Macrophages in Tumor-Associated Adipose Microenvironment Accelerate Tumor progression

Background: Macrophages particularly contribute to the progression of obesity-related tumor. However, the mechanisms that the tumor-adipocyte crosstalk may enable the properties and plasticity of macrophages remain still unclear. Methods: Survival probabilities for recurrence-free survival (RFS) were estimated by the Kaplan–Meier method based on immunohistochemistry and immunouorescence images. 3T3-L1 cells were cocultivated with 4T1 and MDA-MB-231 cells. Then the co-cultivated media were used to treat THP-1/RAW264.7 cells. The markers of macrophages were detected by immunouorescence and Western blot. Transwell migration assay, was conducted to determine the migration capability of macrophages. RT-PCR and ELISA were used to detect the expression and secretion level of chemokines, respectively. Immunouorescence imaging and Western blot were used to investigate the role of SOCS6/STAT3 signaling pathway in the polarization of macrophages. microRNA mimic and inhibitor, and xenograft models were used to explore the role of miR-155 in the polarization of macrophages. Results: We demonstrate that CD163-positive macrophages aggregated to surround adipocytes in breast cancer tissues, which was associated with tumor relapse. In tumor-adipocyte microenvironment, CD163-positive macrophages are recruited and polarized via the elevated expression of CCL2 and CCL5. Consistently, the eliminated macrophages partially inhibited adipocytes-induced tumor proliferation. Likewise, inhibiting chemokines and their receptors or suppressing the phosphorylation of STAT3 signicantly decreased tumor burden in vivo. Finally, the source of CCL2 and CCL5 mainly derived from adipocytes. In coculture of tumor cells and adipocytes, the level of exosomal miRNA-155 was high, then it promoted the generation and release of CCL2 and CCL5 from adipocytes through targeting 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. Conclusion: These results suggest a novel target of tumor-adipocyte-macrophage interconnect that could facilitate obesity-induced 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]. Obesity is mainly dependent on excessive fat accumulation in the regional adipose tissues. However, growing evidence from both clinical and preclinical studies indicates that increased adiposity is associated with increases in cancer incidence and mortality [2,3], mechanistic insights explaining these data are notably lacking. In particular, obesity-caused chronic in ammation is considered as a potent driver of cancer initiation and progression through its ability to cultivate a supported microenvironment that is permissive to neoplastic transformation [4][5][6].
Macrophages, key cells in the in ammatory response, also enrich in adipose and tumor microenvironments, where they mediate the development, growth, and progression of cancer [7].
Macrophages in adipose and tumor microenvironments are a diverse population of cells that develop from different origins and express distinct surface markers [8]. Tissue-resident macrophages like adiposetissue macrophages (ATMs) are originated from embryonic precursors or adult haematopoietic stem cells (HSCs). In the in ammatory environments, a relevant proportion of macrophages also derive from monocyte-derived cells. Classically, two main phenotypes of macrophages in adipose tissue are proin ammatory M1 (classically activated) and anti-in ammatory M2 (alternatively activated) [9]. 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 [10]. M1 macrophages produce pro-in ammatory cytokines (TNF-α, interleukin IL-6) while M2 macrophages play an anti-in ammatory role via secreting IL-10 [11]. Moreover, macrophage phenotype can vary in different tumor types and intratumor districts [11].
Speci cally, macrophages can be polarized into an M2-like state or "switching macrophages", which produce both pro-and anti-in ammatory cytokines and have been observed in tumors and adipose tissues [12,13]. Interestingly, the macrophages that envelope around phagocytosis of a dead or dying adipocyte in a con guration termed crown-like structures (CLSs), a histologic biomarker of in ammation [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 human epidermal growth factor receptor-2 [14,15]. 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.
Chemokines are discovered as a family of secreted proteins, whose prominent role is leukocyte chemotaxis and activation [16]. Chemokine (C-C motif) ligand 2 (CCL2) and CCL5 are a member of chemokine branch of the cytokine superfamily. CCL2 attracts circulating macrophages and stimulates their immunosuppression in the tumor microenvironment via binding to its receptor CCR-2 [17].
Meanwhile, CCL5 could recruit CCR5-overexpressing macrophages, subsequently which depose collagens in residual tumors to drive tumor recurrence [18]. However, whether the expressions of CCL2 and CCL5 are elevated in tumor-associated adipose microenvironment, and repolarize the residual macrophages is less well characterized.
Exosomes are a kind of small extracellular vesicles (30-100 nm) derived from cancer cells and stromal cells [19]. This cell-to-cell biological communication is mediated through the exchange of the exosomes content, which consists of proteins, lipids, metabolites, RNA, and DNA [20]. Exosomes are appreciated as essential mediators of cell-cell communication. Our previous research indicated that exosomal miRNAs from the tumour-adipocyte interaction induce beige/brown differentiation and accelerate catabolism in recipient adipocytes to facilitate tumour progression [21][22][23]. In this study, it's evident that CCL2 and CCL5 facilitate tumor growth through recruiting and polarizing M2-like macrophages in tumor-adipose microenvironment. In addition, the results also show that exosomes derived from tumor-adipocyte interaction induce CCL2 and CCL5 secretion from adipocytes in SOCS6/STAT3-depending way.

Page 4/23
Material And Methods Patients A total of 145 formalin-xed para n-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, and the study was approved by the Institutional Ethics Committee of Renmin Hospital of Wuhan University (approval no. 2018K-C09). Patients did not receive nancial 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 rst diagnosis. All methods were performed in accordance with relevant guidelines and local regulations.

Coculture and invasion assays
Mature 3T3-L1 and breast cancer cells were cocultured using Transwell culture plates (0.4 µm pore size; Millipore). Mature 3T3-L1 cells in the bottom chamber of the Transwell system were cultivated in serumfree medium containing 1% bovine serum albumin (Sigma) for 4 h. A total of 3 × 10^5 4T1 or MCF-7 or MDA-MB-231 cells were cultivated in the top chamber in the presence or absence of mature 3T3-L1 cells in the bottom chamber for the indicated times. The conditioned medium (CA-CM) was collected from adipocytes cultivated with tumour cells for 3 days. After 24 h of coculture in the presence of normal medium or CA-CM (supplemented with 10% FBS), macrophages were seeded in the top chamber in the presence of CM, AD-CM or CA-CM (supplemented with 10% FBS) for Matrigel invasion assays.

Exosome isolation and characterization
After cells were cultured with exosome-depleted serum (AusGeneX), the exosomes were puri ed from the conditioned medium according to the instructions [24]. 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, 81260) solution ( nal 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 puri ed using 5% PEG and then stored in 50-100 µl of particle-free PBS (pH 7.4) at − 80 °C.
The average yield was approximately 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 xed 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 quanti cation of exosomes in PBS as speci ed 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 labelled exosomes (20 µl/well) for the indicated time. Images were acquired using the Olympus FluoView FV1000.
The conditioned media were centrifuged at 1000 g for 10 min, incubated with biotinylated antibody working solution at 37℃ for 1.5 h, incubated with enzyme conjugated working solution at 37℃ 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 speci c antibodies (Additional le 1: Table S1).

Neutralizing experiments
Before 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 le 1: Table S2. 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 tumour cells and the staining intensity. The protein expression level of CD163 was described according to the numbers of CD163 + macrophages counted in 10 random elds of each breast cancer specimen at 400 × magni cation. 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 (ROC) analysis was used to determine the optimal cut-off values for all expression levels regarding the survival rate.

Immuno uorescence imaging
Immuno uorescence (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 uorescence 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 de ned 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. Brie y, HEK 293 T cells were seeded in 12-well plates and grew to 70% con uence. 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 In niti reader. The results are described as the ratio of re y luciferase activity compared to Renilla luciferase activity.

Xenograft tumor formation
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. 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. ×0.52 and was measured once every 2-3 days until using a Vernier calliper. Animals were sacri ced when tumor size reached endpoint or signs of obvious discomfort were observed following our Ethical Committee advice. After the mice were sacri ced, all tissues were collected, embedded in para n and stained with IHC or haematoxylin-eosin (HE).

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 quanti ed 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.

Results
The in ltration 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 surrounding by breast cancer has not been thoroughly explored. It is reported that tumor-associated macrophages (TAMs) are con rmed as "switching macrophages", which generate pro-and anti-in ammatory cytokines meantime and are found in tumors [13]. CD68 (macrophage membrane marker) and CD163 (M2-macrophage marker) have been usually regarded as surrogate markers to investigate TAMs polarity in previous studies [15,25]. And the increased in ltration of CD163 + tumor-associated macrophages (TAMs) in CLSs were associated with shorter disease-free survival in node-negative breast cancer patients [26]. We rstly detected the existence and characteristics of macrophages in a cohort of 145 breast cancer specimens using immunohistochemistry (IHC) and QDs-based uorescent 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 (Fig. 1A). Meanwhile, QDs-based uorescent imaging showed that CD68 + CD163 + macrophages remarkably in ltrated in adipocytes adjacent to tumor tissues and in connective tissue (Fig. 1B). In addition, CD11c is M1 and proin ammatory marker of ATMs [27], further D11c + macrophages were also found to constitute the CLSs [28]. However, CD11c was extremely low expression and even no expression in our study (Fig. S1A). 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 in ltration of CD68 + CD163 + macrophages were notably elevated when tumor metastasized into lymph node (P < 0.001). Moreover, the positive expression of CD163 was more common in patients 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 human epithelial growth factor receptor-2 (HER2).
We further analyzed the association between the expression of CD68 + CD163 + macrophages and prognosis of patients with breast cancer. We calculated the in ltration of CD68 + CD163 + macrophages in 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 adipocytes was signi cantly associated with shorter recurrence-free survival (RFS) than that of patients with negative expression (P = 0.0142, Fig. 1C left). Whereas, there was no signi cance in RFS between CD163-negative and -positive patients grouping via the CD163 + macrophages in ltration in tumor tissues (P = 0.316, Fig. 1C right).
These results demonstrated that macrophages surrounded adipocytes polarized towards M2-like phenotype and high level of in ltrated 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 tumoradipose microenvironment and contributed to 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 [22,29]. Driven the fact that macrophages could be recruited to in ammatory regions by chemokines, including CCL2 [30] and CCL5 [18], we supposed that macrophages surrounded by 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 ( Fig. 2A) and contributed to a poor survival (Fig. 2B). 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 CD68 + CD163 + macrophages (Fig. 2C, 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 Transwell system, the mRNA levels of CCL2 and CCL5 in adipocytes are dramatically elevated (Fig. 3A-B). Likewise, the conditioned media (CM) from mature adipocytes in the presence or absence of breast cancer cells (4T1, MDA-MB-231) cultivated for 3 days were collected. And it shows that CCL2 and CCL5 are highly expressed in CM from mature adipocytes co-cultivated with MDA-MB-231 cells via ELISA assay (Fig. 3C). Notably, the migration capabilities of macrophages also increased in co-cultivated group (Fig. 3D). To sum up, these results have indicated that CCL2 and CCL5 play a crucial role on macrophages recruit.
Chemokines CCL2 and CCL5 induced M2-like polarization of macrophages via activating STAT3 phosphorylation Prominently, it has evidence that chemokines CCL2 and CCL5 enable the selective polarization of subsets of macrophages via interacting their chemokine receptors [16].
To determine how macrophages encapsulated and polarized in CLSs, we explore the impact of CCL2 and CCL5 on macrophages polarity. Next we con rmed that both mouse macrophages (RAW264.7 cell) [31] and human monocyte-derived macrophages (THP-1 cell induced by phorbol-12-myristate-13-acetate (PMA) [32]) showed increased M2-like (CD163) polarization upon treatment of CM from adipocytes cocultured with breast cancer cells relative to the single cultivation ( Fig. 3E-F). In terms of mechanism, the expression of pSTAT3 was increased in macrophages with treatment of cocultured CM (Fig. 3G). 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 ( Fig. 3H-I, Fig. S2A).
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 tumour growth To con rm that the observed reliance for macrophages in CLSs depends on the tumor-adipocytes interaction, we examined whether the conditional presence of cancer cells alters the secreted spectrum of adipocytes. Based on our previous work, we speculated that the exosomes derived from tumour cells are important for adipocytes that are converted into cancer-associated adipocytes (CAAs) and excessively secrete chemokines including CCL5 [21][22][23]. Exosomes were isolated from conditioned medium, displaying that typical exosomal morphology and size and contained CD63, TSG101 and CD81 but not HSP70 (Fig. S3A-C), which was consistent with previous reports on exosomes [33]. To observe exosomes uptake by adipocytes, breast cancer-secreted exosomes were labeled with red uorescence. After being treated with exosomes for 4 hours, mature 3T3-L1 cells were densely packed with exosomes ( Fig. S3D), indicating rapid cellular uptake of exosomes by adipocytes. Further, exosomal miR-155 upregulated in cocultured group. Meanwhile, the mature miR-155 also overexpressed in adipocytes in presence of breast cancer cells, but the precursor of miR-155 remains unchanged (Fig. 4A). 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 (Fig. 4B).
This observed increase in exosomal miR-155 prompted us to analyze the effects of tumor-originated miR-155 on the in ammation change of adipocytes. Intriguingly, we found one potential sequence of miR-155 targeting the human suppressor of cytokine signaling 6 (SOCS6) 3'UTR sequence (Fig. 4C). We next established one luciferase reporter, displaying that the wild-type construct showed a signi cant 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 (Fig. 4D). 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 co-cultivated with breast cancer cells (Fig. 4E-F). SOCS6 is negative regulator of Janus activated kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway [34], and the reduction of SOCS6 nuclear localization promoted protein expression of STAT3 [35]. 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 (Fig. 4F-G). This result indicated that the exosome uptake contributes to the upregulation of pSTAT3 in adipocytes undergoing tumour cells. Further, we investigated the possibility that exo-miR-155 would activate pSTAT3 activity via inhibiting SOCS6. The results indicated that the inhibition of SOCS6 activity in adipocytes cocultivated with breast cancer cells was comparable to that of miR-155 overexpression (Fig. 4H-I). This inhibitory effect was achieved through miR-155 knockdown (Fig. 4H-I). Consistently, the administration in co-cultured adipocytes increased the SOCS6-mediated phosphorylation of STAT3 and the knock-downed miR-155 would reverse the expression (Fig. 4H-I). The xenograft models showed consistent results with in vitro assays, indicating that signi cantly increased tumour growth in mice co-injected with 4T1 cells and mature 3T3-L1 cells, whereas downregulation of miR-155 in tumour-derived exosomes signi cantly reduced the tumour volume in xenografts ( Fig. 4J-L). Likewise, the number and the size of metastatic nodules in liver were markedly increased in the group injected with tumour cells cocultivated with mature 3T3-L1 cells, while downregulation of miR-155 in tumour exosomes signi cantly reduced the number of nodules in liver (Fig. 4L). And the number and the size of metastatic nodules in lung were no obvious difference among these groups (data not shown). Altogether, our results demonstrated that exosomal miR-155 derived from breast cancer cells mediated in ammatory adaptive changes in mature adipocytes through SOCS6/STAT3 signaling pathway.

Macrophages de ciency in tumor-adipocyte interaction inhibits tumor growth
Whether macrophages existence endowed breast cancer cells with the proliferated capacity, we speculated that macrophages de ciency might inhibit adipocyte-associated tumor growth. To this end, we employed mouse model without macrophages to assess the impact of de cient 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 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 speci c proportion were orthotopically injected into the mammary fat pad [22]. Likewise, the macrophages in mouse model were particularly eliminated via injection of anti-F4/80 neutralizing antibody as reported [13] (Fig. 5A). As Fig. 5B-C, the tumor injected 4T1 combined with adipocytes grew much rapidly while the eliminated macrophages via anti-F4/80 mAb could signi cantly reduce these tumors growth. To determine whether M2-like phenotype 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 Fig. 5D, the in ltration 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 co-injected group was much higher than those in groups receiving anti-F4/80 mAb treatment. These data collectively indicated that macrophages de ciency 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-speci c neutralizing antibodies as described in Fig. 6A were injected via the tail vein. BMS-813160, the CCR2/CCR5 inhibitor [36], and Stattic, STAT3 inhibitor [37], were also used to estimate the potential therapeutic effect. It found that compared with AT3 cells alone, combined injection of mature 3T3-L1 accelerated the tumor growth of xenografts (P < 0.05; Fig. 6B). CCL2-or CCL5-speci c 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, Fig. 6B-E). Meanwhile, the decreasing in ltration of CD163 + macrophages were discovered among these groups treated as described above (Fig. 6F).
Exosome originated from tumor-adipocyte interplay induce autophagy in macrophages depending on transcription factor 3 (TFE3) translocation Recent study demonstrates that high expression of CD163 is a prominent biomarker of M2c phenotype of macrophages, thus giving rise to their e cient phagocytosis of apoptotic cells [38]. Meanwhile, activated autophagy conduces to enhance phagocytosis of macrophages [39]. Using RAW264.7 cells stably expressing GFP-LC3, we found that the isolated exosomes enhances the level of LC3 II and GFP-LC3 dots, then the expression of GFP-LC3 dots and the lipidation of LC3 was further increased in the presence of the lysosomal inhibitor Ba lomycin A1(Baf-A1) (Fig. S5A-C). Likewise, the exosomes caused a reduction in the abundance of P62, and this effect was blocked by Baf-A1 (Fig. S5B). In addition, the result showed that the extractive exosomes enabled to stimulate the translocation of TFE3 from the cytoplasm to the nucleus (Fig. S5D-F). Further, knockdown of TFE3 with siRNA ( Fig. S5G) partially reduced the formation of GFP-LC3 puncta in response to exosomes (Fig. S5H). Similarly, The TFE3 KD was particularly e cient in reducing exosomes-induced LC3 lipidation (Fig. S5I-J). In sum, the exosomes from tumor-adipocyte coculture stimulated autophagy by the activation of TFE3.

Discussion
Macrophages are particularly important in adipose tissue, and contribute to link obesity-related in ammation and tumor progression. Likewise, macrophages envelope around phagocytosis of a dead or dying adipocyte, this con guration is termed as crown-like structures (CLS) [40]. Notably, the number and the density of CLS is 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 in ammation [26,41]. Hence, macrophages in ltration in adipose tissue around tumor seems to have vital clinical signi cance, especially in macrophage polarization, yet it needs further investigation. The present study describes that exosomal miRNA-155 from the tumor-adipocyte crosstalk increase 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 in this context retard tumor growth.
Macrophages are a complicated kind of cells, which express diverse surface markers and have unique anatomical locations [7,42]. Macrophages are demonstrated to develop from three distinct origins including embryonic precursors, adult haematopoietic stem cells (HSCs) or bone marrow derived monocytes [43]. Likewise, two main phenotypes of macrophages are the classical polarized M1 macrophages and alternatively polarized M2 macrophages [9]. CD11c is discovered as pre-in ammatory biomarkers especially expressed in tissue-resident macrophages [44] while M2 macrophages have an antiin ammatory effect via up-regulating CD163, CD206 and CD204 [11,42]. Meanwhile, it frequently exists macrophages reprogramming from one subset into another, partly explaining this diversity of macrophages [45]. In addition, macrophage phenotype can vary from different cancer types and intratumor districts [11]. Our results have shown that CD163 + macrophages not CD11c + ATMs highly in ltrating in tumor-adipose regions predicted poor survival in patients undergoing breast cancer. It suggests that the macrophages mainly derived from monocyte-derived cells not tissue-resident macrophages. Importantly, M2 macrophages could be activated into an M2c phenotype, which high expression of CD163 and Mer receptor tyrosine kinase (MerTK), thus giving rise to their e cient phagocytosis of apoptotic cells [46]. Described above, macrophages wrap and engulf the dead or dying adipocytes in CLS, suggesting that M2c phenotype might be 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 [47]. Taken together, CD163, one of special surface markers expressed M2c macrophage, may be the optimal biomarker in CLS. Importantly, tissue-resident M2c macrophages in tumor tissues enrich genes associated with tissue remodeling, and have increased expression of genes concerning immunosuppression [38]. By contrast, M2a macrophages mainly exert anti-in ammatory and woundhealing effects [47] while M2b macrophages, functioned as immunoregulation, secrete pro-and antiin ammatory cytokines meantime and are found in tumors [38,48,49]. Finally, M2d macrophages represent a novel M2 subset and constitute the major in ammatory component in neoplastic tissue, contributing to angiogenesis and cancer metastasis [50]. However, adipose tissues and cancer contain macrophages with a mixed subtypes [8,38]. Thus, comprehending the heterogeneity and plasticity of macrophage phenotypes is pivotal for obesity-related cancer.
The chemokines such as CCL2 and CCL5 have the pivotal functions in the recruit and polarization of macrophages in both tumor tissues and fatty tissues [7,10,41]. 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 expression of proin ammatory cytokines including CCL2 and CCL5 [51,52]. The present results indicate that CLL2 and CCL5 overexpress around the adipose tissues and are positive correlated with CD163 + macrophages in ltration in breast cancer specimens, further promoting M2-like polarization of macrophages via activating pSTAT3. Hypertrophic adipocyte-derived chemotactic CCL2/CC chemokine receptor 2 (CCR2) pathway recruits more macrophages to accumulate in the obese adipose tissue. Subsequently, CCL2 polarizes macrophages toward the M2 phenotype [53]. 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 M2related markers in human macrophages [54]. Likewise, CCL5 is elevated and remains high in adipose microenvironment [55], and promotes tumor recurrence by recruiting CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors [18]. Moreover, CCL5 activates AKT signaling to recruit and repolarize TAMs via bounding to its receptor, CCR5 [56]. The CCR5 inhibitors prevent recruitment of monocytes to the tumor and repolarize macrophages with anti-tumoral effects [56,57]. Therefore, CCL2 and CCL5 not only recruit macrophages to the tumor or obesity-induced microenvironment but shape M2-skewed polarization of macrophages.
Exosome has been de ned as a novel way for cell-to-cell communication that interact with a neighbor or distant target cells [19]. Potentially, the exosome-contained speci c 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.
Indeed, these exosomal miRNAs play pivotal roles in mediating the differentiation and functions of macrophages [58][59][60][61]. For example, the up-regulation of miRNA-155 in macrophages increases CCL2 secretion through directly inhibiting B-cell lymphoma-6 (BCL6) expression and the decreased expression of Arginase-1 and Chil3 [58,62]. Additionally, cancer cell-secreted miRNA-155 targets PPARγ and downregulates its level [22,23]. Meanwhile, a PPAR ligand rosiglitazone converts high fat-induced M2 polarization of macrophages toward anti-in ammatory subtype [63]. By contrast, propranolol, a nonselective sympatholytic -blocker, not only alleviates tumor exosome-stimulated cachexia via activating PPARγ [23] but also inhibits the production of IL-10 in M2 macrophages [64]. The speci c effects of PPAR activators in inhibiting M2 macrophages polarization and the e cacy of these compounds in immunologic/in ammatory diseases requires to further be demonstrated. Taken together, exosomal miRNAs originated from tumor cells may adequately reshape adipocytes in fatty microenvironment to convert macrophages into a pro-tumor niche.
Autophagy has multiple effects on macrophages. On the one hand, autophagy activity in macrophages enhance IL-1β secretion [65]. On the other hand, autophagy may render macrophage phagocytosis [66].
The basic helix-loop-helix transcription factor TFE3 (transcription factor 3) belongs to the MiT/TFE family [67]. It has evidence that TFE3 controls lysosomal biogenesis and autophagy by regulating the expression of several lysosomal and autophagic genes [68]. Normally, TFE3 is located in the cytoplasm and translocates to the nucleus in response to various stress [67]. Importantly, TFE3 is crucial for macrophages activation via inducing autophagy, increasing lysosomal biogenesis, and upregulating numerous proin ammatory cytokines in transcriptional way including CCL2 and CCL5 [69]. Transcription factor EB (TFEB) is similar with TFE3 [67,69], and its expression elevates in IL-4 induced M2 macrophages, but reduces in tumor-conditioned medium-treated macrophages. Likewise, TFEB ablation promotes breast tumor growth [70]. Interesting, recent study shows that TFEB reduction was accompanied in some instances by a concomitant increase in TFE3 levels, which might be indicative of a compensatory mechanism between both transcription factors [69]. Consistent with our results, TFE3 but not TFEB promote autophagic ux in activated macrophages.

Conclusion
In summary, as depicted in model (Fig. 7), we put forward a potential mechanism that exosomal miR-155 from tumor-adipocyte interplay could promote the secretion of CCL2 and CCL5 in adjacent adipocytes via targeting SOCS6/STAT3 signaling, subsequently accumulating M2-like subtype of macrophages towards tumor-adipose microenvironment. Together, these studies shed light on targeting strategies for reducing interaction among tumors, stromal cells and immune cells as a method to clari ed adipocytemacrophage-tumor interactions in planning treatment strategies of obesity-associated cancer.