SG-PR-HECL), and band images were acquired using the LAS-4000 imaging system (GE Healthcare Biosciences, Milwaukee, WI, USA).\u003c/p\u003e \u003cp\u003eNanoparticle tracking analysis (NTA) was performed to assess the size and concentration of EV samples, using a 642-nm laser NanoSight LM10 (Malvern Panalytical, Amesbury, UK). EV samples were diluted in PBS and analyzed using NTA 3.2 software (Malvern Panalytical Ltd., Worcestershire, UK). For each sample, at least five videos were recorded and averaged to obtain representative measurements.\u003c/p\u003e \u003cp\u003eEV morphology was further evaluated by transmission electron microscopy (TEM). A 10 \u0026micro;l EV suspension was applied on a carbon-coated grid and allowed to absorb for 1 minute, followed by removal of excess liquid with filter paper. A 2% uranyl acetate solution was then applied for 20 seconds as a negative stain, and excess stain was gently blotted before imaging. Images were acquired using a JEM1010 transmission electron microscope (Jeol Ltd., Tokyo, Japan) operating at 80 kV. All procedures were performed in triplicate to ensure reproducibility.\u003c/p\u003e\n\u003ch3\u003eDH82 cell culture and EV treatment\u003c/h3\u003e\n\u003cp\u003eThe canine macrophage cell line DH82 was purchased from ATCC (ATCC number: CRL-10389, Manassas, VA, USA). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Solbio, Suwon, Republic of Korea, #DME-001) supplemented with 10% FBS and 1% PS, maintained at 37\u0026deg;C in a 5% CO₂ incubator. DH82 cells were seeded for 12 hours, followed by a media change and treated with CIPp-EVs for an additional 36 hours.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eDH82 cell viability following exposure to CIPp-EVs was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan, #CK04), following the manufacturer's protocol. DH82 cells were seeded in 96-well plates at a density of 1.5 \u0026times; 10\u0026sup3; cells/well and cultured overnight to allow attachment. The medium was then replaced, and cells were treated with either fresh medium alone, 20% PBS, 10% EVs (19.5 \u0026micro;g/ml), or 20% EVs (39 \u0026micro;g/ml) for 36 hours. After adding 10 \u0026micro;l of CCK solution and incubating for 2 hours, cell viability was measured using Epoch Microplate Spectrophotometer (BioTek Korea Co., Ltd., Seoul, Republic of Korea).\u003c/p\u003e\n\u003ch3\u003eMorphological observation\u003c/h3\u003e\n\u003cp\u003eDH82 cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in 6-well plates for 12 hours. Seeded DH82 cells were treated with CIPp-EVs (39 \u0026micro;g/ml) for 36 hours. Cell morphology was observed using a Leica DMil microscope (Leica Microsystems, Wetzlar, Germany) to assess EV-induced morphological changes.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (qRT-PCR) of DH82 cells\u003c/h2\u003e \u003cp\u003eDH82 cells were seeded at 2 \u0026times; 10⁵ cells/well in 6-well plates and allowed to adhere for 12 hours before being treated with CIPp-EVs (39 \u0026micro;g/ml) for 36 hours. After treatment, total RNA was extracted using Easy-Blue Total RNA Extraction Kit (Intron Biotechnology, Daejeon, Korea, #17061) according to the manufacturer\u0026rsquo;s instructions, and RNA purity and concentration were measured using a spectrophotometer (NanoPhotometer NP80, Implen GmbH, Munich, Germany). Quantitative PCR was performed on a Qunatstudio 1 (Applied Biosystems, CA, USA) using SYBR Green qPCR Master Mix (SmartGene, #SG-SYBR-ROXH). Target gene expression levels were normalized to GAPDH, which served as the housekeeping control. Primer sequences utilized in this study are provided in Table S1. Triplicate reactions were carried out for all samples.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence assay\u003c/h3\u003e\n\u003cp\u003eTo analyze M1/M2 surface marker expression in DH82 cells, an immunofluorescence assay was performed. DH82 cells were seeded at a density of 2 \u0026times; 10⁴ cells per confocal dish (SPL Life Sciences, Pocheon, Gyeonggi-do, Republic of Korea, #101350) and incubated for 12 hours. Cells were then treated with CIPp-EVs (39 \u0026micro;g/ml) for 36 hours. As a positive control for M1 polarization, cells were exposed to 100 ng/ml Lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA, #L4391) for the same duration. Following treatment, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at 21\u0026deg;C. After additional PBS washes, cells were blocked with 5% BSA for 30 minutes at 21\u0026deg;C to prevent nonspecific binding. FITC-conjugated anti-mouse CD206 antibody (1:100 dilution, BioLegend, San Diego, CA, USA, #141703) and APC-conjugated anti-mouse CD86 antibody (1:10 dilution, Miltenyi Biotec, Bergisch Gladbach, Germany, #130-102-558) were applied to the cells, followed by incubation for 1 hour at 21\u0026deg;C. Cells were then washed three times with PBS, and coverslips were mounted with DAPI-containing mounting solution (Thermo Fisher Scientific, #00-4959-52). Fluorescence images were acquired using an EVOS FL imaging system (Thermo Fisher Scientific).\u003c/p\u003e\n\u003ch3\u003eFlow cytometry analysis\u003c/h3\u003e\n\u003cp\u003eFlow cytometric analysis was performed to evaluate M1 and M2 surface marker expression in DH82 cells. Cells were seeded at a density of 2 \u0026times; 10⁵ cells per well in 6‑well plates and allowed to adhere for 12 hours. The cultures were then divided into three groups: untreated controls, LPS-treated cells (200 ng/mL, 36 hours) to induce M1 polarization, and CIPp-EV-treated cells (39 \u0026micro;g/mL, 36 hours) to assess EV-mediated modulation of macrophage phenotype. Following treatment, cells were collected, washed with PBS, and fixed in 4% paraformaldehyde for 15 min at 21\u0026deg;C. To reduce nonspecific binding, cells were blocked with 5% BSA for 30 minutes. Staining was performed using the same FITC-conjugated CD206 and APC-conjugated CD86 antibodies described in the immunofluorescence assay, using identical dilutions. After a 1 hour incubation at 21\u0026deg;C in the dark, the cells were washed with PBS and resuspended in 200 \u0026micro;L PBS to obtain a single-cell suspension. Samples were analyzed using a FACS Aria II flow cytometer (BD Biosciences, San Jose, CA, USA), and data were processed with FlowJo software (Tree Star, Ashland, OR, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eConditioned media preparation\u003c/h2\u003e \u003cp\u003eDH82 cells were seeded in 100 mm dishes at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in complete DMEM medium for 12 hours. After attachment, the cultures were divided into three groups. The control group was maintained in fresh complete DMEM without EVs for an additional 36 hours. For EV treatment, cells were exposed to CIPp-EVs at 20% (v/v) for either 24 or 36 hours. Following the respective incubation periods, the cells were washed twice with PBS to completely remove any residual CIPp-EVs. Subsequently, the medium was replaced with fresh DMEM medium with 10% Exo-free FBS (Thermo Fisher Scientific, #A2720803) and 1% PS, and the cells were cultured for an additional 24 hours to produce CM. The CM was harvested, centrifuged at 800 x g for 3 minutes to remove residual cells, and the supernatant was stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eCIPp cells were seeded in 12-well plates at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well and cultured until confluence. To inhibit proliferation, cells were treated with 2 \u0026micro;g/ml of mitomycin (Enzo Life Science, Farmingdale, NY, USA, #BML-GR311) for 2 hours. The cell monolayer was then scratched with a 1 ml pipette tip and cell debris was removed by washing twice with PBS. The remaining adherent cells were cultured in complete DMEM supplemented with 50% CM. Wound healing was monitored at 0, 3, and 6 hours under a microscope using a TCapture program (Tucsen Photonics, Fuzhou, Fujian, China). Wound width was quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and relative width closure (%) was calculated using the following formula: [(Relative width at 0 h \u0026ndash; relative width at 3 h or 6 h)/relative width at 0 h] \u0026times;100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR of CIPp cells\u003c/h2\u003e \u003cp\u003eCIPp cells were seeded at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in 6-well plates and cultured for 12 hours. Seeded CIPp cells were then treated with 50% complete DMEM and 50% CM for 48 hours. Following incubation, total RNA was extracted and EMT marker expression was analyzed by qRT-PCR using the same procedure described for DH82 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed using the GraphPad Prism software (version 10.1.2, GraphPad Software Inc., San Diego, CA, USA). For the CCK-8 cell viability assay, data were analyzed using one-way ANOVA with Tukey\u0026rsquo;s multiple comparison test to assess differences among groups. For experiments involving comparisons between two groups (e.g., qRT-PCR, flow cytometry and wound healing), unpaired two-tailed Student\u0026rsquo;s t-tests were used. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of CIPp-EVs\u003c/h2\u003e \u003cp\u003eEVs were isolated from CIPp cells using TFF and the overall isolation process was schematically illustrated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). TEM confirmed that the isolated EVs displayed a typical round morphology with an approximate diameter of 50 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). As expected from the distinct measurement principles, NTA showed that the size distribution of CIPp-EVs was centered around 140 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Western blotting confirmed the enrichment of EV markers CD9 and CD81 in CIPp-EVs compared with CIPp cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Taken together, these findings verify that TFF-based isolation effectively yields structurally intact EVs with uniform characteristics, supporting their suitability for downstream applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImpact of CIPp-EVs on Macrophage Viability and Morphology\u003c/h2\u003e \u003cp\u003eThe effect of CIPp-EVs on the viability of DH82 cells was evaluated using a CCK-8 assay. Cells were exposed to two concentrations of CIPp-EVs (19.5 and 39 \u0026micro;g/ml) for 36 hours. No significant difference in cell viability was observed between the control and EV-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating that CIPp-EVs did not significantly affect cell viability at either dose. Based on these results, the higher concentration (39 \u0026micro;g/ml) was selected for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to evaluating cell viability, morphological changes in DH82 cells were examined after 36 hours of treatment with or without CIPp-EVs. The EV-treated cells exhibited a more elongated shape compared to the control group, suggesting alterations in cell morphology and a shift toward an M2-like phenotype in response to EV exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This observation is consistent with previous studies (McWhorter FY et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Heinrich et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pe et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which showed that M2-polarized macrophages characteristically adopt an elongated morphology, in contrast to the rounded form of M1 or unstimulated M0 macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCIPp-EVs Promote M2 Macrophage Polarization\u003c/h2\u003e \u003cp\u003eTo investigate the effects of CIPp-EVs on macrophage polarization, DH82 cells were co-cultured with CIPp-EVs (39 \u0026micro;g/ml) for 36 hours. The expression of both M1 and M2 macrophage markers was analyzed using qRT-PCR, immunofluorescence, and flow cytometry.\u003c/p\u003e \u003cp\u003eqRT-PCR analysis showed significant upregulation of M2-associated genes (CD206, VEGF-A, IL-10, and COX2) and downregulation of M1-associated genes (iNOS and IL-6) in EV-treated macrophages compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These transcriptional changes are consistent with a shift toward an M2 phenotype. Immunofluorescence staining further supported this finding. Control cells exhibited minimal expression of either marker, while LPS-treated cells (positive control) displayed bright CD86 (red) staining characteristic of M1 polarization. By contrast, CIPp-EV\u0026ndash;treated macrophages showed a pronounced increase in CD206 (green) signal, indicating M2 polarization at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Additionally, flow cytometry was performed to quantify the populations of M1 and M2 macrophages by measuring the surface expression of CD86 and CD206. The percentage of CD86\u003csup\u003e+\u003c/sup\u003e/CD206\u003csup\u003e\u0026minus;\u003c/sup\u003e (M1) macrophages did not differ significantly between control and CIPp-EV-treated groups. However, CIPp-EV-treated cells showed a significant increase in CD86\u003csup\u003e\u0026minus;\u003c/sup\u003e/CD206\u003csup\u003e+\u003c/sup\u003e (M2) macrophages (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming the polarization towards the M2, TAM-like phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these findings demonstrate that CIPp-EVs effectively induce TAM-like M2 macrophage polarization, as indicated by the upregulation of M2 markers at both the gene and protein levels, and the increase in M2 macrophage populations. This shift towards the M2 phenotype may contribute to the immunosuppressive and tumor promoting properties observed in the tumor microenvironment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTAMs Induced by CIPp-EVs Enhance CIPp Tumor Progression\u003c/h2\u003e \u003cp\u003eHaving confirmed that CIPp-EVs induce TAM-like M2 macrophage polarization in DH82 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the impact of these macrophages on CIPp tumor cell behavior was subsequently evaluated. Treatment of CIPp tumor cells with CM derived from DH82 macrophages exposed to CIPp-EVs for 24 hours (CIPp-EV\u0026ndash;DH82-CM) significantly increased the migratory activity of CIPp cells compared to CM from untreated DH82 macrophages (Control DH82-CM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, CM collected from 36-hour EV-treated DH82 cells showed no significant effect (Fig. S1). These findings suggest that 24-hour CM contains a higher level of TAM-associated factors, and was therefore selected for all subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRepresentative images taken at 0, 3, and 6 hours illustrated a pronounced reduction in the wound gap in the CIPp-EV\u0026ndash;DH82-CM group compared to the Control DH82-CM group. Quantitative analysis confirmed a significant increase in relative migration at 3 hours (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and an even greater effect at 6 hours (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To further assess TAM-mediated effects on tumor progression, EMT-related markers were analyzed. The CIPp-EV\u0026ndash;DH82-CM group exhibited marked downregulation of the epithelial marker E-cadherin (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and upregulation of mesenchymal markers such as vimentin, fibronectin, and α-SMA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for vimentin and fibronectin; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for α-SMA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results indicate that TAM-derived factors promote EMT and enhance tumor invasiveness. Collectively, the results demonstrate that CIPp-EVs induce TAM-like polarization in macrophages, which in turn promotes CIPp tumor cell migration and EMT, thereby contributing to a more aggressive tumor microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussions","content":"\u003cp\u003eThis study demonstrates that CIPp-EVs promote M2 polarization of canine macrophage cell line DH82, and that these M2-polarized macrophages, in turn, enhance EMT and migratory potential in tumor cells. Together, these findings suggest that interactions among EVs, macrophages, and tumor cells establish a self-reinforcing feedback loop within the TME, thereby driving tumor malignancy and progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTDEs are recognized as key modulators of the TME, notably by polarizing macrophages toward an M2 TAM phenotype (Baig et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). In both canine and human mammary carcinomas, studies show that infiltration of M2 TAMs is strongly associated with advanced clinical stage, shorter survival, and disrupted extracellular matrix organization (Monteiro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jeong et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Allison et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Garcia et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). TAMs have been shown to promote tumor progression through multiple mechanisms, including the secretion of immunosuppressive cytokines, angiogenic factors, and extracellular matrix\u0026ndash;remodeling enzymes, thereby fostering an invasive and immune-suppressive microenvironment (Yang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e; Bied et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Beyond these correlative findings, mechanistic studies in human oncology have demonstrated that tumor-derived EVs act upstream of macrophage polarization toward the M2 phenotype, thereby promoting the establishment of a tumor-supportive microenvironment and enhancing tumor progression (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Reed et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tian et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). However, whether a comparable EV\u0026ndash;TAM axis exists in canine tumors remains largely unexplored. The present study addresses this gap by providing functional evidence for EV-driven TAM polarization and its downstream effects in a canine mammary tumor model.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that the activation of the STAT3 signaling pathway reprograms macrophages toward a tumor-promoting M2-like phenotype across multiple cancers. (Takaishi et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ham et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Irey et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mohammad et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In our study, CIPp-EVs induced hallmark features of M2 polarization, including elongated morphology, increased CD206 expression, and elevated secretion of cytokines such as VEGF-A and IL-10 (McWhorter et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rőszer \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Given that IL-10 is a known upstream activator of STAT3 in macrophages (Murray \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hutchins et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), it is hypothesized that CIPp-EVs may engage a positive feedback mechanism wherein EV-induced IL-10 secretion reinforces STAT3 signaling, thereby stabilizing the TAM-like phenotype. While the direct activation of this signaling pathway was not experimentally validated in the current study, the observed pattern of IL-10 upregulation provides a strong rationale for future investigations into STAT3-dependent mechanisms for conditioning and the establishment of a protumorigenic microenvironment.\u003c/p\u003e \u003cp\u003eImportantly, M2-polarized macrophages are not only immunosuppressive but also potent inducers of EMT via secretion of IL-10, VEGF, and TGF-β (Feng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These cytokines repress epithelial markers such as E-cadherin while inducing mesenchymal markers including vimentin, N-cadherin, and fibronectin, thereby enhancing tumor cell motility and invasiveness (Ribatti et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Multiple studies have demonstrated that IL-10, VEGF, and TGF-β secreted by TAMs are critical mediators of EMT, and inhibition of each factor effectively attenuates TAM-induced EMT and tumor cell invasiveness (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cai et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, CM from CIPp-EV\u0026ndash;treated macrophages downregulated E-cadherin and upregulated vimentin and fibronectin in CIPp tumor cells, accompanied by enhanced migratory capacity, confirming EMT induction. It should be noted that the term \"TAM-like macrophages\" used in this study is based on marker expression patterns and in vitro functional effects, rather than comprehensive in vivo functional validation. Nevertheless, the collective findings suggest that CIPp-EVs reprogram macrophages toward TAM-like phenotype, potentially via a STAT3\u0026ndash;IL-10 pathway, and these reprogrammed macrophages, in turn, drive EMT through IL-10 and VEGF signaling\u0026mdash;establishing a self-reinforcing EV\u0026ndash;TAM\u0026ndash;tumor axis that promotes tumor progression in canine mammary carcinoma.\u003c/p\u003e \u003cp\u003eIn this study, to ensure the high purity and integrity of EVs required for functional assays, tangential Flow Filtration (TFF) was utilized. This method allows for the scalable isolation of EVs with high yield and purity even from large-volume or diluted samples (Busatto et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Liangsupree et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Subsequent NTA analysis demonstrated that TFF-isolated EVs exhibited a narrow and uniform size distribution, supporting the suitability of this method for elucidating the biological roles of CIPp-EVs.\u003c/p\u003e \u003cp\u003eGiven the central role of the EV\u0026ndash;TAM axis in establishing a tumor-supportive microenvironment, targeting this pathway has emerged as a promising therapeutic strategy in preclinical cancer models (Im et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our findings demonstrate that TDEs are key drivers of macrophage polarization. Therefore, strategies aimed at inhibiting EV biogenesis or secretion could effectively disrupt this pro-tumorigenic crosstalk. While specific inhibitors were not evaluated in this study, the potential to modulate the TME by severing the EV-mediated communication loop suggests that EV-targeted therapies warrant further investigation as a novel treatment approach for canine mammary carcinoma.\u003c/p\u003e \u003cp\u003eThis study has several limitations. As the experiments were conducted in vitro, these conditions cannot fully recapitulate the complexity of the in vivo tumor microenvironment. Notably, while EV dosing in this study was standardized by protein concentration (\u0026micro;g/mL), future in vivo investigations should adopt particle-based dosing strategies to ensure greater physiological relevance. Additionally, the use of single tumor (CIPp) and macrophage (DH82) cell lines limits generalizability. Thus, validation in primary cells or multiple tumor models is required. Finally, although the STAT3 axis was implicated as a potential mediator, the precise intracellular signaling cascades driving M2 polarization warrant further detailed molecular characterization.\u003c/p\u003e \u003cp\u003eIn conclusion, this study highlights the pivotal role of CIPp-EVs in remodeling the TME through the induction of a pro-tumorigenic feedback loop. These EVs promote the polarization of macrophages into TAMs, which in turn secrete factors that enhance tumor cell migration and EMT, thereby increasing invasive and metastatic potential. Collectively, the findings underscore the significance of the EV\u0026ndash;TAM axis in the progression of canine mammary carcinoma. A deeper understanding of this EV\u0026ndash;TAM\u0026ndash;tumor cell axis may provide a conceptual framework for developing EV- or TAM-targeted therapeutic strategies in canine mammary tumors and other human cancer models that share similar microenvironmental mechanism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interest\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompliance with ethical standards\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by the BK 21 Plus Program for Creative Veterinary Science Research, the Research Institute for Veterinary Science, and the College of Veterinary Medicine.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eNa-Kyoung Chi conceived and designed the study, conducted the experiments, and drafted the manuscript. Se-Hoon Kim, Ju-Hyun An and Ga-Hyun Lim contributed to the execution of the assays and data acquisition. Ki-Hoon Song and Min-Ok Ryu critically reviewed and edited the manuscript for intellectual content. Kyoung-Won Seo validated the original data, provided critical revisions, and supervised the overall study. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eSincere appreciation is extended to Dr. Sung Youl Kim at GNG CELL Co., Ltd. (R\u0026amp;D Center, 122 Unjung-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 13466, Republic of Korea) for generously providing the CIPp-derived extracellular vesicles used in the study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAllison E, Edirimanne S, Matthews J, Fuller SJ (2022) Breast cancer survival outcomes and tumor-associated macrophage markers: a systematic review and meta-analysis. 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Int J Biol Sci 20:5109\u0026ndash;5126. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7150/ijbs.99680\u003c/span\u003e\u003cspan address=\"10.7150/ijbs.99680\" 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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Canine mammary tumor, Epithelial-mesenchymal transition (EMT), Extracellular vesicle, Tumor-associated macrophages (TAMs), Tumor microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-9043816/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9043816/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtracellular vesicles (EVs) derived from tumor cells play a crucial role in modulating and reprogramming the tumor microenvironment (TME), thereby promoting tumor progression. While previous studies have investigated the impact of tumor-derived EVs on macrophages in various cancers, such as bladder cancer and osteosarcoma, and in canines, particularly in melanoma, little is known about the effects of canine mammary tumors influence on macrophage polarization. This study aimed to elucidate the functional role of EVs derived from CIPp cells, a canine mammary tumor cell line, in macrophage polarization and tumor progression. CIPp-derived EVs were isolated and characterized, and their effects on macrophage phenotype were examined using the canine macrophage cell line DH82. Exposure to CIPp-EVs induced DH82 macrophages to adopt a tumor-associated macrophage (TAM)-like M2 phenotype, as confirmed by quantitative RT-PCR, immunofluorescence, and flow cytometry. CIPp-EV treatment significantly upregulated M2-associated markers (CD206, VEGF-A, IL-10, and COX2) while downregulating M1-associated markers (iNOS and IL-6). Furthermore, conditioned media (CM) from CIPp-EV\u0026ndash;treated macrophages enhanced CIPp tumor cell migration and induced epithelial\u0026ndash;mesenchymal transition (EMT), evidenced by the reduction of E-cadherin and increased expression of vimentin, fibronectin, and α-SMA. Together, these findings demonstrate that CIPp-derived EVs reprogram the TME by driving TAM-like M2 macrophage polarization, which in turn promotes tumor cell migration and EMT, thereby facilitating tumor progression and metastasis.\u003c/p\u003e","manuscriptTitle":"Extracellular Vesicles from Canine Mammary Tumor Cells Promote Macrophage M2 Polarization and Enhance Tumor Progression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 19:40:45","doi":"10.21203/rs.3.rs-9043816/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2026-03-21T08:20:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-21T06:57:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-17T15:49:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119115014737775228777898309020689240987","date":"2026-03-17T15:38:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70406554844246587180828677159238786395","date":"2026-03-14T06:34:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-11T13:30:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-11T02:46:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-11T02:45:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2026-03-05T19:44:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c59101a0-05bc-45d8-8206-52de111d51bf","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:02:43+00:00","versionOfRecord":{"articleIdentity":"rs-9043816","link":"https://doi.org/10.1007/s11259-026-11179-3","journal":{"identity":"veterinary-research-communications","isVorOnly":false,"title":"Veterinary Research Communications"},"publishedOn":"2026-03-30 15:59:13","publishedOnDateReadable":"March 30th, 2026"},"versionCreatedAt":"2026-03-13 19:40:45","video":"","vorDoi":"10.1007/s11259-026-11179-3","vorDoiUrl":"https://doi.org/10.1007/s11259-026-11179-3","workflowStages":[]},"version":"v1","identity":"rs-9043816","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9043816","identity":"rs-9043816","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}