Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1

Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1 | 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 Article Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1 Ayano Kudo, Shintaro Kamo, Akinori Yamauchi, Sho Yoshimoto, Yuma Harada, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6575028/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that modulate T cell immunity by secreting humoral factors and forming structural barriers. CAFs secrete the chemokine C-X-C motif chemokine ligand 12 (CXCL12), which binds to C-X-C chemokine receptor 4 (CXCR4) on T cells and induces chemotaxis. Transforming growth factor beta 1 (TGF-β1), another CAF-derived humoral factor, is believed to regulate the CXCL12/CXCR4 axis; however, a direct association between them has not been demonstrated in human medicine or veterinary medicine. This study investigated the role of canine CAFs in T cell migration via the CXCL12/CXCR4 axis and the regulatory influence of TGF-β1. CXCL12 and CXCR4 were expressed in the tumor stroma and on T cells, respectively, in dogs with epithelial malignant tumors. Canine CAFs secreted higher levels of CXCL12 and TGF-β1 than normal fibroblasts, and CAF-derived TGF-β1 modulated both CXCL12 secretion by CAFs and CXCR4 expression on T cells. Furthermore, canine CAFs induced T cell migration through the CXCL12/CXCR4 axis. These findings indicate that CAFs regulate T cell migration via the CXCL12/CXCR4 axis under TGF-β1 signaling, highlighting their critical role within the TME. Biological sciences/Cancer/Cancer microenvironment Biological sciences/Cancer/Tumour immunology Biological sciences/Cell biology/Cell migration/Chemotaxis Cancer-associated fibroblasts Tumor microenvironment Dog C-X-C motif chemokine ligand 12 C-X-C chemokine receptor 4 Transforming growth factor-β1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The tumor microenvironment (TME) is a highly intricate structure comprised of tumor and stromal cells, including immune cells, endothelial cells, and fibroblasts. Among these, cancer-associated fibroblasts (CAFs) are recognized as a principal component of the tumor stroma [ 1 ] . CAFs enhance tumor malignancy by promoting angiogenesis, proliferation, invasion, and metastasis. Notably, their role in modulating T cell immunity has garnered significant attention. The inhibition of T cell infiltration into solid tumors by CAFs results from the secretion of various humoral factors and their function as a physical barrier, characterized by dense extracellular matrix fiber networks [ 2 , 3 ] . The C-X-C motif chemokine ligand 12 (CXCL12) is one of the chemokines secreted by CAFs [ 2 ] ,and it interacts with the C-X-C chemokine receptor 4 (CXCR4) on T cell membranes, inducing chemotaxis, cell proliferation and gene expression [ 3 , 4 ] . High expression levels of CXCL12 in the tumor stroma have been detected in various human cancers, including gastric, bladder, and ovarian cancer through immunohistochemistry (IHC), and CXCL12 upregulation correlated with poor prognosis [ 5 – 7 ] . In CXCL12-positive stroma, more T cells infiltrate the stroma than the tumor component, which is suspected to be a factor that limits a patient’s prognosis [ 7 ] . In vitro studies have further demonstrated that human CAFs secrete higher levels of CXCL12 than normal fibroblasts (NFs) [ 5 , 8 , 9 ] and that these CAFs promote T cell migration via CXCL12 [ 8 ] . As for the receptor CXCR4, CD8 + T cells exhibited higher CXCR4 expression in patients of pancreatic ductal adenocarcinoma compared with healthy individuals [ 8 ] . Considering these findings, the combination of CXCL12/CXCR4 inhibitors with an immune checkpoint inhibitor has been examined in mouse models and shown to significantly enhance antitumor effects [ 10 – 12 ] . This strategy is currently being explored in human medicine, with phase Ⅰ-Ⅱ clinical trials underway. Transforming growth factor beta (TGF-β), another humoral factor secreted by CAFs, has been implicated to modulate the CXCL12/CXCR4 axis. Human CAFs secrete higher levels of TGF-β than NFs [ 13 ] , and TGF-β mediated autocrine or paracrine signaling activates CAFs, increasing CXCL12 secretion and subsequent tumor progression [ 13 , 14 ] . Previous studies have reported that TGF-β isoforms increase CXCR4 expression on human CD4 + T cells [ 15 ] and the culture supernatant of human CAFs upregulates CXCR4 on T cells [ 16 ] . Although the relationship between CXCR4 expression and CAF-derived TGF-β remains unclear, these findings suggest that CAFs modulate T cell migration within the TME by regulating the CXCL12/CXCR4 axis through TGF-β. However, direct evidence for this interaction has not been reported. In veterinary medicine, research on the biology of CAFs remains limited. Several studies isolating canine CAFs from epithelial malignancies have shown that canine CAFs share functional similarities with human CAFs, including high expression of alpha smooth muscle actin (α-SMA), a marker of activated fibroblasts, and promotion of tumor cell migration and invasion via humoral factors [ 17 – 19 ] . Serum CXCL12 levels have been reported to be significantly higher in dogs with metastases of mammary carcinoma compared with healthy dogs [ 20 ] . In addition, serum TGF-β levels have been reported to be significantly higher in tumor-bearing dogs than in healthy controls [ 21 ] . However, the association between the presence of CAFs and these serum chemokines in veterinary medicine is unknown. Immunohistochemical analysis of 13 cases of canine mammary gland tumor stroma did not reveal differences in CXCL12 expression between normal stromal regions and the tumor stroma [ 22 ] . However, studies on the CXCL12/CXCR4 axis in canine CAFs have not been reported, and the effect of canine CAFs on T cell migration via the CXCL12/CXCR4 axis remains unexplored. This study aimed to examine the influence of canine CAFs on T cell migration through the CXCL12/CXCR4 axis and elucidate the mechanism underlying TGF-β-mediated regulation of this axis by CAFs, an area that remains underexplored in both veterinary and human medicine. Results CXCL12 expression in the stroma of canine epithelial malignant tumors CXCL12 expression in the stroma of tumor tissues obtained from 70 cases of epithelial malignant tumors was investigated (Fig. 1 A-D). CXCL12 expression was detected in the cytoplasm of stromal cells adjacent to tumor cells. Among the tumor types, CXCL12 expression was detected in cases of thyroid carcinoma (7/10), renal cell carcinoma (6/10), intestinal adenocarcinoma (3/5), prostate adenocarcinoma (5/10), hepatocellular carcinoma (2/5), mammary gland carcinoma (2/5), lung adenocarcinoma (3/10) and transitional cell carcinoma (2/10) (Table 1 ). The CXCL12 expression score for thyroid carcinoma and renal cell carcinoma tended to be higher than that for other tumors. Fibroblasts in the tumor stroma were positive for α-SMA in all cases (Fig. 1 E-H). Hematoxylin and eosin staining was performed to confirm the tumor and stromal regions (Fig. 1 I-L). Table 1 CXCL12 expression in various epithelial malignant tumors. Pathology Positive no. / tested no. Positive rate (%) CXCL12 score in tumor stroma 1 2 3 Thyroid carcinoma 7/10 70 2 1 4 Renal cell carcinoma 6/10 60 2 1 3 Intestinal adenocarcinoma 3/5 60 3 0 0 Prostate adenocarcinoma 5/10 50 3 2 0 Hepatocellular carcinoma 2/5 40 1 0 1 Mammary gland carcinoma 2/5 40 2 0 0 Lung adenocarcinoma 3/10 30 1 1 1 Transitional cell carcinoma 2/10 20 1 1 0 Anal sac gland carcinoma 0/5 0 0 0 0 CXCL12 score in tumor stroma was scored based on the following criteria: 0, no or ≤ 10% of stromal cells stained; 1, > 10% of stromal cells stained with weak intensity; 2, > 10% of stromal cells stained with moderate intensity; and 3, > 10% of stromal cells stained with strong intensity. Isolation of canine CAFs and detection of CXCL12 protein Primary cultures of canine CAFs were established from surgically resected tumor tissues obtained from 10 dogs diagnosed with epithelial malignant tumors. The patient characteristics are summarized in Supplementary Table 1. NFs were also isolated from the skin tissue of the same patients, although primary cultures could not be established in all cases. The cells obtained were purified by passaging once or twice after the initial culture, and each cell had elongated, spindle-shaped fibroblast features (Fig. 2 A). Fibroblasts were identified based on positive staining for vimentin and negative staining for cytokeratin (Fig. 2 B). CAFs showed positive staining for α-SMA, while NFs showed weak expression in only a subset of cells. CXCL12 expression in CAFs was higher than that in NFs, with particularly strong expression observed around the nucleus. TGF-β1-mediated regulation of CXCL12 secretion from canine CAFs CXCL12 protein levels in culture media from canine NFs (NF-CM) and CAFs (CAF-CM) were quantified using enzyme-linked immunosorbent assay (ELISA). Canine CAFs secreted significantly higher amounts of CXCL12 than NFs ( P = 0.0379) (Fig. 3 A). Serum CXCL12 concentrations were also evaluated in healthy dogs and in 24 dogs with epithelial malignant tumors (Supplementary Fig. 1). A significant correlation was observed between CXCL12 levels in CAF-CM and serum CXCL12 levels (rₛ = 0.9286, P = 0.0067) (Fig. 3 B). Next, TGF-β1 protein levels in NF-CM and CAF-CM were quantified. Canine CAFs secreted significantly higher amounts of TGF-β1 than NFs ( P = 0.0007) (Fig. 3 C). A significant correlation was observed between CXCL12 and TGF-β1 levels in CAF-CM (rₛ = 0.7091, P = 0.0268) (Supplementary Fig. 2). To assess the effect of CAF-derived TGF-β1 on CXCL12 secretion, CAFs were cultured in the presence of the TGF-β receptor inhibitor SB431542, which significantly reduced CXCL12 secretion from CAFs ( P = 0.0335) (Fig. 3 D). Increased CXCR4 expression on canine T cells in epithelial malignant tumors Surface expression of CXCR4 on canine T cells was evaluated in 10 healthy dogs and 26 dogs with epithelial malignant tumors via flow cytometry. The types of epithelial malignant tumor included in this study were as follows: thyroid carcinoma (n = 5); renal cell carcinoma (n = 4); hepatocellular carcinoma (n = 4); lung adenocarcinoma (n = 3); squamous cell carcinoma (n = 3); anal sac gland carcinoma (n = 2); salivary gland carcinoma (n = 2); and one case each of perianal adenocarcinoma and transitional cell carcinoma. Representative data from a healthy beagle and a dog with thyroid carcinoma are shown in Fig. 4 A. CXCR4 expression on CD8 − and CD8 + T cells in dogs with epithelial malignant tumors was significantly higher than that in healthy dogs ( P = 0.0005 and P = 0.0402, respectively) (Fig. 4 B). Upregulation of CXCR4 on canine T cells by CAF-derived TGF-β1 The effect of TGF-β1 on CXCR4 expression in canine T cells was evaluated by culturing canine peripheral blood mononuclear cells (cPBMCs) with recombinant TGF-β1 or CAF-CM. CXCR4 expression on CD4 + and CD8 + T cells was significantly elevated by TGF-β1 stimulation ( P < 0.0001 for both), and this upregulation was abolished by the addition of the TGF-β1 inhibitor SB431542 ( P = 0.0002 for both) (Fig. 5 A). Similarly, culturing with CAF-CM significantly elevated CXCR4 expression on CD4 + and CD8 + T cells ( P = 0.0005 and P = 0.0303, respectively), and this effect was suppressed by SB431542 treatment ( P < 0.0001 for both) (Fig. 5 B). In comparison, culturing with NF-CM resulted in a lower increase in CXCR4 expression than that observed with CAF-CM. However, CXCR4 expression on CD4 + T cells still significantly increased ( P = 0.0405), and this effect was suppressed by SB431542 treatment. CAF-derived CXCL12 enhanced migration of canine CD8 + T cells We determined the effect of the CXCL12/CXCR4 axis on the migration of canine CD8 + T cells using migration assays. CD8 + T cell migration towards recombinant CXCL12 was significantly higher compared with the control (Fig. 6 A) ( P = 0.0011). Pretreatment with the CXCR4 antagonist AMD3100 significantly reduced this migration ( P = 0.0018). Moreover, the number of CD8 + T cells that migrated into CAF-CM was significantly higher compared with the control ( P = 0.0013), and treatment with AMD3100 significantly abolished this increased migration (Fig. 6 B) ( P = 0.0004). T cells attached to CAFs rather than cancer cells via the CXCL12/CXCR4 axis T cells, CAFs, and canine cancer cells were co-cultured. The number of T cells attached to CAFs (small spheres) decreased when cultured in the presence of AMD3100 (Fig. 6 C). The density of attached T cells per cm 2 on CAFs was significantly reduced when co-cultured with RCM-SO in the presence of AMD3100 (Fig. 6 D) ( P = 0.0475), whereas no significant decrease was found when co-cultured with NMTCC ( P = 0.0955). Discussion CAFs are a major component of the TME, and understanding how they suppress T cells is crucial for cancer immunology [ 1 , 23 ] . In multiple solid tumors, T cells are often confined to the stroma and are therefore unable to reach cancer cells. Their inability to access cancer cells may negatively affect patient prognosis [ 24 ] . Recently, CAFs have been suggested to attract T cells via the CXCL12/CXCR4 axis [ 7 , 8 , 11 , 25 ] . While there is limited evidence, CAF-derived TGF-β has been reported to modulate this axis [ 13 ] . However, research on canine CAFs is lacking, and the interaction between canine CAFs and T cell immunity remains largely unexplored. In this study, we evaluated the effect of canine CAFs on T cell migration through the CXCL12/CXCR4 axis and further investigated how CAF-derived TGF-β1 regulates this pathway. Elucidating the factors that control the CXCL12/CXCR4 axis within the TME may provide insights into potential therapeutic strategies for addressing the effect of CAFs on T cell migration. CXCL12 expression in tumor stroma has been examined in several human cancers, including bladder, gastric and ovarian cancers, and is often associated with poor prognosis [ 5 , 7 , 26 , 27 ] . In addition, CAFs isolated from human tumor tissues secreted CXCL12, the level of which tended to be higher than that secreted by NFs [ 5 , 28 ] . Consistent with these findings, we detected CXCL12 expression in the stroma of eight out of the nine canine tumors examined. The association between CXCL12 expression in the tumor stroma and prognosis was not investigated in this study owing to the limited sample size and variable tumor types. Future studies should increase the sample size and focus on specific tumor types to better elucidate the prognostic significance of stromal CXCL12 expression in canine cancers. Furthermore, we found that canine CAFs secreted higher levels of CXCL12 than NFs and that the amount of CXCL12 secreted by CAFs significantly correlated with serum CXCL12 concentrations in the same dogs. While most investigations of CXCL12 have focused on its expression in tissues or cultured cells, some clinical studies have evaluated serum CXCL12 as a potential prognostic biomarker in human medicine. For example, elevated serum CXCL12 levels have been reported in patients with advanced gastric carcinoma and head and neck cancers compared with healthy individuals [ 29 , 30 ] . Although CXCL12 levels can also be elevated in conditions such as inflammation, neural injury, and ischemic diseases [ 31 – 33 ] , the observed correlation in our study suggests that increased serum CXCL12 may reflect secretion from CAFs within the TME into the systemic circulation. This study also identified a regulatory mechanism whereby inhibition of TGF-β1 led to a decrease in CXCL12 secretion by CAFs. This is further supported by the strong positive correlation between CXCL12 and TGF-β1 secretion by CAFs. A previous study of human mammary carcinoma-derived CAFs suggested that autocrine TGF-β can upregulate CXCL12 [ 13 ] , and the present study demonstrates that CAF-derived TGF-β regulates CXCL12 secretion by CAFs. Furthermore, given that serum TGF-β levels are higher in tumor-bearing dogs compared with healthy controls [ 21 , 34 ] , and that TGF-β is also secreted by regulatory T cells and cancer cells [ 35 ] , TGF-β from various sources within the TME may contribute to the increased CXCL12 secretion by CAFs. Upregulation of CXCR4 on T cells is associated with various factors, such as hypoxia, TGF-β, vascular endothelial growth factor (VEGF), interleukin (IL)-2, IL-4, IL-7, and IL-15 [ 36 , 37 ] . In this study, TGF-β1-derived canine CAFs upregulated CXCR4 on the surface membrane of T cells. A previous human medicine report showed that culture supernatants from CAFs increased CXCR4 expression on T cells and that treatment with an anti-TGF-β neutralizing antibody did not prevent this increase in CXCR4 expression [ 16 ] . In the present study, the role of TGF-β1 could have been more directly assessed by using an inhibitor of the activin receptor-like kinase 5 (ALK5) pathway, which is downstream of the TGF-β1 receptor. The results of the in vitro experiments suggest that TGF-β1 secretion by CAFs contributes to the increased CXCR4 expression in cancer-bearing dogs. However, additional factors such as hypoxia and other cytokines may be involved in in vivo conditions. Further studies examining the relationship between serum TGF-β1 concentration and CXCR4 expression on T cells will provide valuable insights. The CXCL12/CXCR4 axis has been implicated in the CAF-mediated inhibition of T cell migration. In a human study using CXCL12 knockdown, activated pancreatic stellate cells, which are considered to have a similar function to CAFs, promoted T cell migration via CXCL12 [ 8 ] . Our study demonstrated that the CXCL12/CXCR4 axis facilitates T cell migration induced by canine CAFs, as shown by the inhibitory effects of the CXCR4 antagonist AMD3100. By using AMD3100, an inhibitor of CXCR4, it was possible to directly demonstrate that migration is promoted by the effect of CAF-derived CXCL12 on CXCR4 in T cells. In the co-culture system of T cells, CAFs, and cancer cells, a greater number of T cells adhered to CAFs when co-cultured with RCM-SO, and this adhesion was reduced by CXCR4 inhibition. Co-culture of breast cancer cell lines (MCF-7 and MDA-MB231) and human CAFs resulted in mutual activation, wherein CAFs exhibited an mRNA upregulation of tumor-promoting factors, including CXCL12 [ 38 , 39 ] . In this study, a greater number of T cells adhered to CAFs when co-cultured with RCM-SO than with NMTCC. This may be due to differences in the activation of CAFs by cancer cells depending on the cell type and origin. Our findings highlight the critical role of CAFs in regulating T cell migration via the CXCL12/CXCR4 axis, which is modulated by TGF-β1. A similar mechanism has been identified in wound healing, wherein TGF-β enhances CXCL12 secretion from activated fibroblasts and upregulates CXCR4 on macrophages and T cells. This mechanism may promote immune cell recruitment to the wound site [ 40 – 42 ] . This similarity suggests that the innate immunomodulatory functions of fibroblasts, which are essential for wound healing, may negatively affect the TME by trapping T cells in the stroma and inhibiting their migration to the tumor cells. Thus, therapeutic strategies targeting the CXCL12/CXCR4 axis or TGF-β1 signaling require careful design, as they may lead to delayed wound healing as a potential systemic side effect. Cancer immunotherapy, such as immune checkpoint inhibitor or chimeric antigen receptor-T cell therapy, has emerged as a promising treatment strategy in recent decades. However, the suppression of T cell immunity via the CXCL12/CXCR4 axis may negatively affect the effectiveness of CAR-T therapy in solid tumors [ 43 ] . Previous human studies have reported that CXCR4 is significantly upregulated in patients showing resistance to immunotherapy [ 44 ] . Moreover, blockade of CXCR4 has been shown to enhance the efficacy of anti-PD-1/PD-L1 antibodies, leading to increased T cell infiltration within the TME [ 11 ] . The findings of the present study suggest that CAFs are involved in T cell recruitment via the CXCL12/CXCR4 axis, which may influence the efficacy of tumor immunotherapy. Elucidating the precise role of this axis could lead to targeted interventions that improve the outcomes of cancer immunotherapy by overcoming CAF-mediated T cell suppression. Despite the findings, this study had some limitations. One limitation was the difficulty in integrating the original tumor types of CAFs. Given the diverse biological behavior of canine epithelial malignancies, the properties of CAFs may vary between tumor types. Additionally, performing a subset analysis of CAFs in this study was difficult. In human medicine, CAFs expressing CXCL12 are known as inflammatory CAFs (iCAFs), which are defined by assessing the expression of multiple markers [ 45 – 47 ] . Human studies have begun to subclassify CAFs based on distinct markers using single-cell analysis [ 48 , 49 ] . Future research should aim to identify markers applicable to canine CAFs and evaluate their functional differences. In conclusion, this study demonstrates that CAFs regulate T cell migration through the CXCL12/CXCR4 axis under the influence of TGF-β1 signaling (Fig. 7 ). These results underscore the pivotal role of CAFs in the TME, providing valuable insights applicable to both veterinary and human oncology. Our results contribute to the theoretical foundation for novel therapeutic strategies that may involve targeting the CXCL12/CXCR4 pathway modulated by CAFs, with the ultimate goal of improving cancer outcomes across species. Methods Canine Samples All animal experiments were conducted in accordance with relevant guidelines and regulations, and were approved by the Animal Experiment Committees of Azabu University (approval no. 220316-26). Skin and malignant epithelial tumor tissues were collected from dogs that underwent surgical resection for epithelial malignancies at the Azabu University Veterinary Teaching Hospital (Japan). Resected tissues were submitted for histopathological diagnosis, and formalin-fixed specimens were archived at commercial pathology laboratories (Patho Lab Co., Ltd., Shizuoka, Japan; North Lab, Sapporo, Japan). Peripheral blood was obtained from healthy Beagles (1–6 years old) housed in the Experimental Animal Facility of Azabu University and from client-owned dogs with epithelial malignant tumors treated at the teaching hospital. Written informed consent was obtained from all owners before inclusion of their animals in the study. IHC Formalin-fixed, paraffin-embedded tissue samples were used for IHC analysis. Freshly cut tissue sections (3 µm) were mounted on glass slides, deparaffinized, and subjected to antigen retrieval (CXCL12, microwaving at 98°C for 15 min; α-SMA, autoclaving at 121℃ for 20 min). Primary antibody staining was performed using mouse anti-CXCL12 (clone 79018; diluted 1:100; R&D Systems, Minneapolis, MN, USA) and mouse anti-α-SMA (clone: 1A4, diluted 1:1000, Sigma-Aldrich, St. Louis, MO, USA) monoclonal antibodies, with incubation at 4°C overnight. Histofine simple stain kit MAX-PO (MULTI) (Nichirei Bioscience Inc., Tokyo, Japan) was used as the secondary antibody, and hematoxylin was used as a counterstain. Canine tonsil tissue sections were used as a positive control for CXCL12, and vascular smooth muscle was used as an internal positive control for α-SMA. Sections without primary antibody staining were used as negative controls. All tissue sections were also stained with standard hematoxylin and eosin for histological examination. CXCL12 staining intensity in stromal cells was graded as negative, weak, moderate, or strong based on previous human studies that evaluated CXCL12 expression via IHC [ 5 , 6 ] , and the percentage of stained cells was determined by evaluating at least 10 high-power fields (200× magnification) for each tissue section. Tissue specimens were considered positive for CXCL12 when > 10% of stromal cells were stained and negative when ≤ 10% of stromal cells were stained. The evaluation was performed under the guidance of a pathologist. A CXCL12 staining score was assigned to the tumor stroma based on both staining intensity and percentage of positive cells, as follows: 0, no or ≤ 10% of stromal cells stained; 1, > 10% of stromal cells stained with weak intensity; 2, > 10% of stromal cells stained with moderate intensity; and 3, > 10% of stromal cells stained with strong intensity. Cell Culture NFs and CAFs were isolated from skin and surgically resected tumor tissues using cell dispersion methods [ 19 , 50 ] . Briefly, the tissues were minced into small pieces and digested with 1 mg/mL collagenase IV (Sigma-Aldrich) with 5% bovine serum albumin (BSA; Wako, Osaka, Japan) at 37°C for 2 h with rotation. Undigested tissues were removed via filtration, and the cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Wako) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Wako) at 37°C with 5% CO₂. Fibroblasts and tumor cells were separated based on the differential detachment time using 1 mM EDTA-4Na and 0.25% trypsin (Wako). The isolated fibroblasts were cultured and used for the experiments after 1–2 passages following the primary culture [ 17 , 50 ] . NFs and CAFs were cultured in dishes until 80% confluence, after which the media was replaced with RPMI media containing RPMI-1640 with 10% heat-inactivated FBS, 25 mM HEPES (Thermo Fisher Scientific, Waltham, MA, USA), 55 µM 2-mercaptoethanol (Wako), and 100 units/mL penicillin and 0.1 mg/mL streptomycin (Wako). After 24 hours of incubation, the culture supernatants were collected as NF-CM and CAF-CM. cPBMCs were isolated via density gradient centrifugation from peripheral blood samples obtained from healthy beagle dogs and client-owned dogs with epithelial malignant tumors that visited the Azabu University Veterinary Teaching Hospital. The cPBMCs were cultured in RPMI medium at 37°C with 5% CO 2 . Immunofluorescence Fibroblasts were seeded on chamber slides (8 well NuncTM Lab-TekTM II Chamber SlideTM System; Thermo Fisher Scientific) at a density of 5.0 ×10 4 cells/well and cultured for 48 h. The cells were fixed with paraformaldehyde at room temperature (RT) for 1 h, after which they were permeabilized with 0.5% Triton X-100 (Wako) in phosphate-buffered saline (PBS) for 15 min and blocked with 1% BSA for 1 h. Immunostaining was then performed using a rabbit anti-vimentin antibody (Clone: SP20, 1:1000, Abcam, Cambridge, UK) as a marker for mesenchymal cells, a mouse anti-cytokeratin antibody (Clone: AE1/AE3, 1:200, Novus Biologicals, Centennial, CO, USA) as a marker of epithelial cells, a mouse anti-α-SMA (clone: 1A4, 1:500, Thermo Fisher Scientific), and a mouse anti-CXCL12 antibody (clone: 79018, 1:40, R&D Systems), all diluted in PBS and incubated at 4°C overnight. The cells were subsequently incubated with a goat anti-rabbit (Alexa-Fluor-594, Cell signaling technology, Danvers, MA, USA) or goat anti-mouse (Alexa-Fluor-488, Thermo Fisher Scientific) secondary antibody at RT for 1 h, followed by nuclei counterstaining with DAPI (Dojindo, Kumamoto, Japan) at RT for 15 min. Coverslips were mounted on the glass slides, and images were obtained using a confocal microscope (TCS SP5Ⅱ; Leica, Wetzlar, Germany). Cytokine Quantification The concentration of CXCL12 in cell culture supernatants and serum was measured using a Canine Stromal Cell-Derived Factor 1 ELISA kit (MBS2606294; MyBioSource, San Diego, CA, USA). The concentration of TGF-β1 in cell culture supernatants was measured using a Human/Mouse/Rat/Porcine/Canine TGF-β1 Quantikine ELISA kit (DB100C; R&D Systems). All assays were performed according to the manufacturer’s instructions. Absorbance was measured at 450 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad, Hercules, CA, USA). Flowcytometry The cPBMCs were washed twice in fluorescence-activated cell sorting (FACS) buffer (1% FBS and 0.1% NaN₃ in PBS). T cells were first stained with a biotin-conjugated mouse anti-human CXCR4 antibody (clone: 12G5; Thermo Fisher Scientific) for 30 min at 4˚C. A negative control was stained with an isotype control antibody (clone: eBM2a; Thermo Fisher Scientific). Following primary staining, the cells were washed twice and then stained with a streptavidin (Thermo Fisher Scientific), mouse anti-dog CD3 (clone: CA17.2A12, Bio-Rad), rat anti-dog CD4 (clone: YKIX302.9, Bio-Rad), and rat anti-dog CD8 (clone: YCATE 55.9, Thermo Fisher Scientific) antibodies for 30 min at 4˚C. Data were acquired using a BD FACSCelesta cell analyzer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (version 10; Treestar, Ashland, OR, USA). TGF-β1 treatment assay for evaluating the effects on the CXCL12/CXCR4 axis To evaluate the effect of TGF-β1 on the CXCL12/CXCR4 axis, CAFs were cultured with or without 5 µM of SB431542 (Cayman Chemical, Ann Arbor, MI, USA), a potent inhibitor of the TGF-β1 pathway that inhibits ALK5 [ 51 ] . After 72 h of incubation, the media was replaced with 2% FBS DMEM. The concentration of CXCL12 in the 24-hour incubation culture supernatant was measured using ELISA. cPBMCs obtained from healthy dogs were stimulated using Dynabeads M-280 Tosylactivated (DB14204; Thermo Fisher Scientific) coated with anti-dog CD3 and anti-dog CD28 antibodies (clone: 1C6; Absolute Antibody, Upper Heyford, Somerset, UK) and cultured with 5 ng/mL recombinant human TGF-β1 (PeproTech, Cranbury, NJ, USA) for 5 d. Recombinant human TGF-β1 was used because the amino acid sequence of canine TGF-β1 is 100% identical to that of human TGF-β1. CXCR4 expression was then evaluated via flow cytometry. Similarly, the stimulated cPBMCs were cultured with NF-CM or CAF-CM with or without 5 µM of SB431542 for 5 d, and CXCR4 expression was evaluated. Migration Assay A Boyden chamber assay was performed to evaluate the effect of the CXCL12/CXCR4 axis on the migration of canine CD8 + T cells. Canine CD8 + T cells were magnetically isolated from cPBMCs using an EasySep™ Positive Selection Kit Ⅱ (STEMCELL, Vancouver, Canada) and cultured overnight to promote CXCR4 expression (Supplementary Fig. 2). The separation efficiency and CXCR4 expression were confirmed to be > 90% prior to use. Canine CD8 + T cells with or without pretreatment with 4 µM AMD3100 (Abcam), a CXCR4 antagonist, were added to the upper compartment of the cell culture inserts (pore size, 8 µm; Corning Inc, Corning, NY, USA). The lower compartment was supplemented with either 100 ng/ml of recombinant human CXCL12 (R&D Systems) or CAFs-CM. After 1.5 h of incubation, the number of migrated cells in the lower compartment was counted using a hemocytometer. Co-culture Assay To evaluate the interaction between T cells, canine CAFs, and cancer cell lines, co-culture experiments were performed. CAFs and canine mammary gland tumor (RCM-SO) or canine transitional cell carcinoma (NMTCC) cell lines were seeded separately using a cell culture insert (flexiPERM disc; Sarstedt, Osaka, Japan) on 6 cm petri dishes. Canine CD5 + T cells were magnetically isolated from cPBMCs and cultured overnight to enhance CXCR4 expression, as described above. The cells were then seeded onto CAFs and tumor cells in the presence or absence of 4 µM of AMD3100 at a density of 1.0 × 10 6 cells/dish. After 24 h of incubation, the culture supernatant was removed, and the dish was washed thrice with PBS to remove floating T cells. Microscopic images were obtained from five randomly selected fields using the BZ-X fluorescence microscope (KEYENCE, Osaka, Japan). The density of attached T cells per cm 2 on CAFs or cancer cells was quantified using Hybrid Cell Count Software (BZ-H3C; KEYENCE). To ensure objectivity, two independent researchers performed cell counting separately. Statistical Analysis Data are presented as the mean ± standard deviation or the median with interquartile range (IQR). Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software Inc., Boston, MA, USA). Normality was assessed using the Shapiro–Wilk test with the significance level set at 0.05. For comparisons between two groups, Welch’s t-test was used for normally distributed data and Mann–Whitney U test was used for non-normally distributed data. For comparisons among at least three groups, one- or two-way analysis of variance (ANOVA) was performed, followed by an appropriate post hoc test: Tukey’s test for normally distributed data and Dunn’s test for non-normally distributed data. Correlations between two variables were assessed using Spearman’s rank correlation coefficient. Statistical significance was set at P < 0.05. Declarations ARRIVE guidelines The study is reported in accordance with the ARRIVE guidelines. Acknowledgements This research was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant number JP24KJ2120). We would like to thank Junichi Kamiie for providing advice and technical assistance with pathological examinations. We are grateful to all members of Patho Lab Co., Ltd. for their assistance in the pathological diagnosis. We would also like to express our gratitude to Dr. Yumiko Kagawa of North Lab for providing tissue samples for this research. Author contributions AK, AY, SY, and ST conceived and designed the study. AK, AY, EK, and ST conducted the sample collection. AK, SK and AY performed the experiments. AK, SK, AY, and YH acquired and analyzed the data. AK and ST drafted and revised the manuscript. All authors have read and approved the final version of the manuscript. Competing interests The authors declare no competing interests. Data availability statement All data generated or analyzed during this study are included in this published article and its supplementary information files. Further inquiries can be directed to the corresponding author. References Zhang, H. et al. Define cancer-associated fibroblasts (CAFs) in the tumor microenvironment: New opportunities in cancer immunotherapy and advances in clinical trials. Mol. Cancer . 22 , 159 (2023). Freeman, P. & Mielgo, A. Cancer-associated fibroblast mediated inhibition of CD8 + cytotoxic T cell accumulation in tumours: Mechanisms and therapeutic opportunities. Cancers 12 , 2687 (2020). Gardeta, S. R. et al. Sphingomyelin depletion inhibits CXCR4 dynamics and CXCL12-mediated directed cell migration in human T cells. Front. Immunol. 13 , 925559 (2022). Shi, Y., Riese, D. J. & Shen, J. The role of the CXCL12/CXCR4/CXCR7 chemokine axis in cancer. Front. Pharmacol. 11 , 574667 (2020). Zhang, F. et al. Cancer-associated fibroblasts induce epithelial-mesenchymal transition and cisplatin resistance in ovarian cancer via CXCL12/CXCR4 axis. Future Oncol. 16 , 2619–2633 (2020). Sugihara, H. et al. Cancer-associated fibroblast-derived CXCL12 causes tumor progression in adenocarcinoma of the esophagogastric junction. Med. Oncol. 32 , 618 (2015). Du, Y. et al. Comprehensive analysis of CXCL12 expression reveals the significance of inflammatory fibroblasts in bladder cancer carcinogenesis and progression. Cancer Cell. Int. 21 , 613 (2021). Ene-Obong, A. et al. Activated pancreatic stellate cells sequester CD8 + T cells to reduce their infiltration of the juxtatumoral compartment of pancreatic ductal adenocarcinoma. Gastroenterology 145 , 1121–1132 (2013). Orimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121 , 335–348 (2005). Zeng, Y. et al. Dual blockade of CXCL12-CXCR4 and PD‐1–PD‐L1 pathways prolongs survival of ovarian tumor–bearing mice by prevention of immunosuppression in the tumor microenvironment. FASEB J. 33 , 6596–6608 (2019). Feig, C. et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti–PD-L1 immunotherapy in pancreatic cancer. Proc. Natl Acad. Sci. U. S. A. 110, 20212–20217 (2013). Zhou, W., Guo, S., Liu, M., Burow, M. E. & Wang, G. Targeting CXCL12/CXCR4 axis in tumor immunotherapy. Curr. Med. Chem. (CMC , 26, 3026–3041 (2019). (2019). Kojima, Y. et al. Autocrine TGF-β and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts. Proc. Natl Acad. Sci. U. S. A. 107, 20009–20014 (2010). Wu, T. et al. Targeting HIC1/TGF-β axis-shaped prostate cancer microenvironment restrains its progression. Cell. Death Dis. 13 , 624 (2022). Buckley, C. D. et al. Persistent induction of the chemokine receptor CXCR4 by TGF-β1 on synovial T cells contributes to their accumulation within the rheumatoid synovium. J. Immunol. 165 , 3423–3429 (2000). Gorchs, L., Oosthoek, M., Yucel-Lindberg, T., Moro, C. F. & Kaipe, H. Chemokine receptor expression on T cells is modulated by CAFs and chemokines affect the spatial distribution of T cells in pancreatic tumors. Cancers 14 , 3826 (2022). Yoshimoto, S., Hoshino, Y., Izumi, Y. & Takagi, S. α-Smooth Muscle Actin Expression in Cancerassociated Fibroblasts in Canine Epithelial Tumors, (2017). Król, M. et al. The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs) as a co-culture in vitro. BMC Vet. Res. 8 , 35 (2012). Kudo, A., Yoshimoto, S., Yoshida, H., Izumi, Y. & Takagi, S. Biological features of canine cancer-associated fibroblasts and their influence on cancer cell invasion. J. Vet. Med. Sci. 84 , 784–791 (2022). Szczubiał, M. et al. Plasma levels of chemokines CCL2 and CXCL12 in female dogs with malignant mammary gland tumours without and with metastases. Pol. J. Vet. Sci. 26 , 385–392 (2023). Itoh, H. et al. Evaluation of immunological status in tumor-bearing dogs. Vet. Immunol. Immunopathol. 132 , 85–90 (2009). Ettlin, J., Clementi, E., Amini, P., Malbon, A. & Markkanen, E. Analysis of gene expression signatures in cancer-associated stroma from canine mammary tumours reveals molecular homology to human breast carcinomas. Int. J. Mol. Sci. 18 , 1101 (2017). Koppensteiner, L., Mathieson, L., O’Connor, R. A. & Akram, A. R. Cancer associated fibroblasts - An impediment to effective anti-cancer T cell immunity. Front. Immunol. 13 , 887380 (2022). Galon, J. & Bruni, D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. Nat. Rev. Drug Discov . 18 , 197–218 (2019). Zhang, Z. et al. Cancer-associated fibroblasts-derived CXCL12 enhances immune escape of bladder cancer through inhibiting P62-mediated autophagic degradation of PDL1. J. Exp. Clin. Cancer Res. 42 , 316 (2023). Izumi, D. et al. CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer. Int. J. Cancer . 138 , 1207–1219 (2016). Qin, Y. et al. Cancer-associated fibroblasts in gastric cancer affect malignant progression via the CXCL12-CXCR4 axis. J. Cancer . 12 , 3011–3023 (2021). Teng, F. et al. Cancer-associated fibroblasts promote the progression of endometrial cancer via the SDF-1/CXCR4 axis. J. Hematol. Oncol. 9 , 8 (2016). Lim, J. B. & Chung, H. W. Serum ENA78/CXCL5, SDF-1/CXCL12, and their combinations as potential biomarkers for prediction of the presence and distant metastasis of primary gastric cancer. Cytokine 73 , 16–22 (2015). Lavaee, F., Zareh, S., Mojtahedi, Z., Malekzadeh, M. & Ghaderi, A. Serum CXCL12, but not CXCR4, Is Associated with Head and Neck Squamous Cell Carcinomas. Asian Pac. J. Cancer Prev. 19 , 901–904 (2018). Hassanshahi, G. et al. Temporal expression profile of CXC chemokines in serum of patients with spinal cord injury. Neurochem Int. 63 , 363–367 (2013). Franchini, S. et al. Serum CXCL12 levels on hospital admission predict mortality in patients with severe sepsis/septic shock. Am. J. Emerg. Med. 33 , 1802–1804 (2015). Ferdousie, T. Serum CXCL10 and CXCL12 chemokine levels are associated with the severity of coronary artery disease and coronary artery occlusion. Int. J. Cardiol. 233 , 23–28 (2017). Takeuchi, H. et al. Canine transforming growth factor-β receptor 2-Ig: A potential candidate biologic for melanoma treatment that reverses transforming growth factor-β1 immunosuppression. Front. Vet. Sci. 8 , 656715 (2021). Jensen-Jarolim, E. et al. Crosstalk of carcinoembryonic antigen and transforming growth factor-β via their receptors: Comparing human and canine cancer. Cancer Immunol. Immunother . 64 , 531–537 (2015). Schioppa, T. et al. Regulation of the Chemokine Receptor CXCR4 by hypoxia. J. Exp. Med. 198 , 1391–1402 (2003). Busillo, J. M. & Benovic, J. L. Regulation of CXCR4 signaling. Biochim. Biophys. Acta . 1768 , 952–963 (2007). Eiro, N. et al. Cancer-associated fibroblasts affect breast cancer cell gene expression, invasion and angiogenesis. Cell. Oncol. (Dordr) . 41 , 369–378 (2018). González, L. et al. Gene expression profile of normal and cancer-associated fibroblasts according to intratumoral inflammatory cells phenotype from breast cancer tissue. Mol. Carcinog. 55 , 1489–1502 (2016). Avniel, S. et al. Involvement of the CXCL12/CXCR4 pathway in the recovery of skin following burns. J. Invest. Dermatol. 126 , 468–476 (2006). Chen, S. et al. Transforming growth factor-β1 increases CXCR4 expression, stromal‐derived factor‐1α‐stimulated signalling and human immunodeficiency virus‐1 entry in human monocyte‐derived macrophages. Immunology 114 , 565–574 (2005). Chen, Z. et al. Dysregulation of DPP4-CXCL12 balance by TGF-β1/SMAD pathway promotes CXCR4 + inflammatory cell infiltration in keloid scars. J. Inflamm. Res. 14 , 4169–4180 (2021). Mezzapelle, R. et al. CXCR4/CXCL12 activities in the tumor microenvironment and implications for tumor immunotherapy. Cancers 14 , 2314 (2022). Zhang, F. et al. Dynamics of peripheral T cell clones during PD-1 blockade in non-small cell lung cancer. Cancer Immunol. Immunother . 69 , 2599–2611 (2020). Chen, Z. et al. Single-cell RNA sequencing highlights the role of inflammatory cancer-associated fibroblasts in bladder urothelial carcinoma. Nat. Commun. 11 , 5077 (2020). Elyada, E. et al. Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts. Cancer Discov . 9 , 1102–1123 (2019). Sebastian, A. et al. Single-cell transcriptomic analysis of tumor-derived fibroblasts and normal tissue-resident fibroblasts reveals fibroblast heterogeneity in breast cancer. Cancers 12 , 1307 (2020). Han, C., Liu, T. & Yin, R. Biomarkers for cancer-associated fibroblasts. Biomark. Res. 8 , 64 (2020). Li, X. et al. Single-cell RNA sequencing reveals a pro-invasive cancer-associated fibroblast subgroup associated with poor clinical outcomes in patients with gastric cancer. Theranostics 12 , 620–638 (2022). Du, H. D., Wu, C. P., Zhou, L. & Tian, J. Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line. Acta Oto-Laryngol . 133 , 755–760 (2013). Tan, K. et al. Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration. PLOS One . 14 , e0221126 (2019). Additional Declarations No competing interests reported. Supplementary Files SupplementaryinformationAyanoKudo.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Jun, 2025 Reviews received at journal 02 Jun, 2025 Reviews received at journal 02 Jun, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers invited by journal 13 May, 2025 Editor assigned by journal 13 May, 2025 Editor invited by journal 05 May, 2025 Submission checks completed at journal 02 May, 2025 First submitted to journal 01 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6575028","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":456169079,"identity":"804da721-36f2-497c-96f5-90fd0168d0bd","order_by":0,"name":"Ayano Kudo","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Ayano","middleName":"","lastName":"Kudo","suffix":""},{"id":456169080,"identity":"421d6e82-faae-4308-87ae-7a510db5c7c6","order_by":1,"name":"Shintaro Kamo","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Shintaro","middleName":"","lastName":"Kamo","suffix":""},{"id":456169081,"identity":"699b7e21-a8df-4339-9544-415bfea3dbeb","order_by":2,"name":"Akinori Yamauchi","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Akinori","middleName":"","lastName":"Yamauchi","suffix":""},{"id":456169082,"identity":"4381461d-23f4-4db5-9d74-9b8fd638670b","order_by":3,"name":"Sho Yoshimoto","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Sho","middleName":"","lastName":"Yoshimoto","suffix":""},{"id":456169083,"identity":"e0e253e2-3c33-49e3-a5c6-e6f606c74ded","order_by":4,"name":"Yuma Harada","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Yuma","middleName":"","lastName":"Harada","suffix":""},{"id":456169084,"identity":"a040f494-1366-4507-a9ec-d937cfd83ffa","order_by":5,"name":"Eiichi Kanai","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Eiichi","middleName":"","lastName":"Kanai","suffix":""},{"id":456169085,"identity":"5263d67b-4953-49ec-ac2b-1ba4c19108b3","order_by":6,"name":"Satoshi Takagi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYJACxgYGBh5+IIMZLiSBVwMzRItkA6laGAwOIGvBB+Qb+A9+nFFTK2N8/PjDz4VtDIkN7IcfMFjuwK3F4AAzs+SGY8d5zM4kJEvPBGnhSTNgkDyDR4v8YwbJB2zHeMwOJByQ5m37n9jAkMPAINmGz2HMzD8f/DvGY9z/sPk3L8gW/jf4tTAcYGaT3NhWw2MgkcwmDdYiQcAWoF/MLGf2HeCRuPGMzXrGOQbjNolnBgfw+UW+gfHxzZ5vdfb8/emPbxeUMcj28yc/fCyJJ8Sg4DCCyQbigiKWAKhD5TJ+JKxlFIyCUTAKRg4AAEWwS+TI+COgAAAAAElFTkSuQmCC","orcid":"","institution":"Azabu University","correspondingAuthor":true,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Takagi","suffix":""}],"badges":[],"createdAt":"2025-05-02 03:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6575028/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6575028/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-16312-x","type":"published","date":"2025-08-23T16:29:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82835303,"identity":"5994aed3-db84-47c7-ade3-742f1f43a62e","added_by":"auto","created_at":"2025-05-15 18:41:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2046406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemistry of CXCL12 and α-SMA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e)-(\u003cstrong\u003eD)\u003c/strong\u003e CXCL12, (\u003cstrong\u003eE\u003c/strong\u003e)-(\u003cstrong\u003eH\u003c/strong\u003e) α-SMA, (\u003cstrong\u003eI\u003c/strong\u003e)-(\u003cstrong\u003eL\u003c/strong\u003e) Hematoxylin and eosin staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLeft two columns\u003c/strong\u003e: canine thyroid carcinoma tissue with a CXCL12 score of 3; \u003cstrong\u003eRight two columns\u003c/strong\u003e: canine lung adenocarcinoma tissue with a CXCL12 score of 0.\u003c/p\u003e\n\u003cp\u003eThe left view of each case is at a low power field, and the right view is at a high power field.\u003c/p\u003e","description":"","filename":"MainFigureAyanoKudo1.png","url":"https://assets-eu.researchsquare.com/files/rs-6575028/v1/15594eb5827bffdea6ac3286.png"},{"id":82834989,"identity":"eb2a720c-edd5-4f39-8bcc-60eb1c15d41c","added_by":"auto","created_at":"2025-05-15 18:33:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1005079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation and identification of fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Microscopic image of NFs and CAFs isolated from a dog with lung adenocarcinoma (optical microscope). (\u003cstrong\u003eB\u003c/strong\u003e) Immunofluorescence staining of vimentin, cytokeratin, α-SMA, and CXCL12 in NFs and CAFs isolated from a dog with lung adenocarcinoma. The top panel shows NFs, and the bottom panel shows CAFs (confocal microscope).\u003c/p\u003e","description":"","filename":"MainFigureAyanoKudo2.png","url":"https://assets-eu.researchsquare.com/files/rs-6575028/v1/514a983d1e524ffc87522233.png"},{"id":82834825,"identity":"96e4296b-4e17-4bb6-934f-ef0014bc912a","added_by":"auto","created_at":"2025-05-15 18:25:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of CXCL12 and TGF-β1 secretion from CAFs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) CXCL12 concentrations in NF-CM and CAF-CM. Data are presented as mean ± SD (NF-CM, \u003cem\u003en\u003c/em\u003e = 7; CAF-CM, \u003cem\u003en\u003c/em\u003e = 10). (\u003cstrong\u003eB\u003c/strong\u003e) Scatter plot showing the correlation between CXCL12 levels in CAF-CM and serum CXCL12 concentrations, with Spearman’s rank correlation coefficient (rₛ) and \u003cem\u003eP\u003c/em\u003e value indicated (\u003cem\u003en\u003c/em\u003e = 7). (\u003cstrong\u003eC\u003c/strong\u003e) TGF-β1 concentrations in NF-CM and CAF-CM. Data are presented as median with IQR (NF-CM, \u003cem\u003en\u003c/em\u003e = 7; CAF-CM, \u003cem\u003en\u003c/em\u003e = 10). (\u003cstrong\u003eD\u003c/strong\u003e) CXCL12 secretion from CAFs cultured in the presence of the TGF-β inhibitor SB431542 (\u003cem\u003en\u003c/em\u003e = 5). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"MainFigureAyanoKudo3.png","url":"https://assets-eu.researchsquare.com/files/rs-6575028/v1/b9490ebc64c28e4aac90fe1f.png"},{"id":82834823,"identity":"f2bf65f0-323e-4e30-9273-5a043d36f4a4","added_by":"auto","created_at":"2025-05-15 18:25:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":27944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of CXCR4 expression on canine T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) CXCR4 expression on CD8\u003csup\u003e-\u003c/sup\u003e and CD8\u003csup\u003e+ \u003c/sup\u003ecanine T cells were evaluated via flow cytometry analysis. Representative data from one healthy beagle and a cancer-bearing dog. Plots are gated on \u003cem\u003elymphocyte \u0026gt; single cells \u0026gt; CD3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+ \u003c/em\u003e\u003c/sup\u003e\u003cem\u003ecells \u0026gt; CD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cells\u003c/em\u003e. (\u003cstrong\u003eB\u003c/strong\u003e) CXCR4 expression on CD8\u003csup\u003e+ \u003c/sup\u003eT cells or CD8\u003csup\u003e-\u003c/sup\u003e T cells in healthy dogs and dogs with cancer. Data are presented as median with IQR (healthy, \u003cem\u003en\u003c/em\u003e = 10; Cancer, \u003cem\u003en\u003c/em\u003e = 26). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"MainFigureAyanoKudo4.png","url":"https://assets-eu.researchsquare.com/files/rs-6575028/v1/7a5ab769552a8ca86d173dfe.png"},{"id":82834829,"identity":"16d7c27a-b4da-4a67-8460-af938db0250e","added_by":"auto","created_at":"2025-05-15 18:25:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of TGF-β1 on CXCR4 expression on T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) CXCR4 expression on CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells after being cultured with the medium containing TGF-β1 (5 ng/mL) with or without the TGF-β1 inhibitor SB431542 (5µM) for 5 d. Mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). (\u003cstrong\u003eB\u003c/strong\u003e) CXCR4 expression on CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells after being cultured with NF-CM or CAF-CM for 5 d. Mean ± SD (Control, \u003cem\u003en\u003c/em\u003e = 3; NF-CM, \u003cem\u003en\u003c/em\u003e = 5; CAF-CM, \u003cem\u003en\u003c/em\u003e = 8). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"MainFigureAyanoKudo5.png","url":"https://assets-eu.researchsquare.com/files/rs-6575028/v1/223efecc4e1ff6aaaa6c2a1e.png"},{"id":82834832,"identity":"a884f83e-c388-4b04-aa5e-68091d669430","added_by":"auto","created_at":"2025-05-15 18:25:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":156937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAFs attract CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells via the CXCL12/CXCR4 axis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Ratio of the number of migrated CD8\u003csup\u003e+\u003c/sup\u003e T cells against CXCL12 to control. Mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). (\u003cstrong\u003eB\u003c/strong\u003e) Ratio of the number of migrated CD8\u003csup\u003e+ \u003c/sup\u003eT cells against CAF-CM to control. Mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). (\u003cstrong\u003eC\u003c/strong\u003e) Microscopic image of T cells attached to CAFs (phase-contrast microscope). (\u003cstrong\u003eD\u003c/strong\u003e) Density of attached T cells per cm\u003csup\u003e2\u003c/sup\u003e on cancer cell lines or CAFs after 24 hours of culture (\u003cem\u003en\u003c/em\u003e = 4). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Control, culture media; CXCL12, culture media with recombinant CXCL12 protein (100 ng/mL); AMD3100, treatment with CXCR4 inhibitor; AMD3100 (4 µM).\u003c/p\u003e","description":"","filename":"MainFigureAyanoKudo6.png","url":"https://assets-eu.researchsquare.com/files/rs-6575028/v1/6b7b6ca27cbda938347e103e.png"},{"id":82834830,"identity":"edbf17a9-cf03-4829-931a-6ac2a857cf17","added_by":"auto","created_at":"2025-05-15 18:25:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":78593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme of CAF-mediated regulation of T cell dynamics through the CXCL12/CXCR4 axis and TGF-β1 signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTGF-β1 secreted from CAFs upregulate CXCR4 in T cells and further promotes CXCL12 secretion from CAFs. T cells with increased CXCR4 expression may be attracted to CAF-derived CXCL12 within the tumor microenvironment. These effects suggest the inhibition of T cell migration toward cancer cells.\u003c/p\u003e","description":"","filename":"MainFigureAyanoKudo7.png","url":"https://assets-eu.researchsquare.com/files/rs-6575028/v1/90e20863cd52a1f5be4d5009.png"},{"id":89848502,"identity":"2cd41d12-9c5a-4180-8b2e-43c6bf5b302b","added_by":"auto","created_at":"2025-08-25 16:51:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4277157,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6575028/v1/983b46ee-1466-4e34-b3c5-1a85bdcd451a.pdf"},{"id":82834834,"identity":"03a47215-c250-4c60-8884-2741e05529a1","added_by":"auto","created_at":"2025-05-15 18:25:44","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":329276,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationAyanoKudo.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6575028/v1/af7ca2f9753fd2da770fc776.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe tumor microenvironment (TME) is a highly intricate structure comprised of tumor and stromal cells, including immune cells, endothelial cells, and fibroblasts. Among these, cancer-associated fibroblasts (CAFs) are recognized as a principal component of the tumor stroma\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. CAFs enhance tumor malignancy by promoting angiogenesis, proliferation, invasion, and metastasis. Notably, their role in modulating T cell immunity has garnered significant attention. The inhibition of T cell infiltration into solid tumors by CAFs results from the secretion of various humoral factors and their function as a physical barrier, characterized by dense extracellular matrix fiber networks \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe C-X-C motif chemokine ligand 12 (CXCL12) is one of the chemokines secreted by CAFs\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e,and it interacts with the C-X-C chemokine receptor 4 (CXCR4) on T cell membranes, inducing chemotaxis, cell proliferation and gene expression\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. High expression levels of CXCL12 in the tumor stroma have been detected in various human cancers, including gastric, bladder, and ovarian cancer through immunohistochemistry (IHC), and CXCL12 upregulation correlated with poor prognosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. In CXCL12-positive stroma, more T cells infiltrate the stroma than the tumor component, which is suspected to be a factor that limits a patient\u0026rsquo;s prognosis\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e studies have further demonstrated that human CAFs secrete higher levels of CXCL12 than normal fibroblasts (NFs)\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e and that these CAFs promote T cell migration via CXCL12\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. As for the receptor CXCR4, CD8\u003csup\u003e+\u003c/sup\u003e T cells exhibited higher CXCR4 expression in patients of pancreatic ductal adenocarcinoma compared with healthy individuals\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Considering these findings, the combination of CXCL12/CXCR4 inhibitors with an immune checkpoint inhibitor has been examined in mouse models and shown to significantly enhance antitumor effects\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. This strategy is currently being explored in human medicine, with phase Ⅰ-Ⅱ clinical trials underway.\u003c/p\u003e \u003cp\u003eTransforming growth factor beta (TGF-β), another humoral factor secreted by CAFs, has been implicated to modulate the CXCL12/CXCR4 axis. Human CAFs secrete higher levels of TGF-β than NFs\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, and TGF-β mediated autocrine or paracrine signaling activates CAFs, increasing CXCL12 secretion and subsequent tumor progression\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Previous studies have reported that TGF-β isoforms increase CXCR4 expression on human CD4\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e and the culture supernatant of human CAFs upregulates CXCR4 on T cells\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Although the relationship between CXCR4 expression and CAF-derived TGF-β remains unclear, these findings suggest that CAFs modulate T cell migration within the TME by regulating the CXCL12/CXCR4 axis through TGF-β. However, direct evidence for this interaction has not been reported.\u003c/p\u003e \u003cp\u003eIn veterinary medicine, research on the biology of CAFs remains limited. Several studies isolating canine CAFs from epithelial malignancies have shown that canine CAFs share functional similarities with human CAFs, including high expression of alpha smooth muscle actin (α-SMA), a marker of activated fibroblasts, and promotion of tumor cell migration and invasion via humoral factors\u003csup\u003e[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Serum CXCL12 levels have been reported to be significantly higher in dogs with metastases of mammary carcinoma compared with healthy dogs\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In addition, serum TGF-β levels have been reported to be significantly higher in tumor-bearing dogs than in healthy controls\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. However, the association between the presence of CAFs and these serum chemokines in veterinary medicine is unknown. Immunohistochemical analysis of 13 cases of canine mammary gland tumor stroma did not reveal differences in CXCL12 expression between normal stromal regions and the tumor stroma\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. However, studies on the CXCL12/CXCR4 axis in canine CAFs have not been reported, and the effect of canine CAFs on T cell migration via the CXCL12/CXCR4 axis remains unexplored.\u003c/p\u003e \u003cp\u003eThis study aimed to examine the influence of canine CAFs on T cell migration through the CXCL12/CXCR4 axis and elucidate the mechanism underlying TGF-β-mediated regulation of this axis by CAFs, an area that remains underexplored in both veterinary and human medicine.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCXCL12 expression in the stroma of canine epithelial malignant tumors\u003c/h2\u003e \u003cp\u003eCXCL12 expression in the stroma of tumor tissues obtained from 70 cases of epithelial malignant tumors was investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). CXCL12 expression was detected in the cytoplasm of stromal cells adjacent to tumor cells. Among the tumor types, CXCL12 expression was detected in cases of thyroid carcinoma (7/10), renal cell carcinoma (6/10), intestinal adenocarcinoma (3/5), prostate adenocarcinoma (5/10), hepatocellular carcinoma (2/5), mammary gland carcinoma (2/5), lung adenocarcinoma (3/10) and transitional cell carcinoma (2/10) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The CXCL12 expression score for thyroid carcinoma and renal cell carcinoma tended to be higher than that for other tumors. Fibroblasts in the tumor stroma were positive for α-SMA in all cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-H). Hematoxylin and eosin staining was performed to confirm the tumor and stromal regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-L).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCXCL12 expression in various epithelial malignant tumors.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePathology\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive no. / tested no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eCXCL12 score\u003c/p\u003e \u003cp\u003ein tumor stroma\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThyroid carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal cell carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntestinal adenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProstate adenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHepatocellular carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMammary gland carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLung adenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTransitional cell carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnal sac gland carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eCXCL12 score in tumor stroma was scored based on the following criteria: 0, no or \u0026le;\u0026thinsp;10% of stromal cells stained; 1, \u0026gt;\u0026thinsp;10% of stromal cells stained with weak intensity; 2, \u0026gt;\u0026thinsp;10% of stromal cells stained with moderate intensity; and 3, \u0026gt;\u0026thinsp;10% of stromal cells stained with strong intensity.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation of canine CAFs and detection of CXCL12 protein\u003c/h3\u003e\n\u003cp\u003ePrimary cultures of canine CAFs were established from surgically resected tumor tissues obtained from 10 dogs diagnosed with epithelial malignant tumors. The patient characteristics are summarized in Supplementary Table\u0026nbsp;1. NFs were also isolated from the skin tissue of the same patients, although primary cultures could not be established in all cases. The cells obtained were purified by passaging once or twice after the initial culture, and each cell had elongated, spindle-shaped fibroblast features (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Fibroblasts were identified based on positive staining for vimentin and negative staining for cytokeratin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). CAFs showed positive staining for α-SMA, while NFs showed weak expression in only a subset of cells. CXCL12 expression in CAFs was higher than that in NFs, with particularly strong expression observed around the nucleus.\u003c/p\u003e \n\u003ch3\u003eTGF-β1-mediated regulation of CXCL12 secretion from canine CAFs\u003c/h3\u003e\n\u003cp\u003eCXCL12 protein levels in culture media from canine NFs (NF-CM) and CAFs (CAF-CM) were quantified using enzyme-linked immunosorbent assay (ELISA). Canine CAFs secreted significantly higher amounts of CXCL12 than NFs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0379) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Serum CXCL12 concentrations were also evaluated in healthy dogs and in 24 dogs with epithelial malignant tumors (Supplementary Fig.\u0026nbsp;1). A significant correlation was observed between CXCL12 levels in CAF-CM and serum CXCL12 levels (rₛ = 0.9286, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0067) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eNext, TGF-β1 protein levels in NF-CM and CAF-CM were quantified. Canine CAFs secreted significantly higher amounts of TGF-β1 than NFs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0007) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). A significant correlation was observed between CXCL12 and TGF-β1 levels in CAF-CM (rₛ = 0.7091, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0268) (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eTo assess the effect of CAF-derived TGF-β1 on CXCL12 secretion, CAFs were cultured in the presence of the TGF-β receptor inhibitor SB431542, which significantly reduced CXCL12 secretion from CAFs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0335) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\n\u003ch3\u003eIncreased CXCR4 expression on canine T cells in epithelial malignant tumors\u003c/h3\u003e\n\u003cp\u003eSurface expression of CXCR4 on canine T cells was evaluated in 10 healthy dogs and 26 dogs with epithelial malignant tumors via flow cytometry. The types of epithelial malignant tumor included in this study were as follows: thyroid carcinoma (n\u0026thinsp;=\u0026thinsp;5); renal cell carcinoma (n\u0026thinsp;=\u0026thinsp;4); hepatocellular carcinoma (n\u0026thinsp;=\u0026thinsp;4); lung adenocarcinoma (n\u0026thinsp;=\u0026thinsp;3); squamous cell carcinoma (n\u0026thinsp;=\u0026thinsp;3); anal sac gland carcinoma (n\u0026thinsp;=\u0026thinsp;2); salivary gland carcinoma (n\u0026thinsp;=\u0026thinsp;2); and one case each of perianal adenocarcinoma and transitional cell carcinoma. Representative data from a healthy beagle and a dog with thyroid carcinoma are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. CXCR4 expression on CD8\u003csup\u003e\u0026minus;\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in dogs with epithelial malignant tumors was significantly higher than that in healthy dogs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0402, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\n\u003ch3\u003eUpregulation of CXCR4 on canine T cells by CAF-derived TGF-β1\u003c/h3\u003e\n\u003cp\u003eThe effect of TGF-β1 on CXCR4 expression in canine T cells was evaluated by culturing canine peripheral blood mononuclear cells (cPBMCs) with recombinant TGF-β1 or CAF-CM. CXCR4 expression on CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells was significantly elevated by TGF-β1 stimulation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both), and this upregulation was abolished by the addition of the TGF-β1 inhibitor SB431542 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002 for both) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eSimilarly, culturing with CAF-CM significantly elevated CXCR4 expression on CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0303, respectively), and this effect was suppressed by SB431542 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In comparison, culturing with NF-CM resulted in a lower increase in CXCR4 expression than that observed with CAF-CM. However, CXCR4 expression on CD4\u0026thinsp;+\u0026thinsp;T cells still significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0405), and this effect was suppressed by SB431542 treatment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCAF-derived CXCL12 enhanced migration of canine CD8\u003csup\u003e+\u003c/sup\u003e T cells\u003c/h2\u003e \u003cp\u003eWe determined the effect of the CXCL12/CXCR4 axis on the migration of canine CD8\u003csup\u003e+\u003c/sup\u003e T cells using migration assays. CD8\u003csup\u003e+\u003c/sup\u003e T cell migration towards recombinant CXCL12 was significantly higher compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0011). Pretreatment with the CXCR4 antagonist AMD3100 significantly reduced this migration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0018). Moreover, the number of CD8\u003csup\u003e+\u003c/sup\u003e T cells that migrated into CAF-CM was significantly higher compared with the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0013), and treatment with AMD3100 significantly abolished this increased migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eT cells attached to CAFs rather than cancer cells via the CXCL12/CXCR4 axis\u003c/h3\u003e\n\u003cp\u003eT cells, CAFs, and canine cancer cells were co-cultured. The number of T cells attached to CAFs (small spheres) decreased when cultured in the presence of AMD3100 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The density of attached T cells per cm\u003csup\u003e2\u003c/sup\u003e on CAFs was significantly reduced when co-cultured with RCM-SO in the presence of AMD3100 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0475), whereas no significant decrease was found when co-cultured with NMTCC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0955).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCAFs are a major component of the TME, and understanding how they suppress T cells is crucial for cancer immunology\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In multiple solid tumors, T cells are often confined to the stroma and are therefore unable to reach cancer cells. Their inability to access cancer cells may negatively affect patient prognosis\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Recently, CAFs have been suggested to attract T cells via the CXCL12/CXCR4 axis\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. While there is limited evidence, CAF-derived TGF-β has been reported to modulate this axis\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, research on canine CAFs is lacking, and the interaction between canine CAFs and T cell immunity remains largely unexplored. In this study, we evaluated the effect of canine CAFs on T cell migration through the CXCL12/CXCR4 axis and further investigated how CAF-derived TGF-β1 regulates this pathway. Elucidating the factors that control the CXCL12/CXCR4 axis within the TME may provide insights into potential therapeutic strategies for addressing the effect of CAFs on T cell migration.\u003c/p\u003e \u003cp\u003eCXCL12 expression in tumor stroma has been examined in several human cancers, including bladder, gastric and ovarian cancers, and is often associated with poor prognosis\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In addition, CAFs isolated from human tumor tissues secreted CXCL12, the level of which tended to be higher than that secreted by NFs\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Consistent with these findings, we detected CXCL12 expression in the stroma of eight out of the nine canine tumors examined. The association between CXCL12 expression in the tumor stroma and prognosis was not investigated in this study owing to the limited sample size and variable tumor types. Future studies should increase the sample size and focus on specific tumor types to better elucidate the prognostic significance of stromal CXCL12 expression in canine cancers. Furthermore, we found that canine CAFs secreted higher levels of CXCL12 than NFs and that the amount of CXCL12 secreted by CAFs significantly correlated with serum CXCL12 concentrations in the same dogs. While most investigations of CXCL12 have focused on its expression in tissues or cultured cells, some clinical studies have evaluated serum CXCL12 as a potential prognostic biomarker in human medicine. For example, elevated serum CXCL12 levels have been reported in patients with advanced gastric carcinoma and head and neck cancers compared with healthy individuals \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Although CXCL12 levels can also be elevated in conditions such as inflammation, neural injury, and ischemic diseases \u003csup\u003e[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, the observed correlation in our study suggests that increased serum CXCL12 may reflect secretion from CAFs within the TME into the systemic circulation.\u003c/p\u003e \u003cp\u003eThis study also identified a regulatory mechanism whereby inhibition of TGF-β1 led to a decrease in CXCL12 secretion by CAFs. This is further supported by the strong positive correlation between CXCL12 and TGF-β1 secretion by CAFs. A previous study of human mammary carcinoma-derived CAFs suggested that autocrine TGF-β can upregulate CXCL12\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, and the present study demonstrates that CAF-derived TGF-β regulates CXCL12 secretion by CAFs. Furthermore, given that serum TGF-β levels are higher in tumor-bearing dogs compared with healthy controls\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, and that TGF-β is also secreted by regulatory T cells and cancer cells\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, TGF-β from various sources within the TME may contribute to the increased CXCL12 secretion by CAFs.\u003c/p\u003e \u003cp\u003eUpregulation of CXCR4 on T cells is associated with various factors, such as hypoxia, TGF-β, vascular endothelial growth factor (VEGF), interleukin (IL)-2, IL-4, IL-7, and IL-15\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In this study, TGF-β1-derived canine CAFs upregulated CXCR4 on the surface membrane of T cells. A previous human medicine report showed that culture supernatants from CAFs increased CXCR4 expression on T cells and that treatment with an anti-TGF-β neutralizing antibody did not prevent this increase in CXCR4 expression\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In the present study, the role of TGF-β1 could have been more directly assessed by using an inhibitor of the activin receptor-like kinase 5 (ALK5) pathway, which is downstream of the TGF-β1 receptor. The results of the \u003cem\u003ein vitro\u003c/em\u003e experiments suggest that TGF-β1 secretion by CAFs contributes to the increased CXCR4 expression in cancer-bearing dogs. However, additional factors such as hypoxia and other cytokines may be involved in \u003cem\u003ein vivo\u003c/em\u003e conditions. Further studies examining the relationship between serum TGF-β1 concentration and CXCR4 expression on T cells will provide valuable insights.\u003c/p\u003e \u003cp\u003eThe CXCL12/CXCR4 axis has been implicated in the CAF-mediated inhibition of T cell migration. In a human study using CXCL12 knockdown, activated pancreatic stellate cells, which are considered to have a similar function to CAFs, promoted T cell migration via CXCL12\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Our study demonstrated that the CXCL12/CXCR4 axis facilitates T cell migration induced by canine CAFs, as shown by the inhibitory effects of the CXCR4 antagonist AMD3100. By using AMD3100, an inhibitor of CXCR4, it was possible to directly demonstrate that migration is promoted by the effect of CAF-derived CXCL12 on CXCR4 in T cells.\u003c/p\u003e \u003cp\u003eIn the co-culture system of T cells, CAFs, and cancer cells, a greater number of T cells adhered to CAFs when co-cultured with RCM-SO, and this adhesion was reduced by CXCR4 inhibition. Co-culture of breast cancer cell lines (MCF-7 and MDA-MB231) and human CAFs resulted in mutual activation, wherein CAFs exhibited an mRNA upregulation of tumor-promoting factors, including CXCL12\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In this study, a greater number of T cells adhered to CAFs when co-cultured with RCM-SO than with NMTCC. This may be due to differences in the activation of CAFs by cancer cells depending on the cell type and origin.\u003c/p\u003e \u003cp\u003eOur findings highlight the critical role of CAFs in regulating T cell migration via the CXCL12/CXCR4 axis, which is modulated by TGF-β1. A similar mechanism has been identified in wound healing, wherein TGF-β enhances CXCL12 secretion from activated fibroblasts and upregulates CXCR4 on macrophages and T cells. This mechanism may promote immune cell recruitment to the wound site\u003csup\u003e[\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e–\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. This similarity suggests that the innate immunomodulatory functions of fibroblasts, which are essential for wound healing, may negatively affect the TME by trapping T cells in the stroma and inhibiting their migration to the tumor cells. Thus, therapeutic strategies targeting the CXCL12/CXCR4 axis or TGF-β1 signaling require careful design, as they may lead to delayed wound healing as a potential systemic side effect.\u003c/p\u003e \u003cp\u003eCancer immunotherapy, such as immune checkpoint inhibitor or chimeric antigen receptor-T cell therapy, has emerged as a promising treatment strategy in recent decades. However, the suppression of T cell immunity via the CXCL12/CXCR4 axis may negatively affect the effectiveness of CAR-T therapy in solid tumors\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Previous human studies have reported that CXCR4 is significantly upregulated in patients showing resistance to immunotherapy\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Moreover, blockade of CXCR4 has been shown to enhance the efficacy of anti-PD-1/PD-L1 antibodies, leading to increased T cell infiltration within the TME\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. The findings of the present study suggest that CAFs are involved in T cell recruitment via the CXCL12/CXCR4 axis, which may influence the efficacy of tumor immunotherapy. Elucidating the precise role of this axis could lead to targeted interventions that improve the outcomes of cancer immunotherapy by overcoming CAF-mediated T cell suppression.\u003c/p\u003e \u003cp\u003eDespite the findings, this study had some limitations. One limitation was the difficulty in integrating the original tumor types of CAFs. Given the diverse biological behavior of canine epithelial malignancies, the properties of CAFs may vary between tumor types. Additionally, performing a subset analysis of CAFs in this study was difficult. In human medicine, CAFs expressing CXCL12 are known as inflammatory CAFs (iCAFs), which are defined by assessing the expression of multiple markers\u003csup\u003e[\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e–\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Human studies have begun to subclassify CAFs based on distinct markers using single-cell analysis\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Future research should aim to identify markers applicable to canine CAFs and evaluate their functional differences.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that CAFs regulate T cell migration through the CXCL12/CXCR4 axis under the influence of TGF-β1 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results underscore the pivotal role of CAFs in the TME, providing valuable insights applicable to both veterinary and human oncology. Our results contribute to the theoretical foundation for novel therapeutic strategies that may involve targeting the CXCL12/CXCR4 pathway modulated by CAFs, with the ultimate goal of improving cancer outcomes across species.\u003c/p\u003e \u003c/div\u003e "},{"header":"Methods","content":"\u003ch2\u003eCanine Samples\u003c/h2\u003e\u003cp\u003e All animal experiments were conducted in accordance with relevant guidelines and regulations, and were approved by the Animal Experiment Committees of Azabu University (approval no. 220316-26).\u003c/p\u003e\u003cp\u003eSkin and malignant epithelial tumor tissues were collected from dogs that underwent surgical resection for epithelial malignancies at the Azabu University Veterinary Teaching Hospital (Japan). Resected tissues were submitted for histopathological diagnosis, and formalin-fixed specimens were archived at commercial pathology laboratories (Patho Lab Co., Ltd., Shizuoka, Japan; North Lab, Sapporo, Japan). Peripheral blood was obtained from healthy Beagles (1–6 years old) housed in the Experimental Animal Facility of Azabu University and from client-owned dogs with epithelial malignant tumors treated at the teaching hospital.\u003c/p\u003e\u003cp\u003eWritten informed consent was obtained from all owners before inclusion of their animals in the study.\u003c/p\u003e\u003ch2\u003eIHC\u003c/h2\u003e\u003cp\u003eFormalin-fixed, paraffin-embedded tissue samples were used for IHC analysis. Freshly cut tissue sections (3 µm) were mounted on glass slides, deparaffinized, and subjected to antigen retrieval (CXCL12, microwaving at 98°C for 15 min; α-SMA, autoclaving at 121℃ for 20 min). Primary antibody staining was performed using mouse anti-CXCL12 (clone 79018; diluted 1:100; R\u0026amp;D Systems, Minneapolis, MN, USA) and mouse anti-α-SMA (clone: 1A4, diluted 1:1000, Sigma-Aldrich, St. Louis, MO, USA) monoclonal antibodies, with incubation at 4°C overnight. Histofine simple stain kit MAX-PO (MULTI) (Nichirei Bioscience Inc., Tokyo, Japan) was used as the secondary antibody, and hematoxylin was used as a counterstain. Canine tonsil tissue sections were used as a positive control for CXCL12, and vascular smooth muscle was used as an internal positive control for α-SMA. Sections without primary antibody staining were used as negative controls. All tissue sections were also stained with standard hematoxylin and eosin for histological examination.\u003c/p\u003e\u003cp\u003eCXCL12 staining intensity in stromal cells was graded as negative, weak, moderate, or strong based on previous human studies that evaluated CXCL12 expression via IHC\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, and the percentage of stained cells was determined by evaluating at least 10 high-power fields (200× magnification) for each tissue section. Tissue specimens were considered positive for CXCL12 when \u0026gt; 10% of stromal cells were stained and negative when ≤ 10% of stromal cells were stained. The evaluation was performed under the guidance of a pathologist. A CXCL12 staining score was assigned to the tumor stroma based on both staining intensity and percentage of positive cells, as follows: 0, no or ≤ 10% of stromal cells stained; 1, \u0026gt; 10% of stromal cells stained with weak intensity; 2, \u0026gt; 10% of stromal cells stained with moderate intensity; and 3, \u0026gt; 10% of stromal cells stained with strong intensity.\u003c/p\u003e\u003ch2\u003eCell Culture\u003c/h2\u003e\u003cp\u003eNFs and CAFs were isolated from skin and surgically resected tumor tissues using cell dispersion methods\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Briefly, the tissues were minced into small pieces and digested with 1 mg/mL collagenase IV (Sigma-Aldrich) with 5% bovine serum albumin (BSA; Wako, Osaka, Japan) at 37°C for 2 h with rotation. Undigested tissues were removed via filtration, and the cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Wako) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Wako) at 37°C with 5% CO₂. Fibroblasts and tumor cells were separated based on the differential detachment time using 1 mM EDTA-4Na and 0.25% trypsin (Wako). The isolated fibroblasts were cultured and used for the experiments after 1–2 passages following the primary culture\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. NFs and CAFs were cultured in dishes until 80% confluence, after which the media was replaced with RPMI media containing RPMI-1640 with 10% heat-inactivated FBS, 25 mM HEPES (Thermo Fisher Scientific, Waltham, MA, USA), 55 µM 2-mercaptoethanol (Wako), and 100 units/mL penicillin and 0.1 mg/mL streptomycin (Wako). After 24 hours of incubation, the culture supernatants were collected as NF-CM and CAF-CM.\u003c/p\u003e\u003cp\u003ecPBMCs were isolated via density gradient centrifugation from peripheral blood samples obtained from healthy beagle dogs and client-owned dogs with epithelial malignant tumors that visited the Azabu University Veterinary Teaching Hospital. The cPBMCs were cultured in RPMI medium at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eFibroblasts were seeded on chamber slides (8 well NuncTM Lab-TekTM II Chamber SlideTM System; Thermo Fisher Scientific) at a density of 5.0 ×10\u003csup\u003e4\u003c/sup\u003e cells/well and cultured for 48 h. The cells were fixed with paraformaldehyde at room temperature (RT) for 1 h, after which they were permeabilized with 0.5% Triton X-100 (Wako) in phosphate-buffered saline (PBS) for 15 min and blocked with 1% BSA for 1 h. Immunostaining was then performed using a rabbit anti-vimentin antibody (Clone: SP20, 1:1000, Abcam, Cambridge, UK) as a marker for mesenchymal cells, a mouse anti-cytokeratin antibody (Clone: AE1/AE3, 1:200, Novus Biologicals, Centennial, CO, USA) as a marker of epithelial cells, a mouse anti-α-SMA (clone: 1A4, 1:500, Thermo Fisher Scientific), and a mouse anti-CXCL12 antibody (clone: 79018, 1:40, R\u0026amp;D Systems), all diluted in PBS and incubated at 4°C overnight. The cells were subsequently incubated with a goat anti-rabbit (Alexa-Fluor-594, Cell signaling technology, Danvers, MA, USA) or goat anti-mouse (Alexa-Fluor-488, Thermo Fisher Scientific) secondary antibody at RT for 1 h, followed by nuclei counterstaining with DAPI (Dojindo, Kumamoto, Japan) at RT for 15 min. Coverslips were mounted on the glass slides, and images were obtained using a confocal microscope (TCS SP5Ⅱ; Leica, Wetzlar, Germany).\u003c/p\u003e\u003ch2\u003eCytokine Quantification\u003c/h2\u003e\u003cp\u003eThe concentration of CXCL12 in cell culture supernatants and serum was measured using a Canine Stromal Cell-Derived Factor 1 ELISA kit (MBS2606294; MyBioSource, San Diego, CA, USA). The concentration of TGF-β1 in cell culture supernatants was measured using a Human/Mouse/Rat/Porcine/Canine TGF-β1 Quantikine ELISA kit (DB100C; R\u0026amp;D Systems). All assays were performed according to the manufacturer’s instructions. Absorbance was measured at 450 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e\u003ch2\u003eFlowcytometry\u003c/h2\u003e\u003cp\u003eThe cPBMCs were washed twice in fluorescence-activated cell sorting (FACS) buffer (1% FBS and 0.1% NaN₃ in PBS). T cells were first stained with a biotin-conjugated mouse anti-human CXCR4 antibody (clone: 12G5; Thermo Fisher Scientific) for 30 min at 4˚C. A negative control was stained with an isotype control antibody (clone: eBM2a; Thermo Fisher Scientific). Following primary staining, the cells were washed twice and then stained with a streptavidin (Thermo Fisher Scientific), mouse anti-dog CD3 (clone: CA17.2A12, Bio-Rad), rat anti-dog CD4 (clone: YKIX302.9, Bio-Rad), and rat anti-dog CD8 (clone: YCATE 55.9, Thermo Fisher Scientific) antibodies for 30 min at 4˚C. Data were acquired using a BD FACSCelesta cell analyzer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (version 10; Treestar, Ashland, OR, USA).\u003c/p\u003e\u003ch2\u003eTGF-β1 treatment assay for evaluating the effects on the CXCL12/CXCR4 axis\u003c/h2\u003e\u003cp\u003eTo evaluate the effect of TGF-β1 on the CXCL12/CXCR4 axis, CAFs were cultured with or without 5 µM of SB431542 (Cayman Chemical, Ann Arbor, MI, USA), a potent inhibitor of the TGF-β1 pathway that inhibits ALK5\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. After 72 h of incubation, the media was replaced with 2% FBS DMEM. The concentration of CXCL12 in the 24-hour incubation culture supernatant was measured using ELISA.\u003c/p\u003e\u003cp\u003ecPBMCs obtained from healthy dogs were stimulated using Dynabeads M-280 Tosylactivated (DB14204; Thermo Fisher Scientific) coated with anti-dog CD3 and anti-dog CD28 antibodies (clone: 1C6; Absolute Antibody, Upper Heyford, Somerset, UK) and cultured with 5 ng/mL recombinant human TGF-β1 (PeproTech, Cranbury, NJ, USA) for 5 d. Recombinant human TGF-β1 was used because the amino acid sequence of canine TGF-β1 is 100% identical to that of human TGF-β1. CXCR4 expression was then evaluated via flow cytometry. Similarly, the stimulated cPBMCs were cultured with NF-CM or CAF-CM with or without 5 µM of SB431542 for 5 d, and CXCR4 expression was evaluated.\u003c/p\u003e\u003ch2\u003eMigration Assay\u003c/h2\u003e\u003cp\u003eA Boyden chamber assay was performed to evaluate the effect of the CXCL12/CXCR4 axis on the migration of canine CD8\u003csup\u003e+\u003c/sup\u003e T cells. Canine CD8\u003csup\u003e+\u003c/sup\u003e T cells were magnetically isolated from cPBMCs using an EasySep™ Positive Selection Kit Ⅱ (STEMCELL, Vancouver, Canada) and cultured overnight to promote CXCR4 expression (Supplementary Fig.\u0026nbsp;2). The separation efficiency and CXCR4 expression were confirmed to be \u0026gt; 90% prior to use. Canine CD8\u003csup\u003e+\u003c/sup\u003e T cells with or without pretreatment with 4 µM AMD3100 (Abcam), a CXCR4 antagonist, were added to the upper compartment of the cell culture inserts (pore size, 8 µm; Corning Inc, Corning, NY, USA). The lower compartment was supplemented with either 100 ng/ml of recombinant human CXCL12 (R\u0026amp;D Systems) or CAFs-CM. After 1.5 h of incubation, the number of migrated cells in the lower compartment was counted using a hemocytometer.\u003c/p\u003e\u003ch2\u003eCo-culture Assay\u003c/h2\u003e\u003cp\u003eTo evaluate the interaction between T cells, canine CAFs, and cancer cell lines, co-culture experiments were performed. CAFs and canine mammary gland tumor (RCM-SO) or canine transitional cell carcinoma (NMTCC) cell lines were seeded separately using a cell culture insert (flexiPERM disc; Sarstedt, Osaka, Japan) on 6 cm petri dishes.\u003c/p\u003e\u003cp\u003eCanine CD5\u003csup\u003e+\u003c/sup\u003e T cells were magnetically isolated from cPBMCs and cultured overnight to enhance CXCR4 expression, as described above. The cells were then seeded onto CAFs and tumor cells in the presence or absence of 4 µM of AMD3100 at a density of 1.0 × 10\u003csup\u003e6\u003c/sup\u003e cells/dish. After 24 h of incubation, the culture supernatant was removed, and the dish was washed thrice with PBS to remove floating T cells. Microscopic images were obtained from five randomly selected fields using the BZ-X fluorescence microscope (KEYENCE, Osaka, Japan). The density of attached T cells per cm\u003csup\u003e2\u003c/sup\u003e on CAFs or cancer cells was quantified using Hybrid Cell Count Software (BZ-H3C; KEYENCE). To ensure objectivity, two independent researchers performed cell counting separately.\u003c/p\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData are presented as the mean ± standard deviation or the median with interquartile range (IQR). Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software Inc., Boston, MA, USA). Normality was assessed using the Shapiro–Wilk test with the significance level set at 0.05. For comparisons between two groups, Welch’s t-test was used for normally distributed data and Mann–Whitney U test was used for non-normally distributed data. For comparisons among at least three groups, one- or two-way analysis of variance (ANOVA) was performed, followed by an appropriate post hoc test: Tukey’s test for normally distributed data and Dunn’s test for non-normally distributed data. Correlations between two variables were assessed using Spearman’s rank correlation coefficient. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eARRIVE guidelines\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study is reported in accordance with the ARRIVE guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant number JP24KJ2120). We would like to thank Junichi Kamiie for providing advice and technical assistance with pathological examinations. We are grateful to all members of Patho Lab Co., Ltd. for their assistance in the pathological diagnosis. We would also like to express our gratitude to Dr. Yumiko Kagawa of North Lab for providing tissue samples for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAK, AY, SY, and ST conceived and designed the study. AK, AY, EK, and ST conducted the sample collection. AK, SK and AY performed the experiments. AK, SK, AY, and YH acquired and analyzed the data. AK and ST drafted and revised the manuscript. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. Further inquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang, H. et al. Define cancer-associated fibroblasts (CAFs) in the tumor microenvironment: New opportunities in cancer immunotherapy and advances in clinical trials. \u003cem\u003eMol. Cancer\u003c/em\u003e. \u003cb\u003e22\u003c/b\u003e, 159 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreeman, P. \u0026amp; Mielgo, A. Cancer-associated fibroblast mediated inhibition of CD8\u0026thinsp;+\u0026thinsp;cytotoxic T cell accumulation in tumours: Mechanisms and therapeutic opportunities. \u003cem\u003eCancers\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 2687 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGardeta, S. R. et al. Sphingomyelin depletion inhibits CXCR4 dynamics and CXCL12-mediated directed cell migration in human T cells. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 925559 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, Y., Riese, D. J. \u0026amp; Shen, J. The role of the CXCL12/CXCR4/CXCR7 chemokine axis in cancer. \u003cem\u003eFront. Pharmacol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 574667 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, F. et al. Cancer-associated fibroblasts induce epithelial-mesenchymal transition and cisplatin resistance in ovarian cancer via CXCL12/CXCR4 axis. \u003cem\u003eFuture Oncol.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 2619\u0026ndash;2633 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSugihara, H. et al. Cancer-associated fibroblast-derived CXCL12 causes tumor progression in adenocarcinoma of the esophagogastric junction. \u003cem\u003eMed. Oncol.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 618 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu, Y. et al. Comprehensive analysis of CXCL12 expression reveals the significance of inflammatory fibroblasts in bladder cancer carcinogenesis and progression. \u003cem\u003eCancer Cell. Int.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 613 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEne-Obong, A. et al. Activated pancreatic stellate cells sequester CD8\u0026thinsp;+\u0026thinsp;T cells to reduce their infiltration of the juxtatumoral compartment of pancreatic ductal adenocarcinoma. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cb\u003e145\u003c/b\u003e, 1121\u0026ndash;1132 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e121\u003c/b\u003e, 335\u0026ndash;348 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng, Y. et al. Dual blockade of CXCL12-CXCR4 and PD‐1\u0026ndash;PD‐L1 pathways prolongs survival of ovarian tumor\u0026ndash;bearing mice by prevention of immunosuppression in the tumor microenvironment. \u003cem\u003eFASEB J.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 6596\u0026ndash;6608 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeig, C. et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti\u0026ndash;PD-L1 immunotherapy in pancreatic cancer. \u003cem\u003eProc. Natl Acad. Sci. U. S. A.\u003c/em\u003e 110, 20212\u0026ndash;20217 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, W., Guo, S., Liu, M., Burow, M. E. \u0026amp; Wang, G. Targeting CXCL12/CXCR4 axis in tumor immunotherapy. \u003cem\u003eCurr. Med. Chem. (CMC\u003c/em\u003e, 26, 3026\u0026ndash;3041 (2019). (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKojima, Y. et al. Autocrine TGF-β and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts. \u003cem\u003eProc. Natl Acad. Sci. U. S. A.\u003c/em\u003e 107, 20009\u0026ndash;20014 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, T. et al. Targeting HIC1/TGF-β axis-shaped prostate cancer microenvironment restrains its progression. \u003cem\u003eCell. Death Dis.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 624 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuckley, C. D. et al. Persistent induction of the chemokine receptor CXCR4 by TGF-β1 on synovial T cells contributes to their accumulation within the rheumatoid synovium. \u003cem\u003eJ. Immunol.\u003c/em\u003e \u003cb\u003e165\u003c/b\u003e, 3423\u0026ndash;3429 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorchs, L., Oosthoek, M., Yucel-Lindberg, T., Moro, C. F. \u0026amp; Kaipe, H. Chemokine receptor expression on T cells is modulated by CAFs and chemokines affect the spatial distribution of T cells in pancreatic tumors. \u003cem\u003eCancers\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 3826 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshimoto, S., Hoshino, Y., Izumi, Y. \u0026amp; Takagi, S. α-Smooth Muscle Actin Expression in Cancerassociated Fibroblasts in Canine Epithelial Tumors, (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKr\u0026oacute;l, M. et al. The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs) as a co-culture in vitro. \u003cem\u003eBMC Vet. Res.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 35 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKudo, A., Yoshimoto, S., Yoshida, H., Izumi, Y. \u0026amp; Takagi, S. Biological features of canine cancer-associated fibroblasts and their influence on cancer cell invasion. \u003cem\u003eJ. Vet. Med. Sci.\u003c/em\u003e \u003cb\u003e84\u003c/b\u003e, 784\u0026ndash;791 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzczubiał, M. et al. Plasma levels of chemokines CCL2 and CXCL12 in female dogs with malignant mammary gland tumours without and with metastases. \u003cem\u003ePol. J. Vet. Sci.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 385\u0026ndash;392 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eItoh, H. et al. Evaluation of immunological status in tumor-bearing dogs. \u003cem\u003eVet. Immunol. Immunopathol.\u003c/em\u003e \u003cb\u003e132\u003c/b\u003e, 85\u0026ndash;90 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEttlin, J., Clementi, E., Amini, P., Malbon, A. \u0026amp; Markkanen, E. Analysis of gene expression signatures in cancer-associated stroma from canine mammary tumours reveals molecular homology to human breast carcinomas. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 1101 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoppensteiner, L., Mathieson, L., O\u0026rsquo;Connor, R. A. \u0026amp; Akram, A. R. Cancer associated fibroblasts - An impediment to effective anti-cancer T cell immunity. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 887380 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalon, J. \u0026amp; Bruni, D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. \u003cem\u003eNat. Rev. Drug Discov\u003c/em\u003e. \u003cb\u003e18\u003c/b\u003e, 197\u0026ndash;218 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Z. et al. Cancer-associated fibroblasts-derived CXCL12 enhances immune escape of bladder cancer through inhibiting P62-mediated autophagic degradation of PDL1. \u003cem\u003eJ. Exp. Clin. Cancer Res.\u003c/em\u003e \u003cb\u003e42\u003c/b\u003e, 316 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIzumi, D. et al. CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer. \u003cem\u003eInt. J. Cancer\u003c/em\u003e. \u003cb\u003e138\u003c/b\u003e, 1207\u0026ndash;1219 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin, Y. et al. Cancer-associated fibroblasts in gastric cancer affect malignant progression via the CXCL12-CXCR4 axis. \u003cem\u003eJ. Cancer\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, 3011\u0026ndash;3023 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeng, F. et al. Cancer-associated fibroblasts promote the progression of endometrial cancer via the SDF-1/CXCR4 axis. \u003cem\u003eJ. Hematol. Oncol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 8 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim, J. B. \u0026amp; Chung, H. W. Serum ENA78/CXCL5, SDF-1/CXCL12, and their combinations as potential biomarkers for prediction of the presence and distant metastasis of primary gastric cancer. \u003cem\u003eCytokine\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e, 16\u0026ndash;22 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLavaee, F., Zareh, S., Mojtahedi, Z., Malekzadeh, M. \u0026amp; Ghaderi, A. Serum CXCL12, but not CXCR4, Is Associated with Head and Neck Squamous Cell Carcinomas. \u003cem\u003eAsian Pac. J. Cancer Prev.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 901\u0026ndash;904 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassanshahi, G. et al. Temporal expression profile of CXC chemokines in serum of patients with spinal cord injury. \u003cem\u003eNeurochem Int.\u003c/em\u003e \u003cb\u003e63\u003c/b\u003e, 363\u0026ndash;367 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranchini, S. et al. Serum CXCL12 levels on hospital admission predict mortality in patients with severe sepsis/septic shock. \u003cem\u003eAm. J. Emerg. Med.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 1802\u0026ndash;1804 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerdousie, T. Serum CXCL10 and CXCL12 chemokine levels are associated with the severity of coronary artery disease and coronary artery occlusion. \u003cem\u003eInt. J. Cardiol.\u003c/em\u003e \u003cb\u003e233\u003c/b\u003e, 23\u0026ndash;28 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeuchi, H. et al. Canine transforming growth factor-β receptor 2-Ig: A potential candidate biologic for melanoma treatment that reverses transforming growth factor-β1 immunosuppression. \u003cem\u003eFront. Vet. Sci.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 656715 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJensen-Jarolim, E. et al. Crosstalk of carcinoembryonic antigen and transforming growth factor-β via their receptors: Comparing human and canine cancer. \u003cem\u003eCancer Immunol. Immunother\u003c/em\u003e. \u003cb\u003e64\u003c/b\u003e, 531\u0026ndash;537 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchioppa, T. et al. Regulation of the Chemokine Receptor CXCR4 by hypoxia. \u003cem\u003eJ. Exp. Med.\u003c/em\u003e \u003cb\u003e198\u003c/b\u003e, 1391\u0026ndash;1402 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBusillo, J. M. \u0026amp; Benovic, J. L. Regulation of CXCR4 signaling. \u003cem\u003eBiochim. Biophys. Acta\u003c/em\u003e. \u003cb\u003e1768\u003c/b\u003e, 952\u0026ndash;963 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEiro, N. et al. Cancer-associated fibroblasts affect breast cancer cell gene expression, invasion and angiogenesis. \u003cem\u003eCell. Oncol. (Dordr)\u003c/em\u003e. \u003cb\u003e41\u003c/b\u003e, 369\u0026ndash;378 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez, L. et al. Gene expression profile of normal and cancer-associated fibroblasts according to intratumoral inflammatory cells phenotype from breast cancer tissue. \u003cem\u003eMol. Carcinog.\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e, 1489\u0026ndash;1502 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvniel, S. et al. Involvement of the CXCL12/CXCR4 pathway in the recovery of skin following burns. \u003cem\u003eJ. Invest. Dermatol.\u003c/em\u003e \u003cb\u003e126\u003c/b\u003e, 468\u0026ndash;476 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, S. et al. Transforming growth factor-β1 increases CXCR4 expression, stromal‐derived factor‐1α‐stimulated signalling and human immunodeficiency virus‐1 entry in human monocyte‐derived macrophages. \u003cem\u003eImmunology\u003c/em\u003e \u003cb\u003e114\u003c/b\u003e, 565\u0026ndash;574 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, Z. et al. Dysregulation of DPP4-CXCL12 balance by TGF-β1/SMAD pathway promotes CXCR4\u0026thinsp;+\u0026thinsp;inflammatory cell infiltration in keloid scars. \u003cem\u003eJ. Inflamm. Res.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 4169\u0026ndash;4180 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMezzapelle, R. et al. CXCR4/CXCL12 activities in the tumor microenvironment and implications for tumor immunotherapy. \u003cem\u003eCancers\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 2314 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, F. et al. Dynamics of peripheral T cell clones during PD-1 blockade in non-small cell lung cancer. \u003cem\u003eCancer Immunol. Immunother\u003c/em\u003e. \u003cb\u003e69\u003c/b\u003e, 2599\u0026ndash;2611 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, Z. et al. Single-cell RNA sequencing highlights the role of inflammatory cancer-associated fibroblasts in bladder urothelial carcinoma. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 5077 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElyada, E. et al. Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts. \u003cem\u003eCancer Discov\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, 1102\u0026ndash;1123 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSebastian, A. et al. Single-cell transcriptomic analysis of tumor-derived fibroblasts and normal tissue-resident fibroblasts reveals fibroblast heterogeneity in breast cancer. \u003cem\u003eCancers\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 1307 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan, C., Liu, T. \u0026amp; Yin, R. Biomarkers for cancer-associated fibroblasts. \u003cem\u003eBiomark. Res.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 64 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, X. et al. Single-cell RNA sequencing reveals a pro-invasive cancer-associated fibroblast subgroup associated with poor clinical outcomes in patients with gastric cancer. \u003cem\u003eTheranostics\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 620\u0026ndash;638 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu, H. D., Wu, C. P., Zhou, L. \u0026amp; Tian, J. Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line. \u003cem\u003eActa Oto-Laryngol\u003c/em\u003e. \u003cb\u003e133\u003c/b\u003e, 755\u0026ndash;760 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan, K. et al. Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration. \u003cem\u003ePLOS One\u003c/em\u003e. \u003cb\u003e14\u003c/b\u003e, e0221126 (2019).\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cancer-associated fibroblasts, Tumor microenvironment, Dog, C-X-C motif chemokine ligand 12, C-X-C chemokine receptor 4, Transforming growth factor-β1","lastPublishedDoi":"10.21203/rs.3.rs-6575028/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6575028/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that modulate T cell immunity by secreting humoral factors and forming structural barriers. CAFs secrete the chemokine C-X-C motif chemokine ligand 12 (CXCL12), which binds to C-X-C chemokine receptor 4 (CXCR4) on T cells and induces chemotaxis. Transforming growth factor beta 1 (TGF-β1), another CAF-derived humoral factor, is believed to regulate the CXCL12/CXCR4 axis; however, a direct association between them has not been demonstrated in human medicine or veterinary medicine. This study investigated the role of canine CAFs in T cell migration via the CXCL12/CXCR4 axis and the regulatory influence of TGF-β1. CXCL12 and CXCR4 were expressed in the tumor stroma and on T cells, respectively, in dogs with epithelial malignant tumors. Canine CAFs secreted higher levels of CXCL12 and TGF-β1 than normal fibroblasts, and CAF-derived TGF-β1 modulated both CXCL12 secretion by CAFs and CXCR4 expression on T cells. Furthermore, canine CAFs induced T cell migration through the CXCL12/CXCR4 axis. These findings indicate that CAFs regulate T cell migration via the CXCL12/CXCR4 axis under TGF-β1 signaling, highlighting their critical role within the TME.\u003c/p\u003e","manuscriptTitle":"Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 18:25:39","doi":"10.21203/rs.3.rs-6575028/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-03T06:24:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-03T00:26:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-02T20:36:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253785183841754739875220172529582904275","date":"2025-05-13T20:54:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72728460552576315208016247586190894067","date":"2025-05-13T18:55:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T13:52:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-13T12:50:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-05T05:16:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-02T10:02:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-02T02:55:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"321c84ea-c3db-4cb2-848a-477216df12dc","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48489908,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":48489909,"name":"Biological sciences/Cancer/Tumour immunology"},{"id":48489910,"name":"Biological sciences/Cell biology/Cell migration/Chemotaxis"}],"tags":[],"updatedAt":"2025-08-25T16:38:00+00:00","versionOfRecord":{"articleIdentity":"rs-6575028","link":"https://doi.org/10.1038/s41598-025-16312-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-23 16:29:52","publishedOnDateReadable":"August 23rd, 2025"},"versionCreatedAt":"2025-05-15 18:25:39","video":"","vorDoi":"10.1038/s41598-025-16312-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-16312-x","workflowStages":[]},"version":"v1","identity":"rs-6575028","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6575028","identity":"rs-6575028","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}