Tartrate-resistant acid phosphatase 5 regulates the metabolic flexibility of macrophages in the tumor microenvironment, thereby influencing their functional fate and modulating tumor growth

Tartrate-resistant acid phosphatase 5 regulates the metabolic flexibility of macrophages in the tumor microenvironment, thereby influencing their functional fate and modulating tumor growth | 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 Tartrate-resistant acid phosphatase 5 regulates the metabolic flexibility of macrophages in the tumor microenvironment, thereby influencing their functional fate and modulating tumor growth Ming-Shen Dai, Yu-Guang Chen, Shu-Han Yu, Diego Miguel, Pei-Yao Liu, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6656922/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Macrophages play a crucial role in anti-tumor immunity, and their dysfunction within the tumor microenvironment contributes to tumor growth and distant metastasis, posing a significant obstacle to cancer immunotherapies. Understanding the molecular checkpoints in macrophages could provide potential therapeutic strategies for cancer treatment. Tartrate-resistant acid phosphatase 5 (ACP5), also known as TRAP5, is an enzyme primarily expressed by osteoclasts and certain immune cells, involved in bone remodeling and immune regulation. Recent research has indicated that ACP5 promotes tumor growth and metastasis in various cancers. However, its specific role within the tumor microenvironment, particularly regarding its effect on macrophages, remains unclear. This study shows that ACP5 is highly expressed in macrophages within breast cancer tissues, as identified through single-cell RNA sequencing. Using ACP5-deficient mice, we observed a significant reduction in tumor burden, metastatic potential, and epithelial-mesenchymal transition in both orthotopic and spontaneous breast cancer models. Mechanistically, ACP5 regulates macrophage polarization, promoting an anti-inflammatory (M2) phenotype that aids tumor progression and metastasis. Notably, ACP5 deletion in bone marrow-derived macrophages impairs AMPK phosphorylation, shifting their metabolism toward glycolysis. This metabolic shift enhances their pro-inflammatory (M1) phenotype, increasing anti-tumor activity against cancer cells. Our findings underscore the vital role of ACP5 in macrophage-mediated immunosuppression and tumor progression, presenting a promising therapeutic target for breast cancer treatment. Biological sciences/Cancer/Cancer microenvironment Health sciences/Pathogenesis/Inflammation/Chronic inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Chronic inflammation increases cancer risk, a concept first recognized by Virchow in the 19th century 1 . Even in tumors not traditionally linked to inflammation, an inflammatory component is a crucial part of the malignant microenvironment 2 .In early tumor formation, immune cells effectively eliminate abnormal cells, but at later stages, they often fail to suppress cancer and instead contribute to tumor progression and metastasis. 3 , 4 . Macrophages, key players in the innate immune system, are crucial for homeostasis, inflammation, and phagocytosis. In the tumor microenvironment (TME), macrophages are the most abundant immune cells and essential for modulating tumor growth and progression. Based on their morphology, phenotype, genetic features and function, macrophages are classified into two main subtypes-M1 macrophages, which mediate pro-inflammatory responses and contribute to antitumor immunity, while M2 macrophages, which exhibit pro-tumor properties, promoting tumor growth and metastasis 5 . A mix of M2 macrophages and a small subset of M1 macrophages, known as tumor-associated macrophages (TAMs), are highly diverse immune cells in the TME 6 , and essential for tumor progression. Tumor cells secrete chemokines and growth factors to attract and convert macrophages into tumor-promoting M2 types. Increased TAM infiltration correlates with poor clinical outcomes and reduced response to standard treatments. 7 . Moreover, dynamic shifts in macrophage subsets influence the effectiveness of immunotherapy, making TAM modulation a growing area of interest for cancer therapy. 8 Therefore, targeting TAMs or modifying the TME to increase the presence of M1 macrophages while decreasing M2 macrophages has been suggested as a potential therapeutic approach.. 9 , 10 , 11 , 12 . Tartrate-resistant acid phosphatase (TRAP), also known as acid phosphatase 5 (ACP5), is a glycosylated monomeric metalloprotease expressed in mammals. It is characterized by its resistance to tartrate inhibition and exhibits optimal enzymatic activity under acidic conditions. TRAP is initially synthesized as a latent proenzyme (TRAP5a), which possesses low phosphatase activity. Upon proteolytic cleavage in the intervening loop domain, it is converted into its active form (TRAP5b), which exhibits high phosphatase activity. 13 , 14 . Under normal conditions, ACP5 is highly expressed in osteoclasts and activated macrophages. However, its expression is elevated under certain pathological conditions, including hairy cell leukemia, osteoclastoma, osteoporosis, and metabolic bone diseases. 15 , 16 . Janckila et al. conducted a comprehensive immunohistochemical analysis using the TRAP 5-specific antibody mAb220, which revealed that macrophages in tissues associated with chronic antigenic stimulation or affected by chronic inflammatory conditions were the primary ACP5-expressing cells. 17 . Interestingly, ACP5 has also been detected in various cancer cells and tissues, including ovarian, cervical cancers, and malignant melanoma, with its expression levels correlating with tumor severity. 18 , 19 , 20 , 21 . High ACP5 expression is linked to poorer survival outcomes in various cancers. It correlates with reduced tumor-free and metastasis-free survival in malignant melanoma, 22 , lower overall survival in lung 23 and hepatocellular carcinoma 24 , and is an independent risk factor for gastric cancer peritoneal transmission 25 . Additionally, ACP5 promotes breast cancer metastasis by interacting with TRIP-1, activating TGFβR2 and Smad2/3 signaling 26 . Although these studies suggest that ACP5 in cancer cells promotes tumor growth and disease progression, its role in macrophages within the tumor-associated microenvironment—the most abundant infiltrating immune cells in tumor tissues—, remains unclear 27 . To investigate the impact of ACP5 on macrophages and the tumor microenvironment, we performed bulk and single-cell RNA sequencing using several public datasets. Our analysis showed that ACP5 is highly expressed in macrophages within breast cancer tissues, but not in breast cancer cells. Using a genetically modified mouse model implanted with breast cancer cells, we observed a significant reduction in tumor size in ACP5-deficient mice compared to wild-type mice, both in the orthotopic fat pad injection model and the spontaneous genetic breast cancer model. Mechanistically, ACP5 deletion led to a notable shift in macrophage polarization, increasing M1-like macrophages while reducing M2-like macrophages in the tumor-associated microenvironment. Unbiased RNA sequencing and immunoblotting of M1-polarized bone marrow-derived macrophages (BMDMs) from ACP5-deficient mice revealed that AMPK-related genes and AMPK activity were upregulated. Functionally, ACP5 KO BMDMs exhibited increased glycolytic activity compared to ACP5 WT BMDMs, leading to enhanced proinflammatory function and increased phagocytic activity against cancer cells. In an orthotopic breast cancer model, co-injection of 4T1 cells with ACP5 KO BMDMs significantly impeded tumor growth, progression, and distant metastasis compared to 4T1 cells co-injected with ACP5 WT BMDMs. Collectively, Our findings indicate that ACP5 in activated macrophages plays a suppressive role in macrophage-mediated inflammatory responses by regulating metabolic flexibility. The absence of ACP5 promotes macrophage reprogramming and enhances their tumor-eradicating capacity. Results ACP5 is mainly expressed on macrophages within breast cancer tissues. Several studies have demonstrated that high ACP5 expression is a negative prognostic factor for overall survival (OS) in patients with lung adenocarcinoma, liver cancer breast cancer, and colon cancer 21 , 23 , 24 , 28 , 29 . Additionally, ACP5 serves as an important downstream hub in CCL18/CCR8 signalling axis for modulating glioma cell growth and proliferation 30 . However, the specific ACP5-expressing cells and the detailed mechanistic regulation of ACP5 in breast cancer patients remain unclear. To address this, we set out to examine the expression levels of ACP5 in breast cancer samples using publicly available RNA expression data from the UCSC Xena platform ( https://xena.ucsc.edu/ ) 31 . As shown in Fig. 1 A, we found elevated ACP5 expression in breast cancer tissues compared to normal breast tissues from the GTEx database and the corresponding adjacent normal tissues from the TCGA dataset. To decipher the functional and molecular regulation of ACP5 in breast cancer, a functional enrichment analysis was performed based on the differentially expressed genes between high and low ACP5 expression groups in TCGA BRCA to assess the different biological processes, molecular functions, and cellular components influenced by ACP5. Interestingly, we observed that the genes associated with leukocyte function, cytokine production, cell-cell interactions, and leukocyte proliferation were predominantly upregulated in breast cancer patients with high ACP5 expression compared to those with low expression (Fig. 1 B, SF1D and E). To identify the main source of ACP5-expressing cells, we examined the differential expression of ACP5 in different cell types in breast cancer tissues by using a public single cell RNA sequencing (scRNA-seq) dataset (GSE176078) 32 , 33 . After applying the same quality control criteria initially used, a total of 100,064 cells from breast cancer tissues were used for this analysis. We found that ACP5 was predominantly expressed in myeloid cells, particularly in macrophages within tumor tissues, showing not only a high proportion of ACP5 positivity but also high expression levels (Fig. 1 C-E, SF1D). ACP5 has been described to be overexpressed in lung adenocarcinoma cells where it exerted pro-tumorigenic functions. Thus, attenuation of ACP5 drastically impaired the proliferation, migration, and invasion in A549 and H1975 cells 28 . On this basis, we examined these functions in human and murine breast cancer cells. Intriguingly, we could not detect any impact on cell viability in breast cancer cells either overexpressing ACP5 or transfected with a dominant-negative mutant version of the protein (SF1A-B). On the other hand, there was no positive correlation between ACP5 and Ki-67 transcripts in TCGA BRCA dataset (SF1C). Collectively, these findings indicate that in breast cancer tissues, ACP5 is predominantly expressed in macrophages rather than in breast cancer cells. Moreover, ACP5 expression in breast cancer cells does not influence their proliferation or growth. This result also highlights that ACP5 in breast cancer tissue is primarily expressed in macrophages, directing our focus to the impact of ACP5 in macrophages within the cancer tissue, rather than in the tumor cells themselves. The ACP5-deficient microenvironment suppresses cancer cell growth and reduces distant metastatic potential in both murine orthotopic and spontaneous breast cancer models. To further investigate the impact of ACP5 in breast cancer progression, we generated an inducible ACP5-deficient mice. The design and knock-out (KO) strategy is shown in supplementary Fig. 2A. First, the knockout efficiency of ACP5 was confirmed by assessing the expression of ACP5 in serum, spleen and kidney samples from ACP5 heterozygous and homozygous mice (SF2B) and by performing IHC analysis on bone marrow samples (SF2D). In addition, the functional effect of ACP5 absence in bone marrow-derived macrophages (BMDMs) harvested from ACP5-deficient mice was analyzed by performing a LPS stimulation experiment (SF2C). Next, to assess the role of ACP5 expression in the tumor microenvornment at promoting cancer progression, we used these ACP5 KO mice to generate an orthotopic murine breast cancer model by injecting the murine breast cancer cell line 4T1 (1x10 5 cells/mouse) into the fat pads of ACP5-proficient and deficient female mice, respectively (Fig. 2 A). Strikingly, the deletion of ACP5 significantly reduced the tumor burden (Fig. 2 B,C). In parallel, we created an autochthonous breast cancer model by backcrossing ACP5 KO mice with MMTV-PyMT mice, which spontaneously develop breast tumors at approximately 9–10 weeks of age 34 , 35 . Consistently, genetic ablation of ACP5 led to a reduction in breast tumor size in age-matched ACP-deficient MMTV-PyMT mice (supplementary Fig. 3A-C). According to our previous findings from bulk and scRNA-sequencing data, we hypothesized that these differences in tumor growth may be attributed to differences in the cellular composition of the tumor microenvironment, particularly macrophages, between ACP5-proficient and ACP5-deficient mice. Thus, we used multiplex immunohistochemistry staining to characterize macrophage subtypes in breast cancer tissues obtained from the orthotopic breast cancer model, and we observed a striking increase in MHC II-positive cells, a marker of M1 macrophages, in ACP5-deficient mice compared to WT mice. Conversely, Arg1-positive immune cells were less abundant in the tumor tissues of ACP5-deficient mice (Fig. 2 D, E). Collectively, these findings strongly suggest that ACP5 is essential for maintaining the balance between pro-inflammatory (M1) and anti-inflammatory (M2) macrophages within the breast cancer-associated microenvironment. Since M2 macrophages play a key role in promoting tumor growth, progression, and epithelial-mesenchymal transition (EMT)—a process that facilitates cancer metastasis—and given that ACP5 has also been shown to induce EMT in lung cancer cells via P53/Smad3 signaling and contribute to the pathogenesis of idiopathic pulmonary fibrosis 28 , 36 , we examined the expression of EMT-related proteins in tumor tissues. Notably, key EMT markers, including MMP-9, Snail, and VEGF, were downregulated in tumor tissues from ACP5-deficient mice, while E-cadherin expression was increased (Fig. 2 F). These results suggest that metastatic potential was also reduced in ACP5-deficient mice following murine breast cancer implantation. ACP5 suppresses proinflammatory reactions in bone marrow-derived macrophage. Tumor cells influence surrounding and infiltrating immune cells through secretion of cyto-/chemokines, enhancing pro-tumorigenic or modulating anti-tumorigenic responses for their own benefit. In this line, given our findings of how ACP5 influences the homeostasis of macrophages within the tumor-associated microenvironment and affects tumor growth in vivo, we examined differences in BMDMs harvested from ACP5-proficient and ACP5-deficient mice following classical (IFN-γ/LPS) or alternative (IL-4/IL-13) activation. To confirm ACP5 activity in BMDMs derived from WT and ACP5-deficient mice, ACP5 staining was examined upon LPS stimulation. As shown in Supplementary Fig. 4, ACP5 activity was completely absent regardless of classical, or tumor-associated medium stimulation. These results confirm that ACP5 activity is entirely absent in BMDMs from ACP5-deficient mice. Notably, we observed an increase in proinflammatory cyto-/chemokines, including IL-1β, TNF, IL-6, and IL-12a/b, in ACP5-deficient BMDMs compared to WT BMDMs following IFN-γ and LPS stimulation. Additionally, genes associated with M1 macrophages, such as iNOS and CD86, were more expressed in ACP5 KO BMDMs than in WT BMDMs upon classical cyto-/chemokine stimulation. In contrast, genes linked to M2 anti-inflammatory macrophages were downregulated in ACP5 KO BMDMs compared to WT BMDMs (Fig. 3 A, B). Furthermore, we examined the expression of M1- and M2-associated genes in WT and ACP5-deficient BMDMs after incubation with tumor-conditioned medium. Most proinflammatory genes, as well as M1 macrophage-secreted cyto-/chemokines and iNOS, were upregulated in ACP5 KO BMDMs (Fig. 3 C). Conversely, M2 macrophage-associated genes, such as Arg1 and CD206, were downregulated (Fig. 3 D). However, the expression of few genes associated with immunosuppressive cyto-/chemokines (such as IL-10) was inconsistent with the result obtained from ACP5 deficient BMDM upon IL-4/IL-13 stimulation. This result suggested that ACP5 deletion promotes the proinflammatory reaction and suppresses the anti-inflammatory function in BMDM. Immunoblotting analysis further confirmed an increased expression of iNOS, while the expression of Arg1 was attenuated in ACP5-deficient BMDMs following classical activation or incubation with tumor-conditioned medium (Fig. 3 E). To validate the immunosuppressive role of ACP5 in BMDMs, we analyzed ACP5 expression in M1 and M2 macrophages using a previously published scRNA-seq dataset from breast cancer patients (GSE176078) 33 . Expression of CXCL10 was used as markers for M1 macrophages, while expression of ERG1 served as marker for M2 macrophages. Notably, ACP5 expression was significantly higher in ERG1-positive macrophages than in CXCL10-positive macrophages. Additionally, since M2 macrophages mainly depend on fatty acid oxidation to support their functions 37 , macrophages expressing FABP5 or APOE—markers of lipid-associated tumor-associated macrophages—are characterized as immunosuppressive macropahges 38 . Notably, we observed an increased expression of ACP5 in these subpopulations within breast cancer tissues (Fig. 3 F). To unbiasedly investigate the underlying mechanisms and genetic differences between ACP5-proficient and ACP5-deficient BMDMs, we performed RNA-seq on these cells. Functionally, GSEA analysis revealed that gene signatures associated with innate immunity, T cell-derived cytotoxicity, and the TNF inflammatory signaling axis were significantly enriched in ACP5-deficient macrophages, whereas immunosuppressive gene signatures were correlated with ACP5-proficient macrophages (Fig. 3 G). Next, we molecularly characterized M1 BMDMs from ACP5-proficient and ACP5-deficient mice using comprehensive RNA-seq analysis. As shown in Fig. 3 H, pathway analysis revealed that genes related to the PI3K-Akt pathway, cAMP pathway, AMPK pathway, AGE-RAGE pathway, and several cellular metabolic pathways were upregulated in ACP5 KO M1 macrophages. Since AMPK is a crucial protein regulating autophagy, cell death, and glucose/fatty acid metabolism, we examined the phosphorylation of AMPK in ACP5 WT and KO BMDMs. Interestingly, an upregulation of pAMPK (Ser174) was observed in ACP5 KO BMDMs compared to WT BMDMs. We further treated ACP5 WT and KO BMDMs with metformin (an AMPK activator) and compound C (an AMPK inhibitor). Even after treatment, pAMPK levels remained higher in ACP5 KO BMDMs compared to ACP5 WT BMDMs (Fig. 3 I). Overall, our results indicate fundamental differences in immune responses and molecular regulation between ACP5-proficient and ACP5-deficient BMDMs. More importantly, ACP5 in macrophages promotes a metabolic shift that drives macrophages toward an immunosuppressive phenotype. ACP5 expression in BMDMs restricts glycolytic activity and modulates macrophage reprogramming. Recent studies have shown that macrophages can be polarized into distinct subpopulations, such as M1 or M2 macrophages, in response to environmental cues like cyto-/chemokines within tumor tissues 38 .Furthermore, based on our findings (Figs. 1 and 2 ), ACP5-deficient mice exhibit a different macrophage composition in orthotopic murine breast cancer tissues compared to ACP5 WT mice. Given this, we investigated the role of ACP5 in macrophage reprogramming. BMDMs harvested from WT and ACP5-deficient mice were first polarized into M1-like macrophages using a classical activation protocol. These M1-like macrophages were then subjected to alternative activation with IL-4/IL-13 stimulation to induce M2-like polarization. As shown in Fig. 4 A, iNOS expression persisted in ACP5-deficient M1-like BMDMs following alternative activation, whereas it was lost in WT M1-like BMDMs. To further validate these findings, Raw264.7 cells were transfected with either ACP5 WT plasmid or an ACP5 dominant-negative mutation plasmid (ACP52m-OE) to assess whether ACP5 influences macrophage polarization in a similar manner. Figure 4 B validates ACP5 activity in Raw264.7 cells transfected with different plasmids. The expression patterns of iNOS and Arg1 in Raw264.7 cells transfected with ACP52m-OE mirrored those observed in ACP5-deficient macrophages upon classical or alternative activation (Fig. 4 C). Notably, polarized M2-like Raw264.7 cells transfected with ACP52m-OE failed to reprogram into M1-like macrophages, as confirmed by immunoblotting for iNOS and Arg1 (Fig. 4 D). Together, our results demonstrate that ACP5 is essential for macrophage reprogramming. Furthermore, our data demonstrated that ACP5 KO BMDMs exhibited increased expression of pAMPK, along with enrichment of AMPK-related genes and metabolic pathways compared to ACP5 WT BMDMs (Fig. 3 ). Since glycolysis in macrophages positively correlates with M1 proinflammatory function 39 , we further investigated mitochondrial metabolism by examining the extracellular acidification rate (ECAR), a measure of glycolysis. To do this, we assessed ECAR following the sequential addition of 0.5 µM Rotenone/Antimycin A (Rot/AA) and 50 mM 2-DG in ACP5 WT and KO BMDMs. As shown in Fig. 4 E, basal glycolysis, physiological rates, and proton efflux rates were significantly higher in ACP5 KO M1 and M2 BMDMs compared to ACP5 WT BMDMs. However, this difference was not observed in naïve BMDMs (without stimulation). Importantly, no significant differences were observed in oxygen consumption rates (OCR), including basal respiration rates and ATP-production coupled respiration rates, between ACP5 KO BMDMs and ACP5 WT BMDMs (Supplementary Fig. 5). These findings suggest that ACP5 plays a crucial role in metabolic reprogramming in macrophages. Interestingly, phagocytosis of CFSE-labeled human Jurkat cells was significantly higher in ACP5 KO M1 BMDMs compared to WT M1 BMDMs (Fig. 5 E). A similar increase in phagocytic ability was observed in ACP5-deficient BMDMs incubated with tumor-conditioned medium. Collectively, these findings indicate that ACP5-deficient M1 BMDMs exhibit enhanced proinflammatory activity, leading to more effective tumor cell eradication compared to ACP5-proficient BMDMs. Considering that phagocytosis is essential for tumor cell clearance, our results collectively suggest that ACP5 in macrophages disrupts tumor growth. ACP5 expression in macrophages enhances breast cancer cell migration and invasiveness. Functionally, different macrophage subpopulations within tumor tissues influence tumor cell invasion, migration, and distant metastasis. Given that ACP5 deletion enhances the proinflammatory function of macrophages and alters their polarization, we investigated whether ACP5-deficient macrophages impact breast cancer growth and progression. As shown in Fig. 5 A and 5 B, a transwell invasion assay revealed that ACP5 WT M2 BMDMs promoted the invasiveness of 4T1 breast cancer cells, whereas co-culture with WT M1 BMDMs significantly reduced their invasiveness. Interestingly, ACP5-KO M2 BMDMs drastically suppressed invasion, bringing it to nearly the same low level observed in WT M1 BMDMs. Since previous studies have reported that M2 macrophage-derived secretomes facilitate tumor cell migration 40 , we further examined 4T1 cell migration after incubation with conditioned medium derived from WT or ACP5-KO BMDMs. WT M2 BMDM-conditioned medium significantly enhanced 4T1 breast cancer cell migration, shortening the artificial wound gap after 9 hours of incubation. In contrast, ACP5-KO M2 BMDM-conditioned medium impaired 4T1 cell migration (Fig. 5 C and 5 D). Together with our previous findings (Figs. 3 and 4 ), ACP5-deficient BMDMs enhance proinflammatory responses and innate immune reactions, resulting in a significant anti-tumor effect that disrupts tumor cell progression, migration and invasiveness. ACP5 deletion in M2 macrophages reduces distant metastasis in breast cancer. Since ACP5-deficient M1/M2 BMDMs exhibited a stronger proinflammatory response and demonstrated potential tumor-eradicating functions compared to ACP5-proficient BMDMs, and given that polarized M2 macrophages play a crucial role in tumor cell migration and invasion (Fig. 5 ), we next investigated the role of ACP5 in a metastatic mouse model of breast cancer. In an orthotopic breast cancer model, co-injecting polarized ACP5 KO M2 BMDMs with 4T1 tumor cells into the mammary fat pad of BALB/c mice significantly reduced tumor size and formation (Fig. 6 A and B). After 21 days, the number of metastatic nodules in lung tissues, quantified by microscopy, was noticeably decreased in this group (Fig. 6 C). Mechanistically, downregulation of MMP9 and Snail, along with upregulation of E-cadherin, in tumor tissues from ACP5-KO M2 macrophage-injected mice suggests that ACP5 deficiency attenuates the epithelial-mesenchymal transition (EMT) process in breast cancer growth in vivo (Fig. 6 D). In a metastatic breast cancer model, similar to the orthotopic model findings, co-injection of 4T1 cells and polarized ACP5 KO M2 macrophages via tail vein injection into BALB/c mice led to a striking reduction in metastatic nodules within lung tissues (Fig. 6 F-H). These results indicate that ACP5-KO M2 BMDMs lose their pro-tumorigenic functions, thereby inhibiting tumor growth and distant metastasis. Collectively, this study demonstrates that ACP5 modulates inflammatory responses in macrophages and plays a critical role in macrophage polarization. Furthermore, ACP5 deletion in M2 macrophages within breast cancer tissues markedly impairs tumor growth and distant metastasis (Fig. 7 ). Discussion Macrophages play a crucial role in regulating tumor growth within the tumor microenvironment 41 , 42 . Tumor cells, endothelial cells, and cancer-associated fibroblasts secrete various factors, including cytokines and chemokines, that recruit macrophages to the TME. Subsequently, macrophages can polarize toward the M2 phenotype, which supports cancer progression and resistance to therapy 42 . This has led to increased interest in strategies that would shift macrophages toward M1 polarization while reducing the presence of M2 macrophages as a potential cancer therapy 43 , 44 . ACP5, a glycosylated metalloprotease mainly expressed in osteoclasts and activated macrophages, is crucial for regulating several physiological processes, such as inflammation and bone remodeling 28 . Furthermore, our previous data suggest that serum ACP5 expression may serve as a predictive and prognostic marker for assessing disease progression and treatment response in breast cancer patients with bone or visceral metastasis. This effect may be linked to increased ACP5 expression in macrophages within the TME. 21 Given this, it is important to explore the role of ACP5 in macrophages within tumor tissue to determine if targeting ACP5 could offer a viable therapeutic approach for cancer treatment. Our study uncovers a novel role of ACP5 in macrophages by regulating metabolic flexibility within the TME. We demonstrate that ACP5 acts as an inhibitory checkpoint for macrophages in the TME by downregulating glucose metabolism through glycolysis, thereby impairing their cytotoxic functions. These findings not only enhance our understanding of ACP5 biology but also position ACP5 as a potential target for future cell therapies in anti-cancer strategies. To gain a better understanding of the role of ACP5 in macrophages within the tumor microenvironment (TME), we performed bulk and single-cell RNA sequencing on breast cancer tissues, revealing the significant involvement of ACP5 in macrophages within the tumor. While previous studies have highlighted the crucial role of ACP5 in tumor growth and progression in various cancer types 21 , 23 , 26 , 28 , 36 , 45 , 46 , 47 , our findings in this study show that the proliferation of both human and murine breast cancer cell lines was unaffected by the overexpression or downregulation of ACP5. Importantly, in an orthotopic breast cancer model, co-injection of ACP5-deficient macrophages with cancer cells resulted in reduced tumor growth, progression, and distant metastasis. This data represents a key difference from prior research that the deletion of ACP5 in macrophages influences breast cancer growth, rather than having a direct effect on the breast cancer cells themselves. Interestingly, our scRNA analysis also identified ACP5 expression in various immune cells and tumor-associated microenvironment cells, such as cancer-associated fibroblasts. This raises the question of whether immune cells like cytotoxic T cells, other myeloid cells, or NK cells contribute to the observed anti-tumor effect. Although co-injection of ACP5-deficient M2 macrophages and tumor cells significantly reduced tumor growth and distant metastasis, highlighting the crucial role of ACP5 in macrophages, the role of ACP5 in these cells within the tumor microenvironment still requires further investigation and assessment. Seeking to shed light on the impact of ACP5 in the function of macrophages, we conducted an unbiased RNA sequencing analysis comparing ACP5-proficient and -deficient BMDMs, validating the findings through various functional analyses. Given that anaerobic metabolic pathways, such as glycolysis, promote proinflammatory responses with strong antitumor effects in macrophages 48 , 49 , 50 , we found that several genes related to catabolic signaling pathways, including cAMP, cGMP-PKG, and AMPK pathways, were enriched in ACP5-deficient macrophages, which was not mentioned before. ACP5-KO macrophages exhibited an intriguing metabolic profile, marked by an increase in ECAR without a corresponding rise in OCR. This suggests a shift towards anaerobic glycolysis, typically linked to elevated ECAR, while mitochondrial oxidative phosphorylation (indicated by OCR) appears unaffected. Additionally, ACP5-KO macrophages exhibited elevated expression of pAMPK, a key regulator of cellular energy balance, as well as macrophage function and polarization 51 , 52 . An elevated pAMPK expression, driving the immune cells to rely more on glycolysis to sustain ATP levels, which explains the increased ECAR (a hallmark of glycolytic activity). This altered metabolic state may result from a disrupted balance between glycolysis and oxidative phosphorylation, regulated by AMPK signaling, enabling ACP5-KO macrophages to adapt to energy deficits while maintaining cellular function. 53 . This metabolic shift could contribute to the altered functional responses of ACP5-deficient macrophages in various physiological or disease contexts. Previous studies have suggested that metabolic shifts in macrophages not only control the balance between proinflammatory and anti-inflammatory responses but also regulate macrophage polarization 38 , 51 , 53 . Interestingly, ACP5 deficiency led to a shift in macrophage polarization, characterized by an increase in M1-like macrophages and a decrease in M2-like macrophages. This change in macrophage phenotype was accompanied by heightened proinflammatory activity and improved anti-tumor functions, indicating that ACP5 may act as a suppressor of macrophage-mediated inflammatory responses. The most plausible conclusion we can draw from our findings is that ACP5 in macrophages plays a critical role in shaping the inflammatory environment of the TME, and its deletion promotes macrophage reprogramming to enhance tumor elimination. There are still several unanswered questions in our study. First, we have yet to investigate the detailed molecular regulation and downstream signaling in ACP5-deficient macrophages. Additionally, the mechanism by which ACP5 regulates AMPK phosphorylation remains unclear. In cancer cells, ACP5 participates in various signaling pathways that influence cell behavior. It interacts with the PI3K/Akt pathway, which was often deregulated in cancers, promoting cell survival, growth, and resistance to apoptosis 29 . The MAPK/ERK pathway is another key route where ACP5 might support cancer cell proliferation and survival. In a pulmonary fibrotic model, ACP5 has been linked to Wnt/β-catenin signaling, which regulates cell proliferation and migration—critical processes in cancer progression 36 . In our GSEA analysis, we found that genes associated with the PI3K-Akt pathway were enriched in ACP5-KO M1 macrophages compared to ACP5-WT M1 macrophages, which appears to contradict recent findings. Recent studies suggest that AMPK and AKT have antagonistic roles under metabolic stress 54 . Our findings may be explained by a compensatory increase in the AKT/PI3K pathway, driven by significantly elevated AMPK phosphorylation and excessive energy consumption in ACP5-deficient macrophages. Interestingly, Other studies suggest that AMPK enhances Akt activation and its associated biological functions through the activation of the growth factor EGF and its downstream Ca2+/Calmodulin-dependent kinase 55 . Future research could focus on further investigating the detailed molecular regulation and interaction between ACP5 and AMPK. Additionally, we did not explore whether the tumor-eradicating effect in ACP5-deficient mice was driven by the influence of other immune cells activated by cytokines and chemokines within the tumor-associated microenvironment. In glioma cells, ACP5 regulates AKT phosphorylation in the CCL18/CCR8 signaling axis, where its deletion reduces AKT phosphorylation 30 . Spatial transcriptomics and conditional deletion mice would be necessary to decipher the detailed function and interaction of ACP5 between tumor cells and various immune cells. Third, although we observed several functional impacts, such as increased phagocytosis of cancer cells, disruption of tumor migration/invasiveness, and reduced distant metastasis due to the influence of ACP5-deficient macrophages in tumor tissues, it remains unclear whether these phenotypes are linked to AMPK activation. Since macrophage polarization, metabolic shifts, and glycolysis are regulated by multiple pathways 38 , 51 , it is challenging to determine whether a single signaling pathway directly contributes to these tumor-eradicating effects. Future studies will be necessary to identify the specific pathways that drive tumor eradication in the absence of ACP5. This study emphasizes the crucial role of ACP5 in regulating macrophage polarization and function within the TME. Our findings also reveal that the absence of ACP5 leads to increased glycolytic activity in macrophages, which is associated with enhanced proinflammatory functions. Furthermore, ACP5-deficient macrophages exhibited increased phagocytic activity against cancer cells, supporting the notion that ACP5 modulates macrophage reprogramming to enhance anti-tumor immunity. By influencing the inflammatory properties of macrophages, ACP5 affects tumor progression, and its deletion shifts macrophages toward a more pro-inflammatory, anti-tumor phenotype. These results suggest novel therapeutic strategies, especially those aimed at reprogramming macrophages to boost their tumor-eradicating potential. At present, ACP5-specific inhibitors like NaAuCl4, AubipyOMe, and CBK289001 have the potential to inhibit ACP5 activity within the tumor, possibly transforming the tumor microenvironment from a pro-tumor to an anti-tumor state 56 , 57 . Although the complete molecular regulation of ACP5 in macrophages is still not fully understood, our study provides new insights into immune cell checkpoints, particularly in macrophages, for anti-cancer therapies. The impact of ACP5 on macrophage populations within the TME offers valuable perspectives for cancer immunology and potential therapeutic approaches. Data availability The datasets generated and/or analyzed in this study are not publicly available but can be obtained from the corresponding author upon reasonable request. Materials and methods Plasmids and antibodies The mouse ACP5 cDNA was amplified from pLenti-ACP5-Myc-DDK (Origene, MR204798L1V), and a double mutation (G217R / M266K) of mouse ACP5 (ACP5-2m) was introduced using a site-directed mutagenesis kit (Promega). Both wild-type and mutant ACP5 cDNAs were then cloned into the pLAS3w.c.Ppuro vector (Sinica, ROC). A stable ACP5-overexpressing Raw264.7 cell line was established via lentiviral infection, followed by puromycin selection. ACP5 enzyme activity was assessed using the ACP5 Active Kit, and antibodies were used to analyze macrophage polarization and cancer metastasis. Animals C57BL/6 wild-type and BALB/c wild-type mice (6–8 weeks old) were purchased from the National Laboratory Animal Center (Taiwan, ROC). ACP5 knock-out (ACP5-KO) mice on a 129SV background were generously provided by Dr. Janckila Anthony. These ACP5-KO mice were back-crossed into a homogeneous BALB/c background for at least six generations.The presence of the ACP5 knockout allele in offspring was confirmed by PCR using specific primers. All mice were bred and maintained at the Laboratory Animal Center of the National Defense Medical Center, Taipei, Taiwan. Cell Lines The 4T1 murine breast tumor cell line was generously provided by Dr. Nan-Shih Liao (Institute of Molecular Biology, Academia Sinica, Taiwan, ROC),** while the RAW264.7 mouse monocyte/macrophage cell line was a gift from Dr. Yi-Ping Chuang (Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan).Both cell lines were originally sourced from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml). For 4T1-BMDM co-culture experiments, transwell inserts with porous polycarbonate membrane filters (0.4 µm pore size) in 6-well plastic culture plates were used. Bone marrow-derived macrophage (BMDM) culture ACP5-WT and ACP5-KO mice (6 to 8 weeks old) will be euthanized, and the femur and tibia from each leg will be excised. Bone marrow will be flushed out using a 23 G needle and a 1 mL syringe, followed by incubation in red cell lysis buffer (0.155 M ammonium chloride in PBS) to remove erythrocytes.The cells will then be cultured in a sterile petri dish containing 10 mL of RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, and L929-conditioned medium for 6 days. After this period, the medium containing non-adherent cells will be removed, and the remaining adherent cells will be further incubated in L929-conditioned medium for an additional 2 days. Generation and functional characterization of M1/M2-polarized macrophages Bone marrow-derived macrophages (BMDMs) were generated by culturing bone marrow cells, isolated from the femur and tibia of mice, in DMEM supplemented with 15% FBS and 20% L929-conditioned medium for 11 days to allow maturation.For macrophage polarization, BMDMs were expanded and treated with: IFNγ (10 ng/mL) and LPS (100 ng/mL) for M1 polarization; IL-4 (10 ng/mL) and IL-13 (10 ng/mL) for M2 polarization Protein extraction, electrophoresis and western blotting The indicated cells were washed with PBS and resuspended in 30–60 µL of lysis buffer containing 1X Complete™ protease inhibitors (Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitors (Cocktail I and II, Sigma). The cell suspension was incubated on ice for 30–40 minutes, followed by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatant (lysate) was carefully transferred to a fresh tube, ensuring the pellet remained undisturbed, and stored at −20°C for further analysis. The lysates were then incubated with 20 mM DTT and 1X sample loading buffer at 95°C for 10 minutes. After this step, the samples were equilibrated to match the least concentrated lysate before being denatured again with reducing sample buffer and DTT at 95°C for another 10 minutes.Protein separation was performed using 4–15% Mini-PROTEAN® TGX™ Precast Protein Gels and TGX buffer (Bio-Rad). The proteins were subsequently transferred onto a 0.2 µm nitrocellulose membrane using Bio-Rad Trans-Blot Turbo Mini Nitrocellulose Transfer Packs.The membranes were blocked with 2.5% milk in PBS containing 0.05% Tween-20 (PBS/Tween) for 1 hour, followed by a brief PBS wash. They were then incubated overnight at 4°C with primary antibodies.The next day, the membranes were washed three times with PBS/Tween for 10 minutes each, followed by incubation with HRP-conjugated secondary antibodies for 45 minutes. Afterward, the membranes underwent three additional PBS/Tween washes (5–10 minutes each).Antibody binding was detected using enhanced chemiluminescence (ECL) substrate, and signals were visualized using X-ray film (Scientific Laboratory Supplies). RNA extraction, cDNA synthesis and quantitative real-time PCR (qRT-PCR) Cells from each well were lysed and collected separately using TRIzol reagent, followed by RNA extraction using the miRNeasy kit (Qiagen, Mat. No. 1071023). RNA purity was assessed using NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA).cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). For quantitative real-time PCR (qRT-PCR), a reaction mix was prepared containing sample cDNA, primers, TaqMan primer-probes, and TaqMan Gene Expression Master Mix (all from Applied Biosystems). qRT-PCR was conducted using the ABI 7900HT Sequencing Detection System (Applied Biosystems). Primer sequences are listed in supplementary table 1, with β-actin used as an internal reference. The relative expression of target genes was calculated using the 2^−ΔΔCt method. All PCR reactions were performed in triplicate. Library construction and RNA-sequencing Next-generation sequencing (NGS) libraries were prepared following the manufacturer’s protocol. The sequencing data were processed and analyzed by GENEWIZ (South Plainfield, NJ, USA). Invasion assay Tumor cell invasion assays were conducted using a Transwell system (Millipore, USA) with 8 µm-pore polycarbonate filter membranes coated with Matrigel. For the migration assay, 1 × 10⁴ 4T1 cells were seeded into the upper chamber, which was then inserted into the lower chamber containing different polarized BMDMs in DMEM with 15% FBS. After 24 hours of incubation, non-migrated cells on the interior surface of the upper chamber were removed. The polycarbonate membranes were then stained with 0.1% crystal violet for 10 minutes, and the number of migrating cells was quantified by examining entire fields under a microscope. Cell migration assay The wound-healing assay was used to assess 4T1 cell migration. 4T1 cells were seeded at a density of 5 × 10⁵ cells per well in a 6-well plate containing 10% FBS/DMEM and cultured until reaching 80% confluence.A scratch was carefully made in the cell monolayer using the tip of a 1 mL pipette, after which the medium was replaced with 50% conditioned medium from different polarized BMDMs. Images were captured at 0 and 9 hours using a microscope at 100× magnification with a camera. This assay was performed at least three times. Phagocytosis assay by Flow cytometric analysis For the phagocytosis assay, Jurkat cells were labeled with 5 mM CFSE for 30 minutes at 37°C. After two washes, the cells were resuspended at 5 × 10⁵ cells/mL in complete medium. BMDMs were harvested and washed as described previously. 5 × 10⁵ macrophages were then incubated with 1.5 × 10⁶ CFSE-labeled Jurkat cells for 60 minutes at 37°C. After incubation, non-adherent cells were removed by washing with PBS, and macrophages were detached using Trypsin-EDTA. Macrophages were then stained with anti-CD11b and analyzed by flow cytometry. Phagocytosis was quantified as the percentage of CD11b and CFSE double-positive cells relative to the total number of CD11b-positive macrophages, calculated as: (CD11b and CFSE double-positive cells/Total CD11b-positive cells)×100 Tumor induction animal experiment For primary tumor growth, 1 × 10⁶ 4T1 cancer cells and 2.5 × 10⁴ M2-polarized BMDMs were orthotopically injected into the inguinal mammary fat pad, following a previously established protocol. For the experimental lung metastasis model, 1 × 10⁵ 4T1 cells and 2.5 × 10⁴ M2-polarized BMDMs were intravenously injected via the tail vein. Mice were euthanized on day 16 for histological analysis. Explore the cell composition of macrophages in murine breast cancer tissues by opal multiplex immunofluorescence staining Murine breast cancer tissues obtained from fat pad orthotopic murine breast cancer cells injection in ACP5 WT and ACP5 KO mice, were proceed for further experiments. An innovative multiplexed immunohistochemistry (IHC) imaging technique using the Opal 6-Plex Manual Detection Kit (Akoya Biosciences®) were utilized. Tissue pre-treatment and antibody incubation were performed following previously published protocols, with the secondary antibody replaced by the EnVision Plus Detection System (Dako #K50070). After washing, the designated tyramide signal amplification dye (Opal 6-Color Kit, Akoya Biosciences®) was applied for 10 minutes, following the manufacturer’s instructions. The slides were then heated in a retrieval buffer using a steamer to remove primary and secondary antibodies, preparing them for the next staining target. This process was followed by cooling, blocking, and repeating antibody and Opal dye incubation for five additional staining cycles. For the Macropahges’ Panel, the following primary antibodies were used for separate incubations: F4/80 (CST, Cat#70076, RRID:, 1:1500), Arginase 1 (CST, Cat#98668, RRID:, 1:100), MHC II (Invitrogen, Cat#BS-8481R, RRID:, 1:100), DAPI (PerkinElmer, Cat# FP1490, 1:500), PanCK (DAKO, Cat#11237709, RRID:, 1:100). Analysis of cell bioenergetics The oxygen consumption rate (OCR), the extracellular acidification rate (ECAR) and Real-Time ATP rate were measured using a Seahorse XFe96 Analyzer (Agilent, California, CA, USA) with Seahorse XF Cell Mito Stress Test Kit (Agilent, cat#: 103015-100), Seahorse XF Glycolytic Rate Assay Kit (Agilent, cat#: 103344-100) and Seahorse XF Real-Time ATP Rate Assay kit (Agilent, cat#: 103592-100), respectively, following the manufacturer’s instructions. Briefly, macrophages were seeded in 8-well Seahorse assay plates at a concentration of 2 × 10 4 cells/well, and cultured overnight for attachment. Prior to the assay, cells were washed, and the medium was replaced with Seahorse XF RPMI for macropahges or supplemented with 20 mM glucose, 2 mM L-glutamine and 1 mM sodium pyruvate. Presto Blue™ assays (Thermo Fisher Scientific) were used to evaluate cell viability and normalize readings from the Seahorse XF Analyzer. Public dataset retrieved and reanalysis Normalized RNA-seq data from different cancer types was obtained from the publicly available database TCGA (The Cancer Genomic Atlas) and GTEx (The Genotype-Tissue Expression). According to this database, all data was obtained by using the Illumina HiSeq platform and retrieved by the bioinformatics tool UCSC Xena browser (URL: https://xenabrowser.net/). The phenotypic cohort including survival data and clinical parameters were also retrieved from the patients diagnosed of breast cancer(BRCA). Samples without detailed information in terms of normalized mRNA expression, survival status and overall times were removed before enrollment. The single-cell RNA-seq dataset was downloaded from GSE176078. We performed scRNA-seq analysis using the R package "Seurat" (version 4.1.0). Cells with fewer than 200 detected genes or more than 50% mitochondrial reads were excluded from the analysis.A shared nearest neighbor (SNN) graph was then constructed, and uniform manifold approximation and projection (UMAP) embedding was generated using the top 20 principal components. The main cell types were identified based on annotations consistent with the original literature 33 . To further classify macrophages, we applied M1/M2 macrophage signatures from published studies and performed subclustering using the R package "CelliD". The proportions of each subpopulation were calculated, and immune-related signaling pathway activity scores were computed using the Broad Institute’s Hallmark collection. Statistical analysis All statistical analyses were performed using GraphPad PRISM 10 software (GraphPad Software, Inc. La Jolla, CA). Statistical comparisons were performed by two-way ANOVA with Bonferroni post-test by Student's t-test or by Mann–Whitney test for non-parametric distributions with small sample size. Probability values of p ≤ 0.05 were considered statistically significant. Declarations Acknowledgement We would like to express our gratitude to all members of Professor Chen Ying-Chuan's group for their valuable scientific discussions. Our thanks also go to the Cancer Registry Group at Tri-Service General Hospital. We appreciate BioRender’s support in creating some of the figures for this article. The bioenergy analysis was made through the technical services provided by the Instrument Center of the National Defense Medical Center. This research was further supported by several grants from the Tri-Service General Hospital (TSGH-E-112206/TSGH-E-113225/TSGH-E-114227) and the National Defense Medical Centre (MND-MAB-D-111073/MND-MAB-D-114084) awarded to YGC. Funding This research was further supported by several grants from the Tri-Service General Hospital (TSGH-E-112206/TSGH-E-113225/TSGH-E-114227) and the National Defense Medical Centre (MND-MAB-D-111073/MND-MAB-D-114084) awarded to YGC. The bioenergy analysis and RNA-seq were funded by a grant from the National Science and Technology Council (NSTC113-2320-B-016-007), awarded to YCC. Author information The project was conceived by MS Dai. The research was designed by YGC, DDM, YCC,CHT, and PY Liu, and they collaborated on writing the manuscript. SFT, HCL, CYB, LJC and YGC conducted the majority of the experiments in collaboration with others. Multiplex IHC were carried out by CHY, TLL, CTC and SHY. ER and PK contributed to the cell death experiments. Bioinformatic and RNA-seq analysis using TCGA datasets were performed by YGC, FYT and SSJ. Histopathological analysis of tumor tissues was conducted in a blinded manner by AJ, TYC, JCY and CYW. Ethic declaration (A) Competing interests: The authors declare no competing interests. (B) Ethics approval for animal experiment for this research All mice were genotyped by PCR and were provided with food ad libitum. All animal experiments were carried out under the appropriate NDMC project license, in compliance with Taiwan's home office regulations for animal welfare as outlined in the Animal (Scientific Procedures) Act 1986 (ASPA). The relevant Animal Ethics Committee approved all experiments involving ACP5-deficient mouse crosses, which were maintained under the necessary licenses. References Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet 2001, 357 (9255) : 539-545. Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. 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Zhao Y, Hu X, Liu Y, Dong S, Wen Z, He W , et al. ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway. Mol Cancer 2017, 16 (1) : 79. Han F, Li CF, Cai Z, Zhang X, Jin G, Zhang WN , et al. The critical role of AMPK in driving Akt activation under stress, tumorigenesis and drug resistance. Nat Commun 2018, 9 (1) : 4728. Boorsma CE, van der Veen TA, Putri KSS, de Almeida A, Draijer C, Mauad T , et al. A Potent Tartrate Resistant Acid Phosphatase Inhibitor to Study the Function of TRAP in Alveolar Macrophages. Sci Rep 2017, 7 (1) : 12570. Reithmeier A, Lundback T, Haraldsson M, Frank M, Ek-Rylander B, Nyholm PG , et al. Identification of inhibitors of Tartrate-resistant acid phosphatase (TRAP/ACP5) activity by small-molecule screening. Chem Biol Drug Des 2018, 92 (1) : 1255-1271. Additional Declarations (Not answered) Supplementary Files SupplementaryTable1humanandmiceimmunecheckpointmarkerQPCRprimers.xlsx SP table 1 FigureSF1.pdf Supplementary Figure 1. ACP5 did not affect tumor cells growth. (A-B) Growth of the indicated cells stably transfected with overexpressing ACP5 or a dominant-negative mutant version of the protein. Data are expressed as means ± SEM (n = 3). (C) A correlation plot generated using the Spearman method visually represents the relationship between the transcripts of ACP5 and MKI 67 in TCGA breast cancer dataset (D.E) Bubble plot of GO enrichment analysis related to molecular function and cellular component for DEGs showing upregulation in breast cancer patients with high ACP5 expression compared to those with low ACP5 expression. "Count" represents the number of DEGs enriched in the pathway, "GeneRatio" indicates the ratio of enriched DEGs to background genes, and "p.adj" shows the p-value adjusted using the Benjamini-Hochberg (BH) method. FigureSF2.pdf Supplementary Figure 2. The design of ACP5 KO mice and the subsequent functional analysis conducted in these mice. (A) A schematic diagram of the ACP5 deletion strategy used to generate ACP5-deficient mice. (B) ACP5 enzymatic activity was assessed in various organs from the genetically modified mice using a commercial kit. (C) BMDMs from ACP5 WT and KO mice were treated with LPS for 24 hours, and ACP5 enzymatic activity was assessed using the ACP5 staining kit (COSMO BIO CO Cat: AK04F). NaAuCl4, a specific ACP5 inhibitor, was used as a negative control. (D) The bone marrow samples from ACP5 WT and KO mice were stained for ACP5 and neuron-specific enolase (NSE), a specific marker for all granulocytes. Representative results from at least three independent replicates are shown. FigureSF3.pdf Supplementary Figure 3. ACP5 deletion attenuated tumor growth in a spontaneous murine breast cancer model. (A-C) Tumor sizes and representative gross images were compared between MMTV/ACP5 WT and MMTV/ACP5 KO mice. The results from (B-C) are shown as representative examples. (D) Total protein was extracted from tumor tissues of these mice and analyzed by Western blot for the indicated proteins. Sf4.pdf Supplementary Figure 4. ACP5 activity in ACP5 WT and KO BMDMs treated under the specified conditions. ACP5 enzymatic activity was measured in BMDMs from both ACP5 WT and KO mice under the indicated conditions. Representative results from at least three independent replicates are shown. SF5.pdf Supplementary Figure 5. Deletion of ACP5 in BMDM did not affect mitochondria respiration. Oxygen consumption rate (OCR) of the specified BMDMs was measured following consecutive injections of oligomycin (1.5 mM), FCCP (1.5 mM), and rotenone/antimycin A (0.5 mM). OCR was normalized to cell numbers. Basal respiration, maximal respiration, ATP production-coupled respiration, and proton leak rates are presented as bar graphs. 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Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Pei-Yao","middleName":"","lastName":"Liu","suffix":""},{"id":463558672,"identity":"89a6071f-5b95-41e8-91da-3c0e619666b3","order_by":5,"name":"Ying-Chuan Chen","email":"","orcid":"https://orcid.org/0000-0001-8031-1893","institution":"National Defense Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Ying-Chuan","middleName":"","lastName":"Chen","suffix":""},{"id":463558673,"identity":"70bc5b46-dd7c-47a3-ad09-1abe3c2b51b7","order_by":6,"name":"Yang-Hong Dai","email":"","orcid":"","institution":"Tri-Service General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yang-Hong","middleName":"","lastName":"Dai","suffix":""},{"id":463558674,"identity":"87b17e38-27fb-4074-a4d7-2ea162591ed5","order_by":7,"name":"Shun-Fu Tseng","email":"","orcid":"","institution":"Division of Hematology/Oncology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Centre, Taipei, Taiwan, Republic of China","correspondingAuthor":false,"prefix":"","firstName":"Shun-Fu","middleName":"","lastName":"Tseng","suffix":""},{"id":463558675,"identity":"b8703494-b981-4c97-ab46-3730ba9d5aa6","order_by":8,"name":"Chih-Hung Ye","email":"","orcid":"","institution":"Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan","correspondingAuthor":false,"prefix":"","firstName":"Chih-Hung","middleName":"","lastName":"Ye","suffix":""},{"id":463558676,"identity":"128f1c9c-a084-4d46-bca7-6cc4d2ab4438","order_by":9,"name":"Thien-Long Le","email":"","orcid":"https://orcid.org/0009-0001-6889-1079","institution":"Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan","correspondingAuthor":false,"prefix":"","firstName":"Thien-Long","middleName":"","lastName":"Le","suffix":""},{"id":463558677,"identity":"07fbfe86-b121-4d2d-8bdf-fd8a20e13515","order_by":10,"name":"Patrick Chun Theng Chong","email":"","orcid":"","institution":"Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"Chun Theng","lastName":"Chong","suffix":""},{"id":463558678,"identity":"453f4f70-9887-4236-8607-21d7432041d4","order_by":11,"name":"Hao-Chan Lo","email":"","orcid":"","institution":"Division of Hematology/Oncology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Centre, Taipei, Taiwan, Republic of Chin","correspondingAuthor":false,"prefix":"","firstName":"Hao-Chan","middleName":"","lastName":"Lo","suffix":""},{"id":463558679,"identity":"5c90ecad-c9b9-4ce6-9704-94c2db73b67a","order_by":12,"name":"Chia-Yu Bai","email":"","orcid":"","institution":"Division of Hematology/Oncology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Centre, Taipei, Taiwan, Republic of China","correspondingAuthor":false,"prefix":"","firstName":"Chia-Yu","middleName":"","lastName":"Bai","suffix":""},{"id":463558680,"identity":"f7032aa7-52a7-4c79-a6c5-4ff58baab61a","order_by":13,"name":"Li-Jia Chen","email":"","orcid":"","institution":"Division of Hematology/Oncology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Centre, Taipei, Taiwan, Republic of China","correspondingAuthor":false,"prefix":"","firstName":"Li-Jia","middleName":"","lastName":"Chen","suffix":""},{"id":463558681,"identity":"30171875-dbd7-4a16-8fa4-86d09a1c1f34","order_by":14,"name":"Chin-Hsien Tsai","email":"","orcid":"","institution":"Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan","correspondingAuthor":false,"prefix":"","firstName":"Chin-Hsien","middleName":"","lastName":"Tsai","suffix":""},{"id":463558682,"identity":"c88886db-4fcb-4ac3-8ae8-8978e7840633","order_by":15,"name":"Chao-Ying Wang","email":"","orcid":"","institution":"Department and Graduate Institute of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan","correspondingAuthor":false,"prefix":"","firstName":"Chao-Ying","middleName":"","lastName":"Wang","suffix":""},{"id":463558683,"identity":"55ac7270-5d00-4e3a-bdf7-d6d6dd70c60d","order_by":16,"name":"Fang-Yu Tsai","email":"","orcid":"","institution":"National Institute of Cancer Research, National Health Research Institutes, Miaoli County 350, Taiwan.","correspondingAuthor":false,"prefix":"","firstName":"Fang-Yu","middleName":"","lastName":"Tsai","suffix":""},{"id":463558684,"identity":"bbc8cc66-7012-4006-85da-53c3d20d4ce1","order_by":17,"name":"Shih Sheng Jiang","email":"","orcid":"","institution":"National Health Research Institutes","correspondingAuthor":false,"prefix":"","firstName":"Shih","middleName":"Sheng","lastName":"Jiang","suffix":""},{"id":463558685,"identity":"b23d3a91-a54c-4a3a-9630-ccd83ab9ed92","order_by":18,"name":"Anthony Janckila","email":"","orcid":"","institution":"Division of Hematology/Oncology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Centre, Taipei, Taiwan, Republic of China","correspondingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"","lastName":"Janckila","suffix":""},{"id":463558686,"identity":"a7916e7e-dcc8-4b94-b3b7-5ccee82b6c0b","order_by":19,"name":"Tsu-Yi Chao","email":"","orcid":"","institution":"nternational Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei City, 11031, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical Unive","correspondingAuthor":false,"prefix":"","firstName":"Tsu-Yi","middleName":"","lastName":"Chao","suffix":""},{"id":463558687,"identity":"3f46bb00-f1d3-4e5d-8006-4fdfad45e482","order_by":20,"name":"Jyh-Cherng Yu","email":"","orcid":"","institution":"Division of Breast Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan","correspondingAuthor":false,"prefix":"","firstName":"Jyh-Cherng","middleName":"","lastName":"Yu","suffix":""},{"id":463558688,"identity":"ac7f0834-dc41-4972-a598-beccd224b30d","order_by":21,"name":"Nan-Shih Liao","email":"","orcid":"https://orcid.org/0000-0003-3707-4145","institution":"Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan","correspondingAuthor":false,"prefix":"","firstName":"Nan-Shih","middleName":"","lastName":"Liao","suffix":""}],"badges":[],"createdAt":"2025-05-13 15:21:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6656922/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6656922/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88360599,"identity":"6e0b3e3f-af9d-44f1-9b60-07d9d706c6e0","added_by":"auto","created_at":"2025-08-05 16:14:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":619163,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACP5 is mainly expressed on macrophages within breast cancer tissues. \u003c/strong\u003e(A) RNA-seq expression data from the TCGA and GTEx databases were analyzed. All normalized expression data were downloaded from the UCSC Xena platform. Transcripts of \u003cem\u003eACP5\u003c/em\u003e in tumor tissues were retrieved from the TCGA database in breast cancer patients. Normal breast tissues were sourced from GTEx and TCGA matched normal samples. Statistical significance was determined using a two-tailed Student’s t-test, with asterisks indicating significance (* p \u0026lt; 0.05, **** p \u0026lt; 0.001). (B) Bubble plot of Gene Ontology (GO) enrichment analysis related to biological process for DEGs showing upregulation in breast cancer patients with high ACP5 expression compared to those with low ACP5 expression. \"Count\" represents the number of DEGs enriched in the pathway, \"GeneRatio\" indicates the ratio of enriched DEGs to background genes, and \"p.adj\" shows the p-value adjusted using the Benjamini-Hochberg (BH) method. (C) Analysis of \u003cem\u003eACP5\u003c/em\u003eexpression at the single-cell level. ACP5 expression levels across different cell types were assessed through dimensional reduction plots (left and middle panels) and dot plots (right panels). The findings indicate that ACP5 is predominantly expressed in immune cells, with higher expression in macrophages. CAF: cancer associated fibroblats; PVL: perivascular-like (PVL) subpopulations (D) The average ACP5 expression across CAF, cancer epithelial cells, normal epithelial cells, and the myeloid series is presented in a violin plot. When compared to the other three cell types in the plot, the average ACP5 expression in myeloid series cells showed statistically significant differences, as determined by an unpaired Student's t-test (P \u0026lt; 0.001).(E) The percentage of ACP5-positive (ACP5-expressing) cells out of the total cell population is shown on the y-axis of this plot, including both immune and epithelial cells.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/8027bcdcde9701d7c92f7493.jpg"},{"id":88360823,"identity":"de808b8b-31d2-4898-919d-6ccefa8d0c86","added_by":"auto","created_at":"2025-08-05 16:23:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":830587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe ACP5-deficient microenvironment suppresses cancer cell growth and reduces distant metastatic potential in both murine orthotopic breast cancer models. \u003c/strong\u003e(A) Schematic diagram of the experimental process (n = 7 per group). A total of 1 × 10^5 4T1 cells were injected into the fat pad of female BALB/c mice and monitored for 28 days. (B) IVIS images of tumor-bearing ACP5-proficient and deficient mice are shown (images taken on Day 21 after tumor injection), along with a quantitative graph of IVIS signals at 7, 14, and 21 days post-injection. Tumor images and weight quantification on Day 21 are presented in (C). Error bars represent the mean ± SEM of this experiment. Statistical significance was assessed by a two-tailed Student’s t-test (*p \u0026lt; 0.05). (D) Multiplex immunohistochemistry identifies the precise locations of immune subsets in murine breast cancer samples (n = 3 per condition: ACP5-deficient and proficient mice). FFPE sections of breast cancer were stained with the ‘Macrophages Panel’ for pan-macrophages (F4/80), M1-like macrophages (MHC II), and M2-like macrophages. Representative images are shown. All images were processed to assess the density of immune cells within defined areas of tumor tissues. The total density was determined as (total cells/area). M1-like cells were distinguished by negative staining for Arginase-1, alongside positive staining for MHC II and F4/80, while M2-like cells were identified by the absence of MHC II expression and the presence of Arginase-1 and F4/80 expression. The density in ACP5 WT and ACP5 KO mice implanted with 4T1 cells was quantified and compared. Error bars represent the mean ± SEM of the experiment. (F) Total protein was extracted from tumor tissues obtained from ACP5-proficient and deficient mice. Western blot analysis for the indicated proteins was performed. Representative results from at least three independent replicates are shown. Additionally, the blot quantification was performed using ImageJ and is presented on the right, showing the relative expression of the indicated proteins normalized to Actin (n = 7 per group). Statistical significance was determined using a two-tailed Student’s t-test, with asterisks indicating statistical significance (*p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/c7979f842a237386c97693d2.jpg"},{"id":88361936,"identity":"0b2dbead-78b9-47c4-bfe8-858cd58b7866","added_by":"auto","created_at":"2025-08-05 16:31:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":726889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACP5 regulates proinflammatory responses in bone marrow-derived macrophages through several pathways. \u003c/strong\u003e(A-D) The indicated transcripts were quantified by RT-PCR and normalized to 18S transcript levels. Expression levels were compared between ACP5-WT and KO M1-like and M2-like macrophages, which were polarized with LPS/IFN-γ and IL-4/IL-13 for 6 hours, respectively. Additionally, transcript levels from ACP5-proficient and deficient macrophages treated with tumor cell-associated conditioned medium (TCM) for 6 hours were also compared.\u003c/p\u003e\n\u003cp\u003e(E) Western blot analysis for the indicated proteins was performed on BMDMs polarized under different conditions from ACP5-proficient and deficient mice. Representative results from at least three independent replicates are shown.\u003c/p\u003e\n\u003cp\u003e(F) The violin plot demonstrates ACP5 expression in M1/M2-like macrophages, characterized by related genes from the scRNA dataset (GEO 176078). FABP5 and APOE genes are represented in macrophages polarized by alternative activation, while CXCL10 and EGR1-positive myeloid cells were defined by M1-like and M2-like macrophages, respectively. Statistical significance was determined using a two-tailed Student’s t-test, with asterisks indicating statistical significance (**p \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e(G-H) RNA sequencing performed on ACP5 WT and KO BMDMs was used for Gene Set Enrichment Analysis of different immunological and inflammatory pathways and GO pathway analysis. (I) Western blot analysis for the indicated proteins was performed on BMDMs polarized under different conditions from ACP5-proficient and deficient mice. Met: metformin, CC: Compound C. Representative results from at least three independent replicates are shown.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/c29fbc27cab8c6eed59a5695.jpg"},{"id":88360630,"identity":"c648d8d2-a93b-4e10-874f-e91ef5f61c0d","added_by":"auto","created_at":"2025-08-05 16:15:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":699247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACP5 expression in BMDMs restricts glycolytic activity and modulates macrophage reprogramming. \u003c/strong\u003e(A) The indicated proteins were analyzed by western blotting. M1 and M2-like macrophages were repolarized again in the indicated BMDMs obtained from ACP5 WT and KO BMDMs respectively. Representative results from at least three independent replicates are shown. (B) ACP5 enzyme activity was assessed in Raw264.7 cells treated under different conditions. Raw264.7/vector: cells stably expressing the vector alone; Raw264.7/ACP5\u003csup\u003eWT-OE\u003c/sup\u003e: cells constitutively expressing the ACP5 overexpression vector; and Raw264.7/ACP5\u003csup\u003e2m-OE\u003c/sup\u003e: cells expressing a loss-of-function ACP5 vector. (C.D) The indicated proteins were analyzed in Raw264.7 cells expressing different vectors. The numbers correspond to Raw264.7 cells transfected with the specified plasmids. Lane 1 in Figure 4C shows Raw264.7 cells transfected with the empty vector without cytokine chemokine stimulation. Lanes 1, 4, and 7 in Figure 4D represent Raw264.7 cells transfected with the indicated plasmids without any polarization protocol (M0). Lanes 2, 5, and 8 in Figure 4D show that Raw264.7 cells transfected with the indicated plasmids were first polarized into M1 macrophages, followed by alternative stimulation, which led to M2 macrophage polarization. Lanes 3, 6, and 9 in the same figures demonstrate that these polarized M2 Raw 264.7 cells were reprogrammed into M1 macrophages. Representative results from at least three independent replicates are shown. (E) ECAR of the indicated BMDMs treated with Rot/AA (0.5 mM) and 2-DG (50 mM) was measured. Basal glycolysis was quantified and shown as bar charts. Data are presented as mean ± SEM. (F) Phagocytosis assay was performed to assess macrophage-mediated tumor cell killing. Effector cells were polarized BMDMs isolated from ACP5 WT and KO mice. The target cells were CFSE-labeled human lymphoma Jurkat cells. Polarized macrophages and CFSE-Jurkat cells (BMDM: Jurkat = 1:10) were incubated for 60 minutes, and BMDMs were labeled with anti-CD11b-PE antibody. The rate of phagocytosis (double positive) was then quantified by flow cytometry.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/9afd125b51bd18eb67a13932.jpg"},{"id":88360816,"identity":"ea101167-4e75-4ce0-84fe-457b33ced72b","added_by":"auto","created_at":"2025-08-05 16:22:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":409242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACP5 expression in macrophages promotes breast cancer cell migration and invasiveness. \u003c/strong\u003e(A) The invasion transwell assay was performed using a non-contact co-culture system. Representative results from at least three independent replicates are shown. (B) OD at 570 nm was measured and quantified under the indicated conditions. (C) A wound healing assay was performed in 4T1 cells cultured with different conditioned media from nonpolarized, M1, and M2 BMDMs. Representative microscopic images are shown at 0 and 9 hours with 100x magnification. (D) The ratio of the width at 9 hours to the basal width (0 hours) was compared in 4T1 cells incubated with the indicated BMDMs obtained from ACP5 WT or KO mice. Statistical significance was determined using a two-tailed Student’s t-test, with asterisks indicating statistical significance (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/d5b9104f93693c406e736e8f.jpg"},{"id":88360815,"identity":"d73c2ecb-1cbf-41f3-98f2-73d76a2abe90","added_by":"auto","created_at":"2025-08-05 16:22:59","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":856472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACP5 deletion in M2 macrophages reduces distant metastasis in breast cancer. \u003c/strong\u003e(A.E) Schematic diagram of the co-injection orthotopic and distant metastatic experimental models (n = 6 per group). (B) Gross images showing tumor weight in mice orthotopically implanted with 4T1 cells and ACP WT or ACP5 KO M2-polarized BMDMs (images taken on Day 21 post-tumor injection). Tumor images and weight quantification on Day 21 are presented. (C) Pulmonary nodules were examined, counted, and quantified. Representative images of pulmonary HE staining are shown. (D) Total protein was extracted from tumor tissues of mice treated under different conditions. Western blot analysis for the indicated proteins was performed. Representative results from at least three independent replicates are shown. Additionally, blot quantification was conducted using ImageJ and is presented on the right, showing the relative expression of the indicated proteins normalized to Actin (n = 6 per group). Gross images of the pulmonary region (F) and representative HE staining images (G) are shown for the tail-vein co-injection pulmonary metastatic model. Pulmonary nodules were quantified (H). Error bars represent the mean ± SEM of the experiment. Statistical significance was determined using a two-tailed Student’s t-test, with asterisks indicating statistical significance (*p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/f1392222d3649d1513130d5f.jpg"},{"id":88361933,"identity":"d1f1663f-6aee-466a-a138-2403022af557","added_by":"auto","created_at":"2025-08-05 16:30:59","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":319580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModels illustrating how ACP5 regulates macrophage polarization, contributing to tumor growth and distant metastasis in breast cancer. \u003c/strong\u003eACP5-proficient macrophages dampen the pro-inflammatory response and inhibit the tumor-eradicating functions of M1-like macrophages. Moreover, ACP5 limits metabolic reprogramming in macrophages within tumor tissue. In contrast, ACP5-deficient macrophages show a significantly enhanced anti-tumor response. Overall, ACP5 acts as a regulator, controlling the pro-inflammatory reaction of macrophages in tumor environments.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/945dd183f74acffe13cc66b2.jpg"},{"id":88361967,"identity":"32118e21-18f5-44c0-94be-11c1b0569915","added_by":"auto","created_at":"2025-08-05 16:31:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6165161,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/0ee7eb34-d6c1-41b3-917c-daf774a4c9b8.pdf"},{"id":88360627,"identity":"c2047087-516a-4e9a-8e6c-8a53a5c1abc2","added_by":"auto","created_at":"2025-08-05 16:15:01","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11358,"visible":true,"origin":"","legend":"SP table 1","description":"","filename":"SupplementaryTable1humanandmiceimmunecheckpointmarkerQPCRprimers.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/46fb1989af231802c9a6156e.xlsx"},{"id":88361931,"identity":"deb75f1f-94d5-453b-89d4-8443cc41c8c4","added_by":"auto","created_at":"2025-08-05 16:30:59","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":944798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. ACP5 did not affect tumor cells growth. \u003c/strong\u003e(A-B) Growth of the indicated cells stably transfected with overexpressing ACP5 or a dominant-negative mutant version of the protein. Data are expressed as means ± SEM (n = 3). (C) A correlation plot generated using the Spearman method visually represents the relationship between the transcripts of \u003cem\u003eACP5\u003c/em\u003e and \u003cem\u003eMKI 67\u003c/em\u003e in TCGA breast cancer dataset (D.E) Bubble plot of GO enrichment analysis related to molecular function and cellular component for DEGs showing upregulation in breast cancer patients with high ACP5 expression compared to those with low ACP5 expression. \"Count\" represents the number of DEGs enriched in the pathway, \"GeneRatio\" indicates the ratio of enriched DEGs to background genes, and \"p.adj\" shows the p-value adjusted using the Benjamini-Hochberg (BH) method.\u003c/p\u003e","description":"","filename":"FigureSF1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/9e7e50932fb8dd102a948f0a.pdf"},{"id":88361932,"identity":"92363300-7573-472d-8967-d170a2836ea0","added_by":"auto","created_at":"2025-08-05 16:30:59","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2039468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. The design of ACP5 KO mice and the subsequent functional analysis conducted in these mice. \u003c/strong\u003e(A) A schematic diagram of the ACP5 deletion strategy used to generate ACP5-deficient mice. (B) ACP5 enzymatic activity was assessed in various organs from the genetically modified mice using a commercial kit. (C) BMDMs from ACP5 WT and KO mice were treated with LPS for 24 hours, and ACP5 enzymatic activity was assessed using the ACP5 staining kit (COSMO BIO CO Cat: AK04F). NaAuCl4, a specific ACP5 inhibitor, was used as a negative control. (D) The bone marrow samples from ACP5 WT and KO mice were stained for ACP5 and neuron-specific enolase (NSE), a specific marker for all granulocytes. Representative results from at least three independent replicates are shown.\u003c/p\u003e","description":"","filename":"FigureSF2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/18382d173ae2e2a1985221ca.pdf"},{"id":88360828,"identity":"c2fd11a1-8ce6-4a6a-96d6-db3aac6409f1","added_by":"auto","created_at":"2025-08-05 16:23:00","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2801788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. ACP5 deletion attenuated tumor growth in a spontaneous murine breast cancer model. \u003c/strong\u003e(A-C) Tumor sizes and representative gross images were compared between \u003cem\u003eMMTV/ACP5\u003c/em\u003eWT and \u003cem\u003eMMTV/ACP5\u003c/em\u003e KO mice. The results from (B-C) are shown as representative examples. (D) Total protein was extracted from tumor tissues of these mice and analyzed by Western blot for the indicated proteins.\u003c/p\u003e","description":"","filename":"FigureSF3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/750f1d62dba3958a39ef3772.pdf"},{"id":88360608,"identity":"b43d004c-723e-4733-a424-e9509298d98e","added_by":"auto","created_at":"2025-08-05 16:14:59","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":7650027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. ACP5 activity in ACP5 WT and KO BMDMs treated under the specified conditions. \u003c/strong\u003eACP5 enzymatic activity was measured in BMDMs from both ACP5 WT and KO mice under the indicated conditions. Representative results from at least three independent replicates are shown.\u003c/p\u003e","description":"","filename":"Sf4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/d7d67540c507d0700dd18bf8.pdf"},{"id":88360819,"identity":"8b932ca0-7f9b-4313-bf36-3cd2f16beb0e","added_by":"auto","created_at":"2025-08-05 16:22:59","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":164400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5. Deletion of ACP5 in BMDM did not affect mitochondria respiration.\u003c/strong\u003e Oxygen consumption rate (OCR) of the specified BMDMs was measured following consecutive injections of oligomycin (1.5 mM), FCCP (1.5 mM), and rotenone/antimycin A (0.5 mM). OCR was normalized to cell numbers. Basal respiration, maximal respiration, ATP production-coupled respiration, and proton leak rates are presented as bar graphs. Data are expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"SF5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/9335e05c924b389ff4fd5deb.pdf"},{"id":88360820,"identity":"867d24ec-8aca-4c8e-b75a-b39e9f3b380b","added_by":"auto","created_at":"2025-08-05 16:23:00","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":757028,"visible":true,"origin":"","legend":"original blot","description":"","filename":"originalblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6656922/v1/df6aaab6294c6a8c425a8d88.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Tartrate-resistant acid phosphatase 5 regulates the metabolic flexibility of macrophages in the tumor microenvironment, thereby influencing their functional fate and modulating tumor growth","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic inflammation increases cancer risk, a concept first recognized by Virchow in the 19th century \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Even in tumors not traditionally linked to inflammation, an inflammatory component is a crucial part of the malignant microenvironment\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.In early tumor formation, immune cells effectively eliminate abnormal cells, but at later stages, they often fail to suppress cancer and instead contribute to tumor progression and metastasis. \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Macrophages, key players in the innate immune system, are crucial for homeostasis, inflammation, and phagocytosis. In the tumor microenvironment (TME), macrophages are the most abundant immune cells and essential for modulating tumor growth and progression. Based on their morphology, phenotype, genetic features and function, macrophages are classified into two main subtypes-M1 macrophages, which mediate pro-inflammatory responses and contribute to antitumor immunity, while M2 macrophages, which exhibit pro-tumor properties, promoting tumor growth and metastasis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. A mix of M2 macrophages and a small subset of M1 macrophages, known as tumor-associated macrophages (TAMs), are highly diverse immune cells in the TME\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, and essential for tumor progression. Tumor cells secrete chemokines and growth factors to attract and convert macrophages into tumor-promoting M2 types. Increased TAM infiltration correlates with poor clinical outcomes and reduced response to standard treatments. \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Moreover, dynamic shifts in macrophage subsets influence the effectiveness of immunotherapy, making TAM modulation a growing area of interest for cancer therapy. \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Therefore, targeting TAMs or modifying the TME to increase the presence of M1 macrophages while decreasing M2 macrophages has been suggested as a potential therapeutic approach.. \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTartrate-resistant acid phosphatase (TRAP), also known as acid phosphatase 5 (ACP5), is a glycosylated monomeric metalloprotease expressed in mammals. It is characterized by its resistance to tartrate inhibition and exhibits optimal enzymatic activity under acidic conditions. TRAP is initially synthesized as a latent proenzyme (TRAP5a), which possesses low phosphatase activity. Upon proteolytic cleavage in the intervening loop domain, it is converted into its active form (TRAP5b), which exhibits high phosphatase activity. \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Under normal conditions, ACP5 is highly expressed in osteoclasts and activated macrophages. However, its expression is elevated under certain pathological conditions, including hairy cell leukemia, osteoclastoma, osteoporosis, and metabolic bone diseases. \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Janckila et al. conducted a comprehensive immunohistochemical analysis using the TRAP 5-specific antibody mAb220, which revealed that macrophages in tissues associated with chronic antigenic stimulation or affected by chronic inflammatory conditions were the primary ACP5-expressing cells. \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Interestingly, ACP5 has also been detected in various cancer cells and tissues, including ovarian, cervical cancers, and malignant melanoma, with its expression levels correlating with tumor severity. \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. High ACP5 expression is linked to poorer survival outcomes in various cancers. It correlates with reduced tumor-free and metastasis-free survival in malignant melanoma, \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, lower overall survival in lung\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and is an independent risk factor for gastric cancer peritoneal transmission\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Additionally, ACP5 promotes breast cancer metastasis by interacting with TRIP-1, activating TGFβR2 and Smad2/3 signaling\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Although these studies suggest that ACP5 in cancer cells promotes tumor growth and disease progression, its role in macrophages within the tumor-associated microenvironment\u0026mdash;the most abundant infiltrating immune cells in tumor tissues\u0026mdash;, remains unclear\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the impact of ACP5 on macrophages and the tumor microenvironment, we performed bulk and single-cell RNA sequencing using several public datasets. Our analysis showed that ACP5 is highly expressed in macrophages within breast cancer tissues, but not in breast cancer cells. Using a genetically modified mouse model implanted with breast cancer cells, we observed a significant reduction in tumor size in ACP5-deficient mice compared to wild-type mice, both in the orthotopic fat pad injection model and the spontaneous genetic breast cancer model. Mechanistically, ACP5 deletion led to a notable shift in macrophage polarization, increasing M1-like macrophages while reducing M2-like macrophages in the tumor-associated microenvironment. Unbiased RNA sequencing and immunoblotting of M1-polarized bone marrow-derived macrophages (BMDMs) from ACP5-deficient mice revealed that AMPK-related genes and AMPK activity were upregulated. Functionally, ACP5 KO BMDMs exhibited increased glycolytic activity compared to ACP5 WT BMDMs, leading to enhanced proinflammatory function and increased phagocytic activity against cancer cells. In an orthotopic breast cancer model, co-injection of 4T1 cells with ACP5 KO BMDMs significantly impeded tumor growth, progression, and distant metastasis compared to 4T1 cells co-injected with ACP5 WT BMDMs. Collectively, Our findings indicate that ACP5 in activated macrophages plays a suppressive role in macrophage-mediated inflammatory responses by regulating metabolic flexibility. The absence of ACP5 promotes macrophage reprogramming and enhances their tumor-eradicating capacity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eACP5 is mainly expressed on macrophages within breast cancer tissues.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSeveral studies have demonstrated that high ACP5 expression is a negative prognostic factor for overall survival (OS) in patients with lung adenocarcinoma, liver cancer breast cancer, and colon cancer\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Additionally, ACP5 serves as an important downstream hub in CCL18/CCR8 signalling axis for modulating glioma cell growth and proliferation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, the specific ACP5-expressing cells and the detailed mechanistic regulation of ACP5 in breast cancer patients remain unclear. To address this, we set out to examine the expression levels of \u003cem\u003eACP5\u003c/em\u003e in breast cancer samples using publicly available RNA expression data from the UCSC Xena platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://xena.ucsc.edu/\u003c/span\u003e\u003cspan address=\"https://xena.ucsc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e31\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, we found elevated \u003cem\u003eACP5\u003c/em\u003e expression in breast cancer tissues compared to normal breast tissues from the GTEx database and the corresponding adjacent normal tissues from the TCGA dataset. To decipher the functional and molecular regulation of \u003cem\u003eACP5\u003c/em\u003e in breast cancer, a functional enrichment analysis was performed based on the differentially expressed genes between high and low \u003cem\u003eACP5\u003c/em\u003e expression groups in TCGA BRCA to assess the different biological processes, molecular functions, and cellular components influenced by ACP5. Interestingly, we observed that the genes associated with leukocyte function, cytokine production, cell-cell interactions, and leukocyte proliferation were predominantly upregulated in breast cancer patients with high ACP5 expression compared to those with low expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, SF1D and E). To identify the main source of ACP5-expressing cells, we examined the differential expression of ACP5 in different cell types in breast cancer tissues by using a public single cell RNA sequencing (scRNA-seq) dataset (GSE176078)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. After applying the same quality control criteria initially used, a total of 100,064 cells from breast cancer tissues were used for this analysis. We found that ACP5 was predominantly expressed in myeloid cells, particularly in macrophages within tumor tissues, showing not only a high proportion of ACP5 positivity but also high expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E, SF1D). ACP5 has been described to be overexpressed in lung adenocarcinoma cells where it exerted pro-tumorigenic functions. Thus, attenuation of ACP5 drastically impaired the proliferation, migration, and invasion in A549 and H1975 cells\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. On this basis, we examined these functions in human and murine breast cancer cells. Intriguingly, we could not detect any impact on cell viability in breast cancer cells either overexpressing ACP5 or transfected with a dominant-negative mutant version of the protein (SF1A-B). On the other hand, there was no positive correlation between \u003cem\u003eACP5\u003c/em\u003e and \u003cem\u003eKi-67\u003c/em\u003e transcripts in TCGA BRCA dataset (SF1C). Collectively, these findings indicate that in breast cancer tissues, ACP5 is predominantly expressed in macrophages rather than in breast cancer cells. Moreover, ACP5 expression in breast cancer cells does not influence their proliferation or growth. This result also highlights that ACP5 in breast cancer tissue is primarily expressed in macrophages, directing our focus to the impact of ACP5 in macrophages within the cancer tissue, rather than in the tumor cells themselves.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe ACP5-deficient microenvironment suppresses cancer cell growth and reduces distant metastatic potential in both murine orthotopic and spontaneous breast cancer models.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the impact of ACP5 in breast cancer progression, we generated an inducible ACP5-deficient mice. The design and knock-out (KO) strategy is shown in supplementary Fig.\u0026nbsp;2A. First, the knockout efficiency of ACP5 was confirmed by assessing the expression of ACP5 in serum, spleen and kidney samples from ACP5 heterozygous and homozygous mice (SF2B) and by performing IHC analysis on bone marrow samples (SF2D). In addition, the functional effect of ACP5 absence in bone marrow-derived macrophages (BMDMs) harvested from ACP5-deficient mice was analyzed by performing a LPS stimulation experiment (SF2C). Next, to assess the role of ACP5 expression in the tumor microenvornment at promoting cancer progression, we used these ACP5 KO mice to generate an orthotopic murine breast cancer model by injecting the murine breast cancer cell line 4T1 (1x10\u003csup\u003e5\u003c/sup\u003e cells/mouse) into the fat pads of ACP5-proficient and deficient female mice, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Strikingly, the deletion of ACP5 significantly reduced the tumor burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB,C). In parallel, we created an autochthonous breast cancer model by backcrossing ACP5 KO mice with MMTV-PyMT mice, which spontaneously develop breast tumors at approximately 9\u0026ndash;10 weeks of age\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Consistently, genetic ablation of ACP5 led to a reduction in breast tumor size in age-matched ACP-deficient MMTV-PyMT mice (supplementary Fig.\u0026nbsp;3A-C). According to our previous findings from bulk and scRNA-sequencing data, we hypothesized that these differences in tumor growth may be attributed to differences in the cellular composition of the tumor microenvironment, particularly macrophages, between ACP5-proficient and ACP5-deficient mice. Thus, we used multiplex immunohistochemistry staining to characterize macrophage subtypes in breast cancer tissues obtained from the orthotopic breast cancer model, and we observed a striking increase in MHC II-positive cells, a marker of M1 macrophages, in ACP5-deficient mice compared to WT mice. Conversely, Arg1-positive immune cells were less abundant in the tumor tissues of ACP5-deficient mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). Collectively, these findings strongly suggest that ACP5 is essential for maintaining the balance between pro-inflammatory (M1) and anti-inflammatory (M2) macrophages within the breast cancer-associated microenvironment. Since M2 macrophages play a key role in promoting tumor growth, progression, and epithelial-mesenchymal transition (EMT)\u0026mdash;a process that facilitates cancer metastasis\u0026mdash;and given that ACP5 has also been shown to induce EMT in lung cancer cells via P53/Smad3 signaling and contribute to the pathogenesis of idiopathic pulmonary fibrosis\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, we examined the expression of EMT-related proteins in tumor tissues. Notably, key EMT markers, including MMP-9, Snail, and VEGF, were downregulated in tumor tissues from ACP5-deficient mice, while E-cadherin expression was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results suggest that metastatic potential was also reduced in ACP5-deficient mice following murine breast cancer implantation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eACP5 suppresses proinflammatory reactions in bone marrow-derived macrophage.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTumor cells influence surrounding and infiltrating immune cells through secretion of cyto-/chemokines, enhancing pro-tumorigenic or modulating anti-tumorigenic responses for their own benefit. In this line, given our findings of how ACP5 influences the homeostasis of macrophages within the tumor-associated microenvironment and affects tumor growth in vivo, we examined differences in BMDMs harvested from ACP5-proficient and ACP5-deficient mice following classical (IFN-γ/LPS) or alternative (IL-4/IL-13) activation. To confirm ACP5 activity in BMDMs derived from WT and ACP5-deficient mice, ACP5 staining was examined upon LPS stimulation. As shown in Supplementary Fig.\u0026nbsp;4, ACP5 activity was completely absent regardless of classical, or tumor-associated medium stimulation. These results confirm that ACP5 activity is entirely absent in BMDMs from ACP5-deficient mice. Notably, we observed an increase in proinflammatory cyto-/chemokines, including IL-1β, TNF, IL-6, and IL-12a/b, in ACP5-deficient BMDMs compared to WT BMDMs following IFN-γ and LPS stimulation. Additionally, genes associated with M1 macrophages, such as iNOS and CD86, were more expressed in ACP5 KO BMDMs than in WT BMDMs upon classical cyto-/chemokine stimulation. In contrast, genes linked to M2 anti-inflammatory macrophages were downregulated in ACP5 KO BMDMs compared to WT BMDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Furthermore, we examined the expression of M1- and M2-associated genes in WT and ACP5-deficient BMDMs after incubation with tumor-conditioned medium. Most proinflammatory genes, as well as M1 macrophage-secreted cyto-/chemokines and iNOS, were upregulated in ACP5 KO BMDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Conversely, M2 macrophage-associated genes, such as Arg1 and CD206, were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, the expression of few genes associated with immunosuppressive cyto-/chemokines (such as IL-10) was inconsistent with the result obtained from ACP5 deficient BMDM upon IL-4/IL-13 stimulation. This result suggested that ACP5 deletion promotes the proinflammatory reaction and suppresses the anti-inflammatory function in BMDM. Immunoblotting analysis further confirmed an increased expression of iNOS, while the expression of Arg1 was attenuated in ACP5-deficient BMDMs following classical activation or incubation with tumor-conditioned medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). To validate the immunosuppressive role of ACP5 in BMDMs, we analyzed ACP5 expression in M1 and M2 macrophages using a previously published scRNA-seq dataset from breast cancer patients (GSE176078)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Expression of CXCL10 was used as markers for M1 macrophages, while expression of ERG1 served as marker for M2 macrophages. Notably, ACP5 expression was significantly higher in ERG1-positive macrophages than in CXCL10-positive macrophages. Additionally, since M2 macrophages mainly depend on fatty acid oxidation to support their functions\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, macrophages expressing FABP5 or APOE\u0026mdash;markers of lipid-associated tumor-associated macrophages\u0026mdash;are characterized as immunosuppressive macropahges\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Notably, we observed an increased expression of ACP5 in these subpopulations within breast cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). To unbiasedly investigate the underlying mechanisms and genetic differences between ACP5-proficient and ACP5-deficient BMDMs, we performed RNA-seq on these cells. Functionally, GSEA analysis revealed that gene signatures associated with innate immunity, T cell-derived cytotoxicity, and the TNF inflammatory signaling axis were significantly enriched in ACP5-deficient macrophages, whereas immunosuppressive gene signatures were correlated with ACP5-proficient macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Next, we molecularly characterized M1 BMDMs from ACP5-proficient and ACP5-deficient mice using comprehensive RNA-seq analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, pathway analysis revealed that genes related to the PI3K-Akt pathway, cAMP pathway, AMPK pathway, AGE-RAGE pathway, and several cellular metabolic pathways were upregulated in ACP5 KO M1 macrophages. Since AMPK is a crucial protein regulating autophagy, cell death, and glucose/fatty acid metabolism, we examined the phosphorylation of AMPK in ACP5 WT and KO BMDMs. Interestingly, an upregulation of pAMPK (Ser174) was observed in ACP5 KO BMDMs compared to WT BMDMs. We further treated ACP5 WT and KO BMDMs with metformin (an AMPK activator) and compound C (an AMPK inhibitor). Even after treatment, pAMPK levels remained higher in ACP5 KO BMDMs compared to ACP5 WT BMDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Overall, our results indicate fundamental differences in immune responses and molecular regulation between ACP5-proficient and ACP5-deficient BMDMs. More importantly, ACP5 in macrophages promotes a metabolic shift that drives macrophages toward an immunosuppressive phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eACP5 expression in BMDMs restricts glycolytic activity and modulates macrophage reprogramming.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRecent studies have shown that macrophages can be polarized into distinct subpopulations, such as M1 or M2 macrophages, in response to environmental cues like cyto-/chemokines within tumor tissues\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.Furthermore, based on our findings (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), ACP5-deficient mice exhibit a different macrophage composition in orthotopic murine breast cancer tissues compared to ACP5 WT mice. Given this, we investigated the role of ACP5 in macrophage reprogramming. BMDMs harvested from WT and ACP5-deficient mice were first polarized into M1-like macrophages using a classical activation protocol. These M1-like macrophages were then subjected to alternative activation with IL-4/IL-13 stimulation to induce M2-like polarization. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, iNOS expression persisted in ACP5-deficient M1-like BMDMs following alternative activation, whereas it was lost in WT M1-like BMDMs. To further validate these findings, Raw264.7 cells were transfected with either ACP5 WT plasmid or an ACP5 dominant-negative mutation plasmid (ACP52m-OE) to assess whether ACP5 influences macrophage polarization in a similar manner. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB validates ACP5 activity in Raw264.7 cells transfected with different plasmids. The expression patterns of iNOS and Arg1 in Raw264.7 cells transfected with ACP52m-OE mirrored those observed in ACP5-deficient macrophages upon classical or alternative activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Notably, polarized M2-like Raw264.7 cells transfected with ACP52m-OE failed to reprogram into M1-like macrophages, as confirmed by immunoblotting for iNOS and Arg1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Together, our results demonstrate that ACP5 is essential for macrophage reprogramming.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, our data demonstrated that ACP5 KO BMDMs exhibited increased expression of pAMPK, along with enrichment of AMPK-related genes and metabolic pathways compared to ACP5 WT BMDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Since glycolysis in macrophages positively correlates with M1 proinflammatory function\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, we further investigated mitochondrial metabolism by examining the extracellular acidification rate (ECAR), a measure of glycolysis. To do this, we assessed ECAR following the sequential addition of 0.5 \u0026micro;M Rotenone/Antimycin A (Rot/AA) and 50 mM 2-DG in ACP5 WT and KO BMDMs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, basal glycolysis, physiological rates, and proton efflux rates were significantly higher in ACP5 KO M1 and M2 BMDMs compared to ACP5 WT BMDMs. However, this difference was not observed in na\u0026iuml;ve BMDMs (without stimulation). Importantly, no significant differences were observed in oxygen consumption rates (OCR), including basal respiration rates and ATP-production coupled respiration rates, between ACP5 KO BMDMs and ACP5 WT BMDMs (Supplementary Fig.\u0026nbsp;5). These findings suggest that ACP5 plays a crucial role in metabolic reprogramming in macrophages. Interestingly, phagocytosis of CFSE-labeled human Jurkat cells was significantly higher in ACP5 KO M1 BMDMs compared to WT M1 BMDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). A similar increase in phagocytic ability was observed in ACP5-deficient BMDMs incubated with tumor-conditioned medium. Collectively, these findings indicate that ACP5-deficient M1 BMDMs exhibit enhanced proinflammatory activity, leading to more effective tumor cell eradication compared to ACP5-proficient BMDMs. Considering that phagocytosis is essential for tumor cell clearance, our results collectively suggest that ACP5 in macrophages disrupts tumor growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eACP5 expression in macrophages enhances breast cancer cell migration and invasiveness.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFunctionally, different macrophage subpopulations within tumor tissues influence tumor cell invasion, migration, and distant metastasis. Given that ACP5 deletion enhances the proinflammatory function of macrophages and alters their polarization, we investigated whether ACP5-deficient macrophages impact breast cancer growth and progression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, a transwell invasion assay revealed that ACP5 WT M2 BMDMs promoted the invasiveness of 4T1 breast cancer cells, whereas co-culture with WT M1 BMDMs significantly reduced their invasiveness. Interestingly, ACP5-KO M2 BMDMs drastically suppressed invasion, bringing it to nearly the same low level observed in WT M1 BMDMs. Since previous studies have reported that M2 macrophage-derived secretomes facilitate tumor cell migration\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, we further examined 4T1 cell migration after incubation with conditioned medium derived from WT or ACP5-KO BMDMs. WT M2 BMDM-conditioned medium significantly enhanced 4T1 breast cancer cell migration, shortening the artificial wound gap after 9 hours of incubation. In contrast, ACP5-KO M2 BMDM-conditioned medium impaired 4T1 cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Together with our previous findings (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), ACP5-deficient BMDMs enhance proinflammatory responses and innate immune reactions, resulting in a significant anti-tumor effect that disrupts tumor cell progression, migration and invasiveness.\u003c/p\u003e \u003cp\u003e \u003cb\u003eACP5 deletion in M2 macrophages reduces distant metastasis in breast cancer.\u003c/b\u003e Since ACP5-deficient M1/M2 BMDMs exhibited a stronger proinflammatory response and demonstrated potential tumor-eradicating functions compared to ACP5-proficient BMDMs, and given that polarized M2 macrophages play a crucial role in tumor cell migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), we next investigated the role of ACP5 in a metastatic mouse model of breast cancer. In an orthotopic breast cancer model, co-injecting polarized ACP5 KO M2 BMDMs with 4T1 tumor cells into the mammary fat pad of BALB/c mice significantly reduced tumor size and formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). After 21 days, the number of metastatic nodules in lung tissues, quantified by microscopy, was noticeably decreased in this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Mechanistically, downregulation of MMP9 and Snail, along with upregulation of E-cadherin, in tumor tissues from ACP5-KO M2 macrophage-injected mice suggests that ACP5 deficiency attenuates the epithelial-mesenchymal transition (EMT) process in breast cancer growth in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In a metastatic breast cancer model, similar to the orthotopic model findings, co-injection of 4T1 cells and polarized ACP5 KO M2 macrophages via tail vein injection into BALB/c mice led to a striking reduction in metastatic nodules within lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H). These results indicate that ACP5-KO M2 BMDMs lose their pro-tumorigenic functions, thereby inhibiting tumor growth and distant metastasis. Collectively, this study demonstrates that ACP5 modulates inflammatory responses in macrophages and plays a critical role in macrophage polarization. Furthermore, ACP5 deletion in M2 macrophages within breast cancer tissues markedly impairs tumor growth and distant metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMacrophages play a crucial role in regulating tumor growth within the tumor microenvironment\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Tumor cells, endothelial cells, and cancer-associated fibroblasts secrete various factors, including cytokines and chemokines, that recruit macrophages to the TME. Subsequently, macrophages can polarize toward the M2 phenotype, which supports cancer progression and resistance to therapy\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. This has led to increased interest in strategies that would shift macrophages toward M1 polarization while reducing the presence of M2 macrophages as a potential cancer therapy\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. ACP5, a glycosylated metalloprotease mainly expressed in osteoclasts and activated macrophages, is crucial for regulating several physiological processes, such as inflammation and bone remodeling\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Furthermore, our previous data suggest that serum ACP5 expression may serve as a predictive and prognostic marker for assessing disease progression and treatment response in breast cancer patients with bone or visceral metastasis. This effect may be linked to increased ACP5 expression in macrophages within the TME.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Given this, it is important to explore the role of ACP5 in macrophages within tumor tissue to determine if targeting ACP5 could offer a viable therapeutic approach for cancer treatment. Our study uncovers a novel role of ACP5 in macrophages by regulating metabolic flexibility within the TME. We demonstrate that ACP5 acts as an inhibitory checkpoint for macrophages in the TME by downregulating glucose metabolism through glycolysis, thereby impairing their cytotoxic functions. These findings not only enhance our understanding of ACP5 biology but also position ACP5 as a potential target for future cell therapies in anti-cancer strategies.\u003c/p\u003e \u003cp\u003eTo gain a better understanding of the role of ACP5 in macrophages within the tumor microenvironment (TME), we performed bulk and single-cell RNA sequencing on breast cancer tissues, revealing the significant involvement of ACP5 in macrophages within the tumor. While previous studies have highlighted the crucial role of ACP5 in tumor growth and progression in various cancer types \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, our findings in this study show that the proliferation of both human and murine breast cancer cell lines was unaffected by the overexpression or downregulation of ACP5. Importantly, in an orthotopic breast cancer model, co-injection of ACP5-deficient macrophages with cancer cells resulted in reduced tumor growth, progression, and distant metastasis. This data represents a key difference from prior research that the deletion of ACP5 in macrophages influences breast cancer growth, rather than having a direct effect on the breast cancer cells themselves. Interestingly, our scRNA analysis also identified ACP5 expression in various immune cells and tumor-associated microenvironment cells, such as cancer-associated fibroblasts. This raises the question of whether immune cells like cytotoxic T cells, other myeloid cells, or NK cells contribute to the observed anti-tumor effect. Although co-injection of ACP5-deficient M2 macrophages and tumor cells significantly reduced tumor growth and distant metastasis, highlighting the crucial role of ACP5 in macrophages, the role of ACP5 in these cells within the tumor microenvironment still requires further investigation and assessment.\u003c/p\u003e \u003cp\u003eSeeking to shed light on the impact of ACP5 in the function of macrophages, we conducted an unbiased RNA sequencing analysis comparing ACP5-proficient and -deficient BMDMs, validating the findings through various functional analyses. Given that anaerobic metabolic pathways, such as glycolysis, promote proinflammatory responses with strong antitumor effects in macrophages\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, we found that several genes related to catabolic signaling pathways, including cAMP, cGMP-PKG, and AMPK pathways, were enriched in ACP5-deficient macrophages, which was not mentioned before. ACP5-KO macrophages exhibited an intriguing metabolic profile, marked by an increase in ECAR without a corresponding rise in OCR. This suggests a shift towards anaerobic glycolysis, typically linked to elevated ECAR, while mitochondrial oxidative phosphorylation (indicated by OCR) appears unaffected. Additionally, ACP5-KO macrophages exhibited elevated expression of pAMPK, a key regulator of cellular energy balance, as well as macrophage function and polarization\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. An elevated pAMPK expression, driving the immune cells to rely more on glycolysis to sustain ATP levels, which explains the increased ECAR (a hallmark of glycolytic activity). This altered metabolic state may result from a disrupted balance between glycolysis and oxidative phosphorylation, regulated by AMPK signaling, enabling ACP5-KO macrophages to adapt to energy deficits while maintaining cellular function.\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. This metabolic shift could contribute to the altered functional responses of ACP5-deficient macrophages in various physiological or disease contexts. Previous studies have suggested that metabolic shifts in macrophages not only control the balance between proinflammatory and anti-inflammatory responses but also regulate macrophage polarization\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Interestingly, ACP5 deficiency led to a shift in macrophage polarization, characterized by an increase in M1-like macrophages and a decrease in M2-like macrophages. This change in macrophage phenotype was accompanied by heightened proinflammatory activity and improved anti-tumor functions, indicating that ACP5 may act as a suppressor of macrophage-mediated inflammatory responses. The most plausible conclusion we can draw from our findings is that ACP5 in macrophages plays a critical role in shaping the inflammatory environment of the TME, and its deletion promotes macrophage reprogramming to enhance tumor elimination.\u003c/p\u003e \u003cp\u003eThere are still several unanswered questions in our study. First, we have yet to investigate the detailed molecular regulation and downstream signaling in ACP5-deficient macrophages. Additionally, the mechanism by which ACP5 regulates AMPK phosphorylation remains unclear. In cancer cells, ACP5 participates in various signaling pathways that influence cell behavior. It interacts with the PI3K/Akt pathway, which was often deregulated in cancers, promoting cell survival, growth, and resistance to apoptosis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The MAPK/ERK pathway is another key route where ACP5 might support cancer cell proliferation and survival. In a pulmonary fibrotic model, ACP5 has been linked to Wnt/β-catenin signaling, which regulates cell proliferation and migration\u0026mdash;critical processes in cancer progression\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In our GSEA analysis, we found that genes associated with the PI3K-Akt pathway were enriched in ACP5-KO M1 macrophages compared to ACP5-WT M1 macrophages, which appears to contradict recent findings. Recent studies suggest that AMPK and AKT have antagonistic roles under metabolic stress\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Our findings may be explained by a compensatory increase in the AKT/PI3K pathway, driven by significantly elevated AMPK phosphorylation and excessive energy consumption in ACP5-deficient macrophages. Interestingly, Other studies suggest that AMPK enhances Akt activation and its associated biological functions through the activation of the growth factor EGF and its downstream Ca2+/Calmodulin-dependent kinase \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Future research could focus on further investigating the detailed molecular regulation and interaction between ACP5 and AMPK. Additionally, we did not explore whether the tumor-eradicating effect in ACP5-deficient mice was driven by the influence of other immune cells activated by cytokines and chemokines within the tumor-associated microenvironment. In glioma cells, ACP5 regulates AKT phosphorylation in the CCL18/CCR8 signaling axis, where its deletion reduces AKT phosphorylation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Spatial transcriptomics and conditional deletion mice would be necessary to decipher the detailed function and interaction of ACP5 between tumor cells and various immune cells. Third, although we observed several functional impacts, such as increased phagocytosis of cancer cells, disruption of tumor migration/invasiveness, and reduced distant metastasis due to the influence of ACP5-deficient macrophages in tumor tissues, it remains unclear whether these phenotypes are linked to AMPK activation. Since macrophage polarization, metabolic shifts, and glycolysis are regulated by multiple pathways\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, it is challenging to determine whether a single signaling pathway directly contributes to these tumor-eradicating effects. Future studies will be necessary to identify the specific pathways that drive tumor eradication in the absence of ACP5.\u003c/p\u003e \u003cp\u003eThis study emphasizes the crucial role of ACP5 in regulating macrophage polarization and function within the TME. Our findings also reveal that the absence of ACP5 leads to increased glycolytic activity in macrophages, which is associated with enhanced proinflammatory functions. Furthermore, ACP5-deficient macrophages exhibited increased phagocytic activity against cancer cells, supporting the notion that ACP5 modulates macrophage reprogramming to enhance anti-tumor immunity. By influencing the inflammatory properties of macrophages, ACP5 affects tumor progression, and its deletion shifts macrophages toward a more pro-inflammatory, anti-tumor phenotype. These results suggest novel therapeutic strategies, especially those aimed at reprogramming macrophages to boost their tumor-eradicating potential. At present, ACP5-specific inhibitors like NaAuCl4, AubipyOMe, and CBK289001 have the potential to inhibit ACP5 activity within the tumor, possibly transforming the tumor microenvironment from a pro-tumor to an anti-tumor state \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Although the complete molecular regulation of ACP5 in macrophages is still not fully understood, our study provides new insights into immune cell checkpoints, particularly in macrophages, for anti-cancer therapies. The impact of ACP5 on macrophage populations within the TME offers valuable perspectives for cancer immunology and potential therapeutic approaches.\u003c/p\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eThe datasets generated and/or analyzed in this study are not publicly available but can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlasmids and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse ACP5 cDNA was amplified from pLenti-ACP5-Myc-DDK (Origene, MR204798L1V), and a double mutation (G217R / M266K) of mouse ACP5 (ACP5-2m) was introduced using a site-directed mutagenesis kit (Promega). Both wild-type and mutant ACP5 cDNAs were then cloned into the pLAS3w.c.Ppuro vector (Sinica, ROC). A stable ACP5-overexpressing Raw264.7 cell line was established via lentiviral infection, followed by puromycin selection. ACP5 enzyme activity was assessed using the ACP5 Active Kit, and antibodies were used to analyze macrophage polarization and cancer metastasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6 wild-type and BALB/c wild-type mice (6–8 weeks old) were purchased from the National Laboratory Animal Center (Taiwan, ROC). ACP5 knock-out (ACP5-KO) mice on a 129SV background were generously provided by Dr. Janckila Anthony. These ACP5-KO mice were back-crossed into a homogeneous BALB/c background for at least six generations.The presence of the ACP5 knockout allele in offspring was confirmed by PCR using specific primers. All mice were bred and maintained at the Laboratory Animal Center of the National Defense Medical Center, Taipei, Taiwan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 4T1 murine breast tumor cell line was generously provided by Dr. Nan-Shih Liao (Institute of Molecular Biology, Academia Sinica, Taiwan, ROC),** while the RAW264.7 mouse monocyte/macrophage cell line was a gift from Dr. Yi-Ping Chuang (Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan).Both cell lines were originally sourced from the American Type Culture Collection (ATCC) (Rockville, MD, USA).\u003c/p\u003e\n\u003cp\u003eCells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml). For 4T1-BMDM co-culture experiments, transwell inserts with porous polycarbonate membrane filters (0.4 µm pore size) in 6-well plastic culture plates were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBone marrow-derived macrophage (BMDM) culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eACP5-WT and ACP5-KO mice (6 to 8 weeks old) will be euthanized, and the femur and tibia from each leg will be excised. Bone marrow will be flushed out using a 23 G needle and a 1 mL syringe, followed by incubation in red cell lysis buffer (0.155 M ammonium chloride in PBS) to remove erythrocytes.The cells will then be cultured in a sterile petri dish containing 10 mL of RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, and L929-conditioned medium for 6 days. After this period, the medium containing non-adherent cells will be removed, and the remaining adherent cells will be further incubated in L929-conditioned medium for an additional 2 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration and functional characterization of M1/M2-polarized macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone marrow-derived macrophages (BMDMs) were generated by culturing bone marrow cells, isolated from the femur and tibia of mice, in DMEM supplemented with 15% FBS and 20% L929-conditioned medium for 11 days to allow maturation.For macrophage polarization, BMDMs were expanded and treated with: IFNγ (10 ng/mL) and LPS (100 ng/mL) for M1 polarization; IL-4 (10 ng/mL) and IL-13 (10 ng/mL) for M2 polarization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein extraction, electrophoresis and western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe indicated cells were washed with PBS and resuspended in 30–60 µL of lysis buffer containing 1X Complete™ protease inhibitors (Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitors (Cocktail I and II, Sigma). The cell suspension was incubated on ice for 30–40 minutes, followed by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatant (lysate) was carefully transferred to a fresh tube, ensuring the pellet remained undisturbed, and stored at −20°C for further analysis.\u003c/p\u003e\n\u003cp\u003eThe lysates were then incubated with 20 mM DTT and 1X sample loading buffer at 95°C for 10 minutes. After this step, the samples were equilibrated to match the least concentrated lysate before being denatured again with reducing sample buffer and DTT at 95°C for another 10 minutes.Protein separation was performed using 4–15% Mini-PROTEAN® TGX™ Precast Protein Gels and TGX buffer (Bio-Rad). The proteins were subsequently transferred onto a 0.2 µm nitrocellulose membrane using Bio-Rad Trans-Blot Turbo Mini Nitrocellulose Transfer Packs.The membranes were blocked with 2.5% milk in PBS containing 0.05% Tween-20 (PBS/Tween) for 1 hour, followed by a brief PBS wash. They were then incubated overnight at 4°C with primary antibodies.The next day, the membranes were washed three times with PBS/Tween for 10 minutes each, followed by incubation with HRP-conjugated secondary antibodies for 45 minutes. Afterward, the membranes underwent three additional PBS/Tween washes (5–10 minutes each).Antibody binding was detected using enhanced chemiluminescence (ECL) substrate, and signals were visualized using X-ray film (Scientific Laboratory Supplies).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction, cDNA synthesis and quantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells from each well were lysed and collected separately using TRIzol reagent, followed by RNA extraction using the miRNeasy kit (Qiagen, Mat. No. 1071023). RNA purity was assessed using NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA).cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). For quantitative real-time PCR (qRT-PCR), a reaction mix was prepared containing sample cDNA, primers, TaqMan primer-probes, and TaqMan Gene Expression Master Mix (all from Applied Biosystems). qRT-PCR was conducted using the ABI 7900HT Sequencing Detection System (Applied Biosystems). Primer sequences are listed in supplementary table 1, with β-actin used as an internal reference. The relative expression of target genes was calculated using the 2^−ΔΔCt method. All PCR reactions were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLibrary construction and RNA-sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext-generation sequencing (NGS) libraries were prepared following the manufacturer’s protocol. The sequencing data were processed and analyzed by GENEWIZ (South Plainfield, NJ, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvasion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor cell invasion assays were conducted using a Transwell system (Millipore, USA) with 8 µm-pore polycarbonate filter membranes coated with Matrigel. For the migration assay, 1 × 10⁴ 4T1 cells were seeded into the upper chamber, which was then inserted into the lower chamber containing different polarized BMDMs in DMEM with 15% FBS. After 24 hours of incubation, non-migrated cells on the interior surface of the upper chamber were removed. The polycarbonate membranes were then stained with 0.1% crystal violet for 10 minutes, and the number of migrating cells was quantified by examining entire fields under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell migration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wound-healing assay was used to assess 4T1 cell migration. 4T1 cells were seeded at a density of 5 × 10⁵ cells per well in a 6-well plate containing 10% FBS/DMEM and cultured until reaching 80% confluence.A scratch was carefully made in the cell monolayer using the tip of a 1 mL pipette, after which the medium was replaced with 50% conditioned medium from different polarized BMDMs. Images were captured at 0 and 9 hours using a microscope at 100× magnification with a camera. This assay was performed at least three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhagocytosis assay by Flow cytometric analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the phagocytosis assay, Jurkat cells were labeled with 5 mM CFSE for 30 minutes at 37°C. After two washes, the cells were resuspended at 5 × 10⁵ cells/mL in complete medium. BMDMs were harvested and washed as described previously. 5 × 10⁵ macrophages were then incubated with 1.5 × 10⁶ CFSE-labeled Jurkat cells for 60 minutes at 37°C. After incubation, non-adherent cells were removed by washing with PBS, and macrophages were detached using Trypsin-EDTA. Macrophages were then stained with anti-CD11b and analyzed by flow cytometry. Phagocytosis was quantified as the percentage of CD11b and CFSE double-positive cells relative to the total number of CD11b-positive macrophages, calculated as: \u003cu\u003e(CD11b\u0026nbsp;and\u0026nbsp;CFSE\u0026nbsp;double-positive\u0026nbsp;cells/Total\u0026nbsp;CD11b-positive\u0026nbsp;cells)×100\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumor induction animal experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor primary tumor growth, 1 × 10⁶ 4T1 cancer cells and 2.5 × 10⁴ M2-polarized BMDMs were orthotopically injected into the inguinal mammary fat pad, following a previously established protocol. For the experimental lung metastasis model, 1 × 10⁵ 4T1 cells and 2.5 × 10⁴ M2-polarized BMDMs were intravenously injected via the tail vein. Mice were euthanized on day 16 for histological analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExplore the cell composition of macrophages in murine breast cancer tissues by opal multiplex immunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMurine breast cancer tissues obtained from fat pad orthotopic murine breast cancer cells injection in ACP5 WT and ACP5 KO mice, were proceed for further experiments. An innovative multiplexed immunohistochemistry (IHC) imaging technique using the Opal 6-Plex Manual Detection Kit (Akoya Biosciences®) were utilized. Tissue pre-treatment and antibody incubation were performed following previously published protocols, with the secondary antibody replaced by the EnVision Plus Detection System (Dako #K50070). After washing, the designated tyramide signal amplification dye (Opal 6-Color Kit, Akoya Biosciences®) was applied for 10 minutes, following the manufacturer’s instructions. The slides were then heated in a retrieval buffer using a steamer to remove primary and secondary antibodies, preparing them for the next staining target. This process was followed by cooling, blocking, and repeating antibody and Opal dye incubation for five additional staining cycles. For the Macropahges’ Panel, the following primary antibodies were used for separate incubations: F4/80 (CST, Cat#70076, RRID:, 1:1500), Arginase 1 (CST, Cat#98668, RRID:, 1:100), MHC II (Invitrogen, Cat#BS-8481R, RRID:, 1:100), DAPI (PerkinElmer, Cat#\u0026nbsp;FP1490, 1:500), PanCK (DAKO, Cat#11237709, RRID:, 1:100).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of cell bioenergetics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe oxygen consumption rate (OCR), the extracellular acidification rate (ECAR) and Real-Time ATP rate were measured using a Seahorse XFe96 Analyzer (Agilent, California, CA, USA) with Seahorse XF Cell Mito Stress Test Kit (Agilent, cat#: 103015-100), Seahorse XF Glycolytic Rate Assay Kit (Agilent, cat#: 103344-100) and Seahorse XF Real-Time ATP Rate Assay kit (Agilent, cat#: 103592-100), respectively, following the manufacturer’s instructions. Briefly, macrophages were seeded in 8-well Seahorse assay plates at a concentration of 2 × 10\u003csup\u003e4\u003c/sup\u003e cells/well, and cultured overnight for attachment. Prior to the assay, cells were washed, and the medium was replaced with Seahorse XF RPMI for macropahges or supplemented with 20 mM glucose, 2 mM L-glutamine and 1 mM sodium pyruvate. Presto Blue™ assays (Thermo Fisher Scientific) were used to evaluate cell viability and normalize readings from the Seahorse XF Analyzer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublic dataset retrieved and reanalysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNormalized RNA-seq data from different cancer types was obtained from the publicly available database TCGA (The Cancer Genomic Atlas) and GTEx (The Genotype-Tissue Expression). According to this database, all data was obtained by using the Illumina HiSeq platform and retrieved by the bioinformatics tool UCSC Xena browser (URL: https://xenabrowser.net/). The phenotypic cohort including survival data and clinical parameters were also retrieved from the patients diagnosed of breast cancer(BRCA). Samples without detailed information in terms of normalized mRNA expression, survival status and overall times were removed before enrollment. The single-cell RNA-seq dataset was downloaded from GSE176078. We performed scRNA-seq analysis using the R package \"Seurat\" (version 4.1.0). Cells with fewer than 200 detected genes or more than 50% mitochondrial reads were excluded from the analysis.A shared nearest neighbor (SNN) graph was then constructed, and uniform manifold approximation and projection (UMAP) embedding was generated using the top 20 principal components. The main cell types were identified based on annotations consistent with the original literature\u003csup\u003e33\u003c/sup\u003e. To further classify macrophages, we applied M1/M2 macrophage signatures from published studies and performed subclustering using the R package \"CelliD\". The proportions of each subpopulation were calculated, and immune-related signaling pathway activity scores were computed using the Broad Institute’s Hallmark collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using GraphPad PRISM 10 software (GraphPad Software, Inc. La Jolla, CA). Statistical comparisons were performed by two-way ANOVA with Bonferroni post-test by Student's t-test or by Mann–Whitney test for non-parametric distributions with small sample size. Probability values of p ≤ 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to all members of Professor Chen Ying-Chuan\u0026apos;s group for their valuable scientific discussions. Our thanks also go to the Cancer Registry Group at Tri-Service General Hospital. We appreciate BioRender\u0026rsquo;s support in creating some of the figures for this article. The bioenergy analysis was made through the technical services provided by the Instrument Center of the National Defense Medical Center. This research was further supported by several grants from the Tri-Service General Hospital (TSGH-E-112206/TSGH-E-113225/TSGH-E-114227) and the National Defense Medical Centre (MND-MAB-D-111073/MND-MAB-D-114084) awarded to YGC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was further supported by several grants from the Tri-Service General Hospital (TSGH-E-112206/TSGH-E-113225/TSGH-E-114227) and the National Defense Medical Centre (MND-MAB-D-111073/MND-MAB-D-114084) awarded to YGC. The bioenergy analysis and RNA-seq were funded by a grant from the National Science and Technology Council (NSTC113-2320-B-016-007), awarded to YCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was conceived by MS Dai. The research was designed by YGC, DDM, YCC,CHT, and PY Liu, and they collaborated on writing the manuscript. SFT, HCL, CYB, LJC and YGC conducted the majority of the experiments in collaboration with others. Multiplex IHC were carried out by CHY, TLL, CTC and SHY. ER and PK contributed to the cell death experiments. Bioinformatic and RNA-seq analysis using TCGA datasets were performed by YGC, FYT and SSJ. Histopathological analysis of tumor tissues was conducted in a blinded manner by AJ, TYC, JCY and CYW.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthic declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) Competing interests: The authors declare no competing interests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(B) Ethics approval for animal experiment for this research\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll mice were genotyped by PCR and were provided with food ad libitum. All animal experiments were carried out under the appropriate NDMC project license, in compliance with Taiwan\u0026apos;s home office regulations for animal welfare as outlined in the Animal (Scientific Procedures) Act 1986 (ASPA). The relevant Animal Ethics Committee approved all experiments involving ACP5-deficient mouse crosses, which were maintained under the necessary licenses.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBalkwill F, Mantovani A. Inflammation and cancer: back to Virchow? \u003cem\u003eLancet\u003c/em\u003e 2001, \u003cstrong\u003e357\u003c/strong\u003e(9255)\u003cstrong\u003e:\u003c/strong\u003e 539-545.\u003c/li\u003e\n\u003cli\u003eMantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. \u003cem\u003eNature\u003c/em\u003e 2008, \u003cstrong\u003e454\u003c/strong\u003e(7203)\u003cstrong\u003e:\u003c/strong\u003e 436-444.\u003c/li\u003e\n\u003cli\u003eNoy R, Pollard JW. 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Understanding the molecular checkpoints in macrophages could provide potential therapeutic strategies for cancer treatment. Tartrate-resistant acid phosphatase 5 (ACP5), also known as TRAP5, is an enzyme primarily expressed by osteoclasts and certain immune cells, involved in bone remodeling and immune regulation. Recent research has indicated that ACP5 promotes tumor growth and metastasis in various cancers. However, its specific role within the tumor microenvironment, particularly regarding its effect on macrophages, remains unclear. This study shows that ACP5 is highly expressed in macrophages within breast cancer tissues, as identified through single-cell RNA sequencing. Using ACP5-deficient mice, we observed a significant reduction in tumor burden, metastatic potential, and epithelial-mesenchymal transition in both orthotopic and spontaneous breast cancer models. Mechanistically, ACP5 regulates macrophage polarization, promoting an anti-inflammatory (M2) phenotype that aids tumor progression and metastasis. Notably, ACP5 deletion in bone marrow-derived macrophages impairs AMPK phosphorylation, shifting their metabolism toward glycolysis. This metabolic shift enhances their pro-inflammatory (M1) phenotype, increasing anti-tumor activity against cancer cells. Our findings underscore the vital role of ACP5 in macrophage-mediated immunosuppression and tumor progression, presenting a promising therapeutic target for breast cancer treatment.\u003c/p\u003e","manuscriptTitle":"Tartrate-resistant acid phosphatase 5 regulates the metabolic flexibility of macrophages in the tumor microenvironment, thereby influencing their functional fate and modulating tumor growth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 16:14:52","doi":"10.21203/rs.3.rs-6656922/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b6f4e3d-a6cc-4a4b-9fce-c0f6b1ded7e5","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49222794,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":49222795,"name":"Health sciences/Pathogenesis/Inflammation/Chronic inflammation"}],"tags":[],"updatedAt":"2025-08-05T16:14:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-05 16:14:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6656922","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6656922","identity":"rs-6656922","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}