Macrophages in Tumor‐Associated Adipose Microenvironment Accelerate Tumor Progression

Adipose‐tissue macrophages (ATMs), a complex ensemble of diverse macrophage subtypes, are prevalent in the tumor‐adipose microenvironment (TAME) and facilitate tumor growth. However, the mechanisms in which the tumor‐adipocyte crosstalk may enable the properties and plasticity of macrophages remain unclear. The single‐cell RNA‐sequence profiling reveals that a subset of macrophages expressed CD163, CCL2, and CCL5 in TAME, exhibiting an immunosuppressive subtype. It is demonstrated that CD163+ macrophages aggregate to surround adipocytes in breast cancer tissues. The expressions of CCL2 and CCL5 are also elevated in TAME and enable the recruitment and polarize macrophages. Mechanically, the level of exosomal miRNA‐155 increased in the coculture of tumor cells and adipocytes, and then it promoted the generation and release of CCL2 and CCL5 from adipocytes by targeting the SOCS6/STAT3 pathway. Inhibition of exosomal miRNA‐155 in tumor cells reduced the CCL2 and CCL5 levels in tumor‐adipocytes coculture and further retarded tumor growth. Finally, the deletion of macrophages partially inhibited adipocyte‐induced tumor proliferation. Likewise, inhibiting chemokines and their receptors or suppressing the phosphorylation of STAT3 decreased tumor burden in preclinical models. These results demonstrate that the niche factors in TAME, such as exosomal miRNA‐155, regulate the function and polarity of macrophages to facilitate tumor progression.


Introduction
Obesity is a growing global epidemic and results in preventable risk factor for cancer incidence and mortality, being responsible for an estimated ≈20% of cancer-related deaths in adults. [1] proportion of macrophages also derive from monocyte-derived cells. And chemokine (C-C motif) ligand 2 (CCL2) and CCL5 play a prominent role in the chemotaxis and activation of monocyte-derived macrophages. CCL2 attracts circulating macrophages and stimulates their immunosuppression in the tumor microenvironment via binding to its receptor CCR2. [14] Meanwhile, CCL5 could recruit CCR5-overexpressing macrophages, subsequently which depose collagens in residual tumors to drive tumor recurrence. [15] The collagen deposition in the breast cancer microenvironment was also related to high stromal expression of CD163. [16] Similarly, stroma stiffness was highest in the more aggressive basal-like and human epidermal growth factor receptor-2 (HER-2) subtypes, and positively correlated with the number of infiltrating tumor-associated macrophages. [17] Classically, two main phenotypes of macrophages in adipose tissue are pro-inflammatory M1 (classically activated) and antiinflammatory M2 (alternatively activated). [18] The macrophages in lean adipose tissue are polarized toward the M2 activation state, whereas in obese adipose tissue, they are toward the M1 activation state. [19] M1 macrophages produce pro-inflammatory cytokines (including TNF-α, interleukin-6) while M2 macrophages play an anti-inflammatory role via secreting IL-10. [20] Moreover, macrophage phenotype can vary in different tumor types and intratumor districts. [20] Specifically, macrophages can be polarized into an M2-like state or "switching macrophages", which produce both pro-and anti-inflammatory cytokines and have been observed in tumors and adipose tissues. [21,22] Interestingly, the macrophages that envelope around phagocytosis of a dead or dying adipocyte in a configuration termed crownlike structures (CLSs), a histologic biomarker of inflammation. [2] In breast cancer patients, the pathologically increase of CLSs in mammary adipose tissue is related to poor prognosis, estrogen receptor negativity, and the overexpression of positive HER-2. [23,24] Moreover, tumor-associated macrophages (TAMs) are important coordinators linking inflammation to cancer progression, the differentiation of which is stimulated by inflammatory factors derived from cancer cells or stromal cells. TAMs facilitated tumor progression including proliferation, extracellular matrix (ECM) remodeling, angiogenesis, and immune escape, either by releasing epidermal growth factor or by degrading ECM proteins. [25] Hence, macrophage collection in adipose tissue nearby the breast cancer lesion appears to have important clinical implications, particularly regarding macrophage properties; however, it has not been thoroughly investigated.
Exosomes are a kind of small extracellular vesicles (30-100 nm) derived from cancer cells and stromal cells. [26] This cell-to-cell biological communication is mediated through the exchange of the exosomes content, which consists of proteins, lipids, metabolites, RNA, and DNA. [27] Exosomes are appreciated as essential mediators of cell-cell communication. Our previous research indicated that exosomal miRNAs from the tumor-adipocyte interaction induce beige/brown differentiation and accelerate catabolism in recipient adipocytes to facilitate tumor progression. [28][29][30] In this study, it's evident that exosomal miRNA-155 enriches in the coculture of the tumor cells and adipocytes, and stimulates the secretion of CCL2 and CCL5 from adipocytes in SOCS6/STAT3-depending way. In addition, these chemokines recruit and polarize M2-like macrophages to facilitate tumor growth.

Patients
A total of 145 formalin-fixed paraffin-embedded (FFPE) tissue samples of breast cancer were obtained from Renmin Hospital of Wuhan University. All patients involved in the study provided a written informed consent. Patients did not receive financial compensation. Clinical information was extracted from medical records and pathology reports, and the detailed clinicopathological characteristics of the patient are shown in Table 1. Patients were all followed-up for at least 5 years from the date of first diagnosis. All methods were performed in

Single-Cell RNA-Seq Data Processing
SnRNA-seq data from 10X Genomics were aligned and quantified using the Cell Ranger software. And we obtained scRNAseq data from GSE161529. Downstream statistical analyses were conducted using the Seurat (V4.0.4) software packages for R. Each sample was individually quality-checked. Cells were filtered by the following criteria: at least 200 and no more than 5000 detected genes, and no more than 20% mitochondrial reads per cell. Cells with extremely high numbers of reads or genes detected were filtered to minimize the occurrence of doublets. And genes expressed in <1% of cells for the individual sample were filtered. Multiple samples were combined using the anchor-based integration method implemented in Seurat. The principal component dimensions 1:30 were used for all dimension reduction and integration steps. The cluster resolutions were set to 0.1. For dimensionality reduction visualizations we used the uniform manifold approximation and projection (UMAP) algorithm. The annotation of clusters was based on the expression of cell markers. iTALK (an R package to characterize and illustrate intercellular communication) was used to visualize the cell-cell communication of chemokine ligands and receptors. We used the FindMarkers function of Seurat to find differential expression genes, and perform kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment.

Cell Culture and Reagents
The

Exosome Isolation and Characterization
After cells were cultured with exosome-depleted serum (Aus-GeneX), the exosomes were purified from the conditioned medium according to the instructions. [31] The medium was centrifuged at 500 g for 5 min and at 2000 g for 30 min at 4 °C to remove cellular debris and large apoptotic bodies. After centrifugation, media was added to an equal volume of a 2 × polyethylene glycol (PEG, MW 6000, Sigma, 81 260) solution (final concentration, 8%). The samples were mixed thoroughly by inversion and incubated at 4 °C overnight. Before the tubes were tapped occasionally and drained for 5 min to remove excess PEG, the samples were further centrifuged at maximum speed (15 000 rpm) for 1 h at 4 °C. The resulting pellets were further purified using 5% PEG and then stored in 50-100 µl of particle-free PBS (pH 7.4) at -80 °C. The average yield was ≈300 µg of exosomal protein from 5 ml of supernatant. Total RNA was extracted by using Trizol reagent (Life Technologies), followed by miRNA assessment by microarrays and RT-PCR described below. Exosomes were analyzed by electron microscopy to verify their presence, by a nanoparticle characterization system to measure their size and concentration, and by western blot to detect their proteins (TSG101, CD63, and CD81).

Electron Microscopy
After being fixed with 2% paraformaldehyde, samples were adsorbed onto nickel formvar-carbon-coated electron microscopy grids (200 mesh), dried at room temperature, and stained with 0.4% (w/v) uranyl acetate on ice for 10 min. The grids were observed under a HITACHI HT7700 transmission electron microscope.

Nanoparticle Characterization System (NanoSight)
The NanoSight (Malvern Zetasizer Nano ZS-90) was used for real-time characterization and quantification of exosomes in PBS as specified by the manufacturer's instructions.

Exosome Uptake Analysis
Exosomes derived from breast cancer cells were labeled by the cell membrane labeling agent PKH26 (Sigma-Aldrich). After being seeded in 96-well plates and allowed to differentiate, mature 3T3-L1 cells were incubated with labeled exosomes (20 µl per well) for the indicated time. Images were acquired using the Olympus FluoView FV1000.

ELISA
The conditioned media were collected to analyze the secretion levels of CCL2 and CCL5 via Mouse MCP-1/CCL2 ELISA kit (Sizhengbai, CME0046) and Mouse CCL5/RANTES ELISA kit (Sizhengbai, CME0048). The conditioned media were centrifuged at 1000 g for 10 min, incubated with biotinylated antibody working solution at 37 °C for 1.5 h, incubated with enzymeconjugated working solution at 37 °C for 0.5 h, and color developing for 10 min.

Western Blotting
After being washed twice with ice-cold PBS, cells were collected with SDS loading buffer and boiled for 10 min. The proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected with specific antibodies (Additional file 1: Table S1, Supporting Information).

Neutralizing Experiments
Before being used to treat macrophages, CA-CM was neutralized by goat serum or CCL2 or CCL5 neutralizing antibodies overnight. Macrophages were treated with the neutralized CA-CM for 3 days, and then were collected for western blot analysis.

RNA Extraction and Quantitative PCR
Gene expression was analyzed using real-time PCR. The mRNA primer sequences are provided in Additional file 1: Table S2, Supporting Information. The miRNA primer kits were purchased from RiboBio (Guang Zhou, China).

Immunohistochemistry
A cohort of 145 human breast cancer specimens was collected from Renmin Hospital of Wuhan University from 2011 to 2013. Immunohistochemistry (IHC) staining was performed, and the staining results were scored by two independent pathologists based on the proportion of positively stained tumor cells and the staining intensity. The protein expression level of CD163 was described according to the numbers of CD163+ macrophages counted in 10 random fields of each breast cancer specimen at 400 × magnification. The intensity of protein expression was scored as 0 (no staining), 1 (weak staining, light brown), 2 (moderate staining, brown), and 3 (strong staining, dark brown). The protein staining score was determined using the following formula: overall score = percentage score × intensity score. Receiver operating characteristic analysis was used to determine the optimal cutoff values for all expression levels regarding the survival rate.

Immunofluorescence Imaging
Immunofluorescence (IF) imaging was performed to investigate the localization of pSTAT3 (Tyr705) (#9145, 1:200, Cell Signaling Technology), CD68, and CD163. Tissue specimens undergoing IF staining were incubated with Alexa Fluor-conjugated secondary antibodies against the primary antibodies for 1 h at room temperature, followed by counterstaining with DAPI for 5 min. Images were captured using a fluorescence microscope (Olympus BX63; Olympus Corporation).

Image Segmentation and Data Analysis
Images were segmented using the EBImage package (available from Bioconductor repository https://www.bioconductor.org) with the R software. The nuclear region was defined using a polygon mask based on the nuclear Hoechst signal, and a second polygon mask was generated using the GFP or RFP signal. For the assessment of autophagic vesicles, a third mask was created on cytoplasmic regions exhibiting a high-intensity signal of GFP corresponding with LC3 aggregates.

Luciferase Assays
The 3′ UTRs of target genes containing predicted miRNA binding sites (gene wt ) were cloned into the GV272 vector (GeneChem Biotechnology, Shanghai, China), and the miRNA binding sites were replaced with a 4-nt fragment to produce a mutated 3' UTR (gene mut ) in the vector. Briefly, HEK 293 T cells were seeded in 12-well plates and grew to 70% confluence. The cells were transfected with gene wt or gene mut , the pre-miRNA expression plasmid, and pRL-SV40, which constitutively expresses Renilla luciferase as an internal control. At 48 h post-transfection, the cells were lysed, and Renilla luciferase activity was assessed by the TECAN Infiniti reader. The results are described as the ratio of firefly luciferase activity compared to Renilla luciferase activity.

Mice Models
Six-week-old female BALB/c mice were purchased from Vital River, Beijing. The animals were handled according to the protocol approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University. All animal experiments were carried out in accordance with relevant guidelines and local regulations. The following cell lines were used to create subcutaneous models: 4 × 10 5 4T1 cells treated with control lentivirus, transfected with miRNA-155 inhibitor lentivirus. Breast cancer cells were injected alone or in combination with mature adipocytes (1 × 10 5 cells). All cell samples were injected with Matrigel (1:1), total volume 100 µl, into the mammary fat pad or the axilla of the mice. For macrophages deletion, mice were treated with blocking antibodies against F4/80 as described.
Six-week-old female wild-type C57BL/6 mice were obtained from Vital River, Beijing. Mouse Mammary Carcinoma AT-3 cells (2 × 10 5 ) alone or in combination with mature adipocytes (1 × 10 5 cells) were injected into C57BL/6 hosts under the mammary gland fat. When tumors became palpable, mice were treated with the STAT3 inhibitor Stattic (10 mg kg −1 , days 0 and 7, Sigma-Aldrich), blocking antibodies against CCL2 (BioXcell, West Lebanon, NH, USA) and/or CCL5 (200 µg, days −1 and 0, and were repeated every 3 d thereafter to maintain depletion or neutralization) (R&D Systems, Minnesota, USA) by intraperitoneal injection, or the CCR2/CCR5 antagonist BMS-813160 (10 mg kg −1 , gavage, days 0, 4, 8 and 12) (MedChemEcpress, Shanghai, China). On the following days, mice well-being and tumor growth were monitored and documented. Tumor volume was defined as (longest diameter) × (shortest diameter) 2 × 0.52 and was measured once every 2-3 days until using a Vernier caliper. Animals were sacrificed when tumor size reached endpoint or signs of obvious discomfort were observed following our Ethical Committee advice. After the mice were sacrificed, all tissues were collected, embedded in paraffin, and stained with IHC or hematoxylin-eosin.

Ethics Statement
All patients involved in the study provided a written informed consent, and the study was approved by the Institutional Ethics Committee of Renmin Hospital of Wuhan University (approval no. 2018K-C09). All experiments involving animals were handled according to the protocol approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University (approval no. 2018K-C09).

Statistical Analysis
All experiments were done independently at least three times. The results are presented as the mean ± SD. The relative increase in protein expression was quantified using Image J software and was normalized to control protein expression in each experiment. Data sets obtained from different experimental conditions were compared with the t-test when comparing only 2 groups. Multiple comparisons between groups were performed using the Mann-Whitney U test or Tukey's multiple comparisons. Survival probabilities for recurrence-free survival (RFS) were estimated using the Kaplan-Meier method and variables were compared using the log-rank test. Pearson's correlation was used to evaluate the correlations among CD163, CCL2, and CCL5 expression levels. In the bar graphs, a single asterisk (*) indicates P < 0.05.

Single-Cell Profiling of Macrophages in the Tumor-Adipocyte Microenvironment Reveals Distinct Immunosuppressive Subsets
We performed scRNA-seq profiles in tumor-adipose juncture from two patients with breast cancer (BC-A and BC-B). We processed ≈8000-9000 cells in each sample. After quality control and filtering, 4093 single cells in the BC-A sample and 5917 single cells in the BC-B sample were retained. And the control group is selected from GEO dataset (GSE161529). The comprehensive flow is shown to map the tumor-immune transcriptional landscape ( Figure 1A). First, the unsupervised clustering analysis was applied on integrated single-cell datasets to define major cell populations ( Figure 1B). This principally defined 7 distinct clusters including B cells, endothelial cells, malignant epithelial cells, fibroblasts, myeloid cells, plasma cells, and T cells based on the expression of conversant genetic markers ( Figure 1B). Further, the chemokines CC chemokine ligand 2 (CCL2) and CCL5 and their receptors were mapped at single-cell resolution, showing that the levels of CCL2 and CCL5 were obviously up-regulated in the tumor-adipose microenvironment compared to that in the control group. Therein, CCL2 and CCL5 are enriched in macrophages, malignant epithelial cells, and fibroblasts; and T cells highly expressed CCL5. Likewise, the receptors, CC chemokine receptor 2 (CCR2) and CCR5, mainly overexpressed in macrophages ( Figure 1B). In addition, T cells were significantly increased in samples from tumor-adipose juncture ( Figure 1C), as the high infiltration of T cells in the boundary is a biomarker for T cell exhaustion. [32] The classical T cell sub-clusters were shown in Figure S1A,B, Supporting Information, and CD8+ T cell is the main type in both low and high expression groups, while CD4+ T cells increased in the high group ( Figure 1J). Because our initial clustering did not resolve distinct macrophage subtypes, we re-clustered macrophages into two subsets based on the expression of CD163 ( Figure 1D,E). Then we detected the chemokines (CCL2 and CCL5) and their receptors within these sub-clusters, indicating that they were enriched in CD163 + macrophages ( Figure 1F). When we adopted differential expression analysis on CD163 + macrophages, the top 20 genes in CD163 + macrophages were associated with neutrophil activation, upregulation of cell chemotaxis and chemokine-mediated signaling pathway, but downregulation of leukocyte cell-cell adhesion and antigen processing and presentation of exogenous antigen ( Figure 1G,H). Finally, CD163 + macrophages secreted multiple chemokines like CCL2, CCL3, CCL4, and CCL7 and inflammatory cytokines like IL6, IL32 ( Figure 1I, Figure S2A-C, Supporting Information). Then they exerted an immunosuppressive effect via recruiting myeloid-derived suppressor cells (MDSCs) and inhibiting the activation of T cells ( Figure 1K). For example, CCL4 could bind their receptor CXCR4 to stimulate the infiltration of MDSC and suppress the anti-tumor function of effector T cells. [33,34] In summary, single-cell profiling revealed that CD163 + macrophages enrich in the tumor-adipose microenvironment and display a predicted immunosuppressive subtype.

The Infiltration of M2-Like Macrophages in Tumor-Surrounding Adipose Tissues were Associated with Poor Prognosis in Patients with Breast Cancer
The heterogeneity and plasticity of macrophages in adipose tissues surrounded by breast cancer has not been thoroughly explored. It is reported that TAMs are confirmed as "switching macrophages", which generate pro-and anti-inflammatory cytokines meantime and are found in tumors. [22] CD68 (macrophage membrane marker) and CD163 (M2-macrophage marker) have been usually regarded as surrogate markers to investigate TAMs polarity in previous studies. [24,35] The increased infiltration of CD163 + TAMs in CLSs were associated with shorter disease-free survival in node-negative breast cancer patients. [24] We first detected the existence and characteristics of macrophages in a cohort of 145 breast cancer specimens using IHC and QDs-based fluorescent imaging technique (Table 1). We detected the expression of CD68 and CD163. Immunohistochemical staining revealed that the expression of CD163 + macrophage is markedly elevated in the surrounding adipose tissue and the center of tumor tissue of breast cancer (Figure 2A). Meanwhile, QDs-based fluorescent imaging showed that CD68 + CD163 + macrophages remarkably infiltrated in adiposes adjacent to tumor tissues and in connective tissue ( Figure 2B). In addition, CD11c is M1 and a proinflammatory marker of adipose-tissue macrohages (ATMs), [36] further D11c + macrophages were also found to constitute the CLSs. [37] However, CD11c was an extremely low expression and even no expression in our study ( Figure S3A). Our data demonstrated that the expression of CD163 in breast cancer specimens was associated with tumor size (P = 0.017), and it was greatly increased in patients with tumor size ≥2 cm. In addition, the infiltration of CD68 + CD163 + macrophages were notably elevated when tumor metastasized into the lymph node (P < 0.001). Moreover, the positive expression of CD163 was more common in patients with overexpressed Ki-67 compared to the control group (P = 0.007). There was no correlation between CD163 and other clinicopathological features, including age, vascular invasion, hormone receptors, and HER-2 (Table 1).
We further analyzed the association between the expression of CD68 + CD163 + macrophages and the prognosis of patients with breast cancer. We calculated the infiltration of CD68 + CD163 + macrophages surrounding the adipocytes adjacent to tumor tissues and in tumor tissues, respectively. The Kaplan-Meier survival analysis showed that the high expression of CD68 + CD163 + macrophages in adiposes was significantly associated with shorterRFS-than that of patients with negative expression (P = 0.0142, Figure 2C up). Whereas, there was no significance in RFS between CD163-negative and -positive patients grouping via the CD163 + macrophages infiltration in tumor tissues (P = 0.316, Figure 2C   polarized toward M2-like phenotype and a high level of infiltrated CD68 + CD163 + macrophages in adipose adjacent to tumor tissues was associated with various clinicopathologic parameters and poor prognosis in breast cancer patients. Taken together, these data indicate that the level of CD163 + macrophages were up-regulated in the tumor-adipose microenvironment and were associated with tumor progression.

Chemokines CCL2 and CCL5 Contribute to Recruit Macrophages
Our previous study and others have shown that the secreted levels of CCL2 and CCL5 elevated in adipocytes cocultured with tumor cells. [29,38] Given the fact that macrophages could be recruited to inflammatory regions by chemokines, including CCL2 [39] and CCL5, [15] we supposed that macrophages surrounding the adipocytes were recruited by CCL2 and CCL5 to adipose tissue. Of 145 breast cancer specimens in our study, we found that the chemokines, both CCL2 and CCL5, overexpressed around the adipose tissues ( Figure 3A) and contributed to a poor survival ( Figure 3B). We next sought to determine whether macrophages might be an important target of CCL2 and CCL5 within breast tumors. Indeed, the proteins expression of both CCL2 and CCL5 are correlated with expression of CD163 + macrophages ( Figure 3C, R = 0.211, P = 0.011; R = 0.261, P = 0.001, respectively), suggesting that CCL2 and CCL5 may attract macrophage migration during the formation of CLSs.
In vitro, after the presence of MDA-MB-231 cultivated for 3 days in the Transwell system, the mRNA levels of CCL2 and CCL5 in adipocytes are dramatically elevated (Figure 4A,B). Likewise, the conditioned media (CM) from mature adipocytes in the presence or absence of breast cancer cells (MDA-MB-231) cultivated for 3 days were collected. And it shows that CCL2 and CCL5 are highly expressed in CM from mature adipocytes cocultivated with MDA-MB-231 cells via ELISA assay ( Figure 4C). Notably, the migration capabilities of macrophages also increased in the cocultivated group ( Figure 4D). To sum up, these results have indicated that CCL2 and CCL5 play a crucial role on macrophages recruitment.

Chemokines CCL2 and CCL5 Induced M2-like Polarization of Macrophages Via Activating STAT3 Phosphorylation
Prominently, there is evidence that the chemokines CCL2 and CCL5 contribute to the selective polarization of macrophage subsets by interacting with their chemokine receptors. [40] To determine how macrophages were recruited and polarized in TAME, we explore the impact of CCL2 and CCL5 on macrophages polarity. Next, we confirmed that both mouse macrophages (RAW264.7 cell) [41] and human monocyte-derived macrophages (THP-1 cell induced by phorbol-12-myristate-13-acetate (PMA) [42] ) showed increased M2-like (CD163) polarization upon treatment of CM from adipocytes cocultured with breast cancer cells relative to the single cultivation ( Figure 4E,F). In terms of mechanism, the expression of pSTAT3 was Adv. Biology 2023, 7, 2200161  increased in macrophages with the treatment of cocultured CM ( Figure 4G). To explore the impact of chemokines on macrophages polarization, the neutralizing antibodies targeted CCL2 and CCL5 respectively were adopted. The results demonstrated that the polarized markers of macrophages and the expression of pSTAT3 were reversed relative to control group ( Figure 4H,I, Figure S4A, Supporting Information). In summary, our results demonstrated that the increase of CCL2 and CCL5 in cocultured CM promoted M2-like polarization of macrophages in vitro through phosphorylating STAT3.

Tumor-Derived Exosomal miR-155 Stimulates CCL2 and CCL5 Secretion by Adipocytes to Accelerate Tumor Growth
To confirm that the observed reliance for macrophages in TAME depends on the tumor-adipocytes interaction, we examined whether the conditional presence of cancer cells alters the secreted spectrum of adipocytes. We speculated that the exosomes derived from tumor cells are important for adipocytes that are converted into cancer-associated adipocytes and excessively secrete chemokines including CCL5. Exosomes were isolated from the conditioned medium collected from adipocytes cultivated with MDA-MB-231 cells for 3 days, displaying that typical exosomal morphology and size and contained CD63, TSG101, and CD81 but not HSP70 ( Figure S5A-C, Supporting Information), which was consistent with previous reports on exosomes. [43] To observe exosomes uptake by adipocytes, breast cancer-secreted exosomes were labeled with red fluorescence. After being treated with exosomes for 4 h, mature 3T3-L1 cells were densely packed with exosomes ( Figure S5D, Supporting Information), indicating rapid cellular uptake of exosomes by adipocytes. Further, the upregulation of exosomal miR-155 in the cocultured group has been demonstrated in our previous work via non-coding RNA sequence technology. [28][29][30] Meanwhile, the mature miR-155 also overexpressed in adipocytes in presence of breast cancer cells, but the precursor of miR-155 remains unchanged ( Figure 5A, Figure S6, Supporting Information). It suggested that exosomal miR-155, the suitable candidate, mainly released from breast cancer cells. What's more, the secretion levels of CCL2 and CCL5 are strongly elevated in CM from mature adipocytes cocultivated with MDA-MB-231 cells, which are reversed by miR-155 knockdown ( Figure 5B). This observed increase in exosomal miR-155 prompted us to analyze the effects of tumor-originated miR-155 on the inflammation change of adipocytes. Intriguingly, we found one potential sequence of miR-155 targeting the human suppressor of cytokine signaling 6 (SOCS6) 3'UTR sequence ( Figure 5C). We next established one luciferase reporter, displaying that the wild-type construct showed a significant decrease in normalized luciferase activity relative to the control in the presence of pre-miR-155, and that mutation of the 3'UTR of human SOCS6 rescued this luciferase activity ( Figure 5D), which was consistent with the previous research. [44] This result shows that SOCS6 is a direct target of miR-155. We also detected the mRNA and protein expression of SOCS6, and it was evident that the generation of SOCS6 was decreased in adipocytes cocultivated with breast cancer cells ( Figure 5E,F, Figure S7A, Supporting Information). SOCS6 is a negative regulator of Janus activated kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, [44] and the reduction of SOCS6 nuclear localization promoted protein expression of STAT3. [45] This was consistent with our results, showing that pSTAT3 level was extremely increased in mature adipocytes in the presence of breast cancer cells, which was reversed by cytochalasin D (CytoD), an endocytosis inhibitor ( Figure 5F,G). This result indicated that the exosome uptake contributes to the upregulation of pSTAT3 in adipocytes undergoing tumor cells. Further, we investigated the possibility that exo-miR-155 would activate pSTAT3 activity via inhibiting SOCS6. The results indicated that a decrease in SOCS6 level was found in adipocytes cocultivated with breast cancer cells or adipocytes with miR-155 overexpression. Meanwhile, miR-155 knockdown partially increased the SOCS6 expression in adipocytes cocultured with malignant cells (Figure 5H,I, Figure S7B, Supporting Information). Consistently, the adipocytes in cocultured with neoplastic cells increased the level of phosphorylated STAT3, and the knock-downed miR-155 reversed the expression ( Figure 5H,I).
The xenograft models showed consistent results with in vitro assays, indicating that significantly increased tumor growth in mice coinjected with 4T1 cells and mature 3T3-L1 cells, whereas downregulation of miR-155 in tumor-derived exosomes significantly reduced the tumor volume in xenografts ( Figure 5J-L). Likewise, the number and the size of metastatic nodules in the liver were markedly increased in the group injected with tumor cells cocultivated with mature 3T3-L1 cells, while downregulation of miR-155 in tumor exosomes significantly reduced the number of nodules in liver ( Figure 5L). And the number and the size of metastatic nodules in the lung were no obvious differences among these groups (data not shown). Altogether, our results demonstrated that exosomal miR-155 derived from breast cancer cells mediated inflammatory adaptive changes in mature adipocytes through SOCS6/STAT3 signaling pathway.

Depleted Macrophages in Tumor-Adipocyte Interaction Inhibits Tumor Growth
Whether macrophages existence endowed breast cancer cells with the proliferated capacity, we speculated that macrophages deficiency might inhibit adipocyte-associated tumor growth. To this end, we employed a mouse model without macrophages to assess the impact of deficient macrophages on breast cancer proliferation. On account that macrophages in adipose and tumor tissues are mixed by tissue-resident macrophages and monocyte-derived cells, anti-F4/80 mAb treatment could delete multifarious macrophages and be used in further study. As in our previous studies, 3T3-L1 cells were induced to differentiate into mature adipocytes. Furthermore, a kind of breast cancer cell from BALB/c mouse 4T1 with or without mature adipocytes in a specific proportion were orthotopically injected into the mammary fat pad. [29] Likewise, the macrophages in the mouse model were particularly eliminated via injection of an anti-F4/80 neutralizing antibody as reported [22] (Figure 6A). As Figure 6B,C, the tumor injected 4T1 combined with adipocytes grew much more rapidly while the eliminated macrophages via anti-F4/80 mAb could retard adipocyte-induced tumor growth (P < 0.05; Figure 6B). To determine whether M2-like phenotype Figure 5. Tumor-derived ExomiR-155 mediates inflammatory files of adipocytes by targeting SOCS6/STAT3 signaling. A) Adipocytes were cocultivated in the presence or absence of 4T1 or MDA-MB-231 cells. After 3 days, RNA was extracted from the adipocytes and mature miRNA-155 and pre miRNA-155 were further verified by qPCR (left and middle). ExomiR-155 was also verified by qPCR (right). B) The secretion levels of CCL2 and CCL5 in different groups. C) The predicted miR-155 binding site in the 3'UTR of the SOCS6 gene from TargetScan. D) The GV272 vector containing the 3'UTR of the target gene harbouring wild-type (wt) or mutated (mt) miRNA binding sites was transfected into HEK 293 T cells stably expressing miRNA or empty vector as a normal control (NC). Luciferase activity was analyzed at 48 h post-transfection, and the ratio of firefly luciferase activity to Renilla luciferase activity is shown. E) The mRNA expression levels of SOCS6 in mature adipocytes in the presence or absence of MDA-MB-231 cells. F,G) Western blot analysis of related protein expression and Immunofluorescence staining for pSTAT3 in different groups. The adipocytes in the cytochalasin D (CytoD) group were treated with CytoD (final concentration, 2 µg ml −1 ) and 20 µg of exosomes purified from CA-CM. H,I) Western blot analysis of related protein expression and Immunofluorescence staining for pSTAT3 in different groups. Breast cancer cells were transfected with miR-155 inhibitor, and mature adipocytes were transfected with miR-155 mimic as the positive control. J,K) miRNA-155-knockdown 4T1 cells were coinjected with mature 3T3-L1 cells into the mammary and axilla fat pads of mice. As a control, normal 4T1 cells transfected with virus without miR-155 inhibitor was served as a negative control, and normal 4T1 cells mixed with or without adipocytes in Matrigel were inoculated into the mammary and axilla fat pads of BALB/c nude mice (5 mice in each group). Tumor size was measured every 5 days stopping at day 25. L) Macro metastatic lesions were observed in the livers (arrow). Data are presented as the mean ± S.D. of at least three independent experiments. * P < 0.05 versus control values.
of macrophages represents a general mechanism for promoting tumor progression, we further evaluated the number and biomarkers of macrophages in this model. From IHC stained images shown in Figure 6D, the infiltration of CD163 + macrophages were remarkably elevated in mice injected 4T1 with adipocytes, by contrast, anti-F4/80 mAb dramatically alleviated the macrophages recruitment. Consistently, pathological grade in samples of the coinjected group was much higher than those in groups receiving anti-F4/80 mAb treatment. These data collectively indicated that macrophages deficiency in adipose tissues inhibited adipocyte-promoted tumor growth.

Blocking the Chemokines/Receptors/STAT3 Axis Prevents Breast Cancer Growth
To investigate the role of CCL2-CCR2 and CCL5-CCR5 axis and their downstream key regulator STAT3 on tumor progression in vivo, we inoculated the mouse mammary carcinoma cells AT3 in the presence or absence of mature adipocytes into the mammary fat pads of C57BL/6 mice (n = 6 per group), and evaluated tumor growth. When the xenografts became palpable, CCL2-or CCL5-specific neutralizing antibodies as described in Figure 7A were injected via the tail vein. BMS-813160, the CCR2/CCR5 inhibitor, [46] and Stattic, STAT3 inhibitor, [47] were also used to estimate the potential therapeutic effect. It found that compared with AT3 cells alone, a combined injection of mature 3T3-L1 accelerated the tumor growth of xenografts (P < 0.05; Figure 7B). CCL2-or CCL5-specific neutralizing antibodies, as well as BMS-813160 and Stattic, markedly retarded the tumor proliferation of xenografts in the malignant AT3 cells plus mature adipocytes inoculation groups (P < 0.05, Figure 7B-E). Meanwhile, the decreasing infiltration of CD163 + macrophages were discovered among these groups treated as described above ( Figure 7F).

Discussion
Macrophages are particularly important in adipose tissue, and contribute to link obesity-related inflammation and tumor progression. Likewise, macrophages envelope around the phagocytosis of a dead or dying adipocyte, this configuration is termed as CLS. [48] Notably, the number and the density of CLS a positively correlated to high body mass index, large adipocyte size, postmenopausal status as well as insulin resistance in obese subjects, suggesting the pathophysiologic role CLS played in adipose tissue inflammation. [24,49] Hence, macrophages infiltration in adipose tissue around tumor seems to have vital clinical significance, especially in macrophage polarization, yet it needs further investigation. The present study describes that exosomal miRNA-155 from the tumor-adipocyte crosstalk increases Adv. Biology 2023, 7, 2200161  Blocking the chemokines/receptors/STAT3 axis prevents breast cancer growth. A) Schematic overview of the in vivo treatment of murine mammary carcinoma AT-3 cells alone or in combination with mature adipocytes, further treated by STAT3 inhibitor Stattic, blocking antibodies against CCL2 and CCL5, or the CCR2/CCR5 antagonist BMS-813160, respectively. B) Growth kinetic of murine mammary carcinoma AT-3 cells was evolving in immunocompetent C57Bl/6 mice, treated as indicated in (A). n ≥ 6 for mice in each group. Results (means ± SD tumor growth curves) are plotted (*P < 0.05, **P < 0.01 versus control; ns, not statistically significant; Student's t-test). C-E) Individual tumor growth curves of mice in AT-3 cells combined with mature adipocytes, respectively treated by STAT3 inhibitor Stattic, blocking antibodies against CCL2 and CCL5, or the CCR2/CCR5 antagonist BMS-813160. the secretion of CCL2 and CCL5 in adipocytes, further the chemokines recruit macrophages around the adipocytes and repolarized macrophages towards M2-like subtype. Not surprisingly, deleting macrophages by F4/80 neutralizing antibody in this context reduce adipocyte-induced tumor growth.
Macrophages are a complicated kind of cells, which express diverse surface markers and have unique anatomical locations. [12,50] Macrophages are demonstrated to develop from three distinct origins including embryonic precursors, adult HSCs or bone marrow-derived monocytes. [50] Likewise, two main phenotypes of macrophages are the classical polarized M1 macrophages and alternatively polarized M2 macrophages. [18] CD11c is discovered as pre inflammatory biomarkers especially expressed in tissue-resident macrophages [51] while M2 macrophages have an anti-inflammatory effect via up-regulating CD163, CD206, and CD204. [20,50] Meanwhile, it frequently exists macrophages reprogramming from one subset into another, partly explaining this diversity of macrophages. [52] Macrophage phenotype can vary from different cancer types and intratumor districts. [20] Our results have shown that CD163 + macrophages not CD11c + ATMs highly infiltrating in tumor-adipose regions predicted poor survival in patients undergoing breast cancer. It suggests that the macrophages mainly derived from monocytederived cells, not tissue-resident macrophages. However, two primary breast cancer samples and two cases from GEO dataset were included in the single-cell sequencing, which had certain data limitations, driven by the individual differences and tumor heterogeneity. Anti-F4/80 mAb neutralizes macrophages in mice, including primary macrophages, M1 and M2 macrophages, indicating that the neutralization lacks specificity. Elimination of macrophages from the tumor microenvironment by the macrophage-depleting agent clodronate liposome impeded tumor growth in the multiple myeloma model. [53] Depletion of macrophages eliminated malignant progression by decreasing exosomes derived from mesenchymal stem cells in breast cancer. [54] Another study demonstrated that specific elimination of TAMs was induced by the application of dichloromethylene diphosphonate (DMDP)-containing liposomes in B16 melanoma model, however, the absence of TAMs induced the infiltration of inflammatory cells into or around the tumors. [55] Therefore, the elimination of macrophages alone did not inhibit tumor growth, probably because more inflammatory cells were infiltrated in the tumor microenvironment.
The M1 macrophages are also involved in the progression of malignancies, through secreting proinflammatory cytokines and increasing cytotoxic activity in antitumor immunity. [25] Importantly, M2 macrophages can be divided into 4 subtypes according to different surface markers and secreted cytokines. [56] M2 macrophages could be activated into an M2c phenotype, which has high expression of CD163 and Mer receptor tyrosine kinase, thus giving rise to their efficient phagocytosis of apoptotic cells. [57] Described above, macrophages wrap and engulf the dead or dying adipocytes in CLS, suggesting that M2c phenotype might be a potential subset of macrophage in this condition. However, M2a macrophages, another subtype of M2 macrophages, express high levels of CD206 but not CD163. These M2a macrophages enrich genes associated with tissue remodeling and wound healing. [58] Taken together, CD163, one of the special surface markers expressed M2c macrophage, may be the optimal biomarker in CLS. Importantly, tissueresident M2c macrophages in tumor tissues enrich genes associated with tissue remodeling, and have increased expression of genes concerning immunosuppression. [59] By contrast, M2a macrophages mainly exert anti-inflammatory and woundhealing effects [58] while M2b macrophages, which functioned as immunoregulation, secrete pro-and anti-inflammatory cytokines meantime and are found in tumors. [59][60][61] Finally, M2d macrophages represent a novel M2 subset and constitute the major inflammatory component in neoplastic tissue, contributing to angiogenesis and cancer metastasis. [62] Moreover, single-cell RNA sequencing of breast cancer samples identified a special subpopulation of macrophages named as lipid-associated macrophages (LAMs). LAMs displayed high expression of CD163, LIPA, LGALS3, and TREM2, indicating that LAMs are a subtype of M2-like macrophages, and involved in lipid accumulation and phagocytosis. [63] Thus, comprehending the heterogeneity and plasticity of macrophage phenotypes is pivotal for obesity-related cancers.
The chemokines such as CCL2 and CCL5 have the pivotal functions in the recruitment and polarization of macrophages in both tumor tissues and fatty tissues. [12,19,49] In the case of obesity, the hypertrophic expansion of adipose tissue has many features in common with the growth of solid tumors. The hypoxia in obese adipose tissue induces expression of the transcription factor HIF-1α, which is consistent with tumor hypoxia, thereby increasing the expression of proinflammatory cytokines including CCL2 and CCL5. [64,65] The present results indicate that CCL2 and CCL5 overexpress around the adipose tissues and are positively correlated with CD163 + macrophages infiltration in breast cancer specimens, further promoting M2-like polarization of macrophages via activating pSTAT3. Hypertrophic adipocyte-derived chemotactic CCL2/CCR2 pathway recruits more macrophages to accumulate in the obese adipose tissue. Subsequently, CCL2 polarizes macrophages toward the M2 phenotype. [66] Additionally, CCL2 enhances the LPS-induced expression of IL-10 in macrophages, while CCL2 blockade results in increased generation of M1 polarization-related genes and cytokines, and decreased generation of M2-related markers in human macrophages. [67] Likewise, CCL5 is elevated and remains high in adipose microenvironment, [68] and promotes tumor recurrence by recruiting CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors. [15] Moreover, CCL5 activates AKT signaling to recruit and repolarize TAMs via bounding to its receptor, CCR5. [69] The CCR5 inhibitors prevent recruitment of monocytes to the tumor and repolarize macrophages with anti-tumoral effects. [69,70] Therefore, CCL2 and CCL5 not only recruit macrophages to the tumor or obesity-induced microenvironment but shape M2-skewed polarization of macrophages. Moreover, T cells also express CCL5. CCL5 promotes the accumulation of various immune cells, including CD8 + T cells, [71] natural killer cells [72] as well as regulatory T cells (Tregs). [73] High level of CCL5 was expressed by tumor-infiltrating myeloid-derived suppressor cells (MDSCs) in B16 melanoma, and expression of CCR5 was detected on Tregs. Thus, CCL5 attracted large numbers of Tregs via CCR5 in vitro. [73] The transcription of CCL5 was induced by focal adhesion kinase in squamous cell carcinoma cells, thereby promoting the exhaustion of CD8 + T cells and recruitment of Tregs. [74] Although T cell influx was enhanced by overexpression of miR-155 in breast cancer cells and thereby delayed tumor growth, the sensitivity of tumors to immune checkpoint blockade therapy was upregulated. [75] Exosome has been defined as a novel way for cell-to-cell communication that interact with a neighbor or distant target cells. [26] Potentially, the exosome-contained specific contents like miRNAs as potential biomarkers and the amount of released exosomes are altered in obesity and cancer. In current results, exosomal miRNA-155 suppresses SOCS6, a negative regulator of the JAK-STAT signaling pathway, further induces the phosphorylation of STAT3 that contributes to the secretion of CCL2 and CCL5. Inhibition of miRNA-155 suppresses the generation of CCL2 and CCL5 and blocks adipocytes-induced tumor growth. Our previous studies have demonstrated that exosomes from the tumor-adipocyte symbiosis contain several specific miRNAs such as miRNA-126, miRNA-144, and miRNA-155. [28][29][30] Indeed, these exosomal miRNAs play pivotal roles in mediating the differentiation and functions of macrophages. [76][77][78][79] For example, the up-regulation of miRNA-155 in macrophages increases CCL2 secretion by directly inhibiting B-cell lymphoma-6 expression and the decreased expression of Arginase-1 and Chil3. [76,80] Additionally, cancer cell-secreted miRNA-155 targets peroxisome proliferator-activated receptor γ (PPARγ) and downregulates its level. [29,30] Meanwhile, a PPARγ ligand rosiglitazone converts high fat-induced M2 polarization of macrophages toward anti-inflammatory subtype. [81] By contrast, propranolol, a non-selective sympatholytic b-blocker, not only alleviates tumor exosome-stimulated cachexia via activating PPARγ [30] but also inhibits the production of IL-10 in M2 macrophages. [82] The specific effects of PPARγ activators in inhibiting M2 macrophages polarization and the efficacy of these compounds in immunologic/inflammatory diseases requires to further be demonstrated. Taken together, exosomal miRNAs originated from tumor cells may adequately reshape adipocytes in a fatty microenvironment to convert macrophages into a pro-tumor niche.

Conclusion
In summary, as depicted in sthe model figure, we put forward a potential mechanism that exosomal miR-155 from tumoradipocyte interplay could promote the secretion of CCL2 and CCL5 in adjacent adipocytes via targeting SOCS6/STAT3 signaling, subsequently accumulating M2-like macrophages towards TAME to expedite tumor growth. Likewise, knockdown exosomal miR-155, inhibited chemokines-associated signaling, and deleting these macrophages could retard the tumor growth. Together, these studies shed light on targeting strategies for blocking the adipocyte-macrophage-tumor interactions as new therapeutic modalities for obesity-associated cancer.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.