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Inhibition of STAT3 by 2-Methoxyestradiol suppresses M2 polarization and protumoral functions of macrophages in breast cancer

BMC Cancer volume 24, Article number: 1129 (2024) Cite this article

Abstract

Background

Breast cancer metastasis remains the leading cause of cancer-related deaths in women worldwide. Infiltration of tumor-associated macrophages (TAMs) in the tumor stroma is known to be correlated with reduced overall survival. The inhibitors of TAMs are sought after for reprogramming the tumor microenvironment. Signal transducer and activator of transcription 3 (STAT3) is well known to contribute in pro-tumoral properties of TAMs. 2-Methoxyestradiol (2ME2), a potent anticancer and antiangiogenic agent, has been in clinical trials for treatment of breast cancer. Here, we investigated the potential of 2ME2 in modulating the pro-tumoral effects of TAMs in breast cancer.

Methods

THP-1-derived macrophages were polarized to macrophages with or without 2ME2. The effect of 2ME2 on macrophage surface markers and anti-inflammatory genes was determined by Western blotting, flow cytometry, immunofluorescence, qRT‒PCR. The concentration of cytokines secreted by cells was monitored by ELISA. The effect of M2 macrophages on malignant properties of breast cancer cells was determined using colony formation, wound healing, transwell, and gelatin zymography assays. An orthotopic model of breast cancer was used to determine the effect of 2ME2 on macrophage polarization and metastasis in vivo.

Results

First, our study found that polarization of monocytes to alternatively activated M2 macrophages is associated with the reorganization of the microtubule cytoskeleton. At lower concentrations, 2ME2 treatment depolymerized microtubules and reduced the expression of CD206 and CD163, suggesting that it inhibits the polarization of macrophages to M2 phenotype. However, the M1 polarization was not significantly affected at these concentrations. Importantly, 2ME2 inhibited the expression of several anti-inflammatory cytokines and growth factors, including CCL18, TGF-β, IL-10, FNT, arginase, CXCL12, MMP9, and VEGF-A, and hindered the metastasis-promoting effects of M2 macrophages. Concurrently, 2ME2 treatment reduced the expression of CD163 in tumors and inhibited lung metastasis in the orthotopic breast cancer model. Mechanistically, 2ME2 treatment reduced the phosphorylation and nuclear translocation of STAT3, an effect which was abrogated by colivelin.

Conclusions

Our study presents novel findings on mechanism of 2ME2 from the perspective of its effects on the polarization of the TAMs via the STAT3 signaling in breast cancer. Altogether, the data supports further clinical investigation of 2ME2 and its derivatives as therapeutic agents to modulate the tumor microenvironment and immune response in breast carcinoma.

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Introduction

Breast cancer is one of the most frequently diagnosed malignancy in women worldwide. An estimated 300,590 new breast cancer cases were diagnosed in 2023 in the USA [1]. The projected number of breast cancer cases in India would be 232,832 in the year 2025 [2]. Conventional systemic chemotherapy and radiotherapy are the most common approaches for breast cancer treatment, however patients may relapse and develop distant metastases. Although early-stage breast cancer is associated with a favorable prognosis, patients diagnosed at an advanced stage suffer from metastasis, therapeutic relapse, and poor outcomes. Metastases to distant organs is responsible for breast cancer related deaths, underscoring the advances in early diagnosis [3]. The breast tumor microenvironment is surrounded by different cell types and signaling molecules. Tumor-associated macrophages (TAMs), which exhibit two major phenotypes, antitumor M1 macrophages and protumoral M2 macrophages, are the most prominent immune cells in the cancer milieu [4,5,6]. Immune effector cells are chemotactically recruited in the tumor microenvironment, skewed by tumor cells toward the M2 phenotype, and in turn, secrete cytokines and growth factors that increase tumor metastasis, angiogenesis, and chemoresistance [7]. In breast cancer, TAMs can comprise up to 50% of stromal cells. Increased infiltration of M2 macrophages is associated with reduced overall survival as well as relapse-free survival in breast cancer [8,9,10,11,12]. As a result, there is an active effort to identify small molecule inhibitors of M2 polarization as monotherapy or in combination with standard chemotherapy, which may improve the disease outcome [13, 14].

Members of the signal transducer and activator of transcription (STAT) family, including STAT3, are considered crucial in promoting the phenotypic changes of macrophages to the M2 subtype [15, 16]. STAT3 phosphorylation (Tyr705) causes dimerization, nuclear translocation, and constitutive activation of the transcription of various protumoral downstream effectors in TAMs, which have been shown to regulate cell proliferation, chemoresistance, and angiogenesis [17, 18]. Therefore, STAT3 signaling is now being recognized as an attractive target for modulating M2 TAMs as well as for improving the therapeutic outcomes of immunotherapy.

2-Methoxyestradiol (2ME2), a metabolite of 17β-estradiol, is an anticancer drug with strong antiangiogenic activity. 2ME2 shows negligible binding affinity to estrogen receptors, which prevents its cytotoxic and estrogenic activities [19]. 2ME2 has undergone clinical trials for the treatment of breast, renal, ovarian, prostate, and multiple myeloma tumors [19,20,21,22,23,24]. The promising antitumor effects of 2ME2 have also led investigators to probe the potential of different formulations of 2ME2, including a nanocrystal colloidal dispersion [25], the development of potent derivatives [26], and its use in combination therapy [27]. Recent studies have now pointed out that in addition to their antiproliferative effects on tumors, these drugs might also modulate the tumor microenvironment. Although previous studies have indicated the immunomodulatory effects of 2-methoxyestradiol [28,29,30], its effect on TAMs remains unknown. Hence, we investigated the effect of 2ME2 on tumor-associated M2 macrophages. The data reveal that 2ME2 inhibits the activation of macrophages to the M2 phenotype and suppresses the metastasis-promoting functions of M2 TAMs in vitro and in vivo. Moreover, we show that the functional modulation of TAMs by 2ME2 is associated with inhibition in the phosphorylation and nuclear localization of STAT3, a key driver of M2 polarization.

Materials and methods

Reagents and chemicals

DMEM, RPMI-1640 medium, penicillin G, streptomycin, DMSO, and trypsin were obtained from Hi-Media (Mumbai, India). 2-Methoxyestradiol, LPS and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Aldrich (St. Louis, MO, USA). STAT3 inhibitor, WP1066, PVDF membrane and ECL chemiluminescence kit were purchased from Millipore (Millipore Corp, USA). Human recombinant interleukins IL-4, IL-13 and IFN-γ were purchased from Peprotech. Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Alexa Fluor antibodies, SYBR Green, and TRIzol Reagent were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against acetylated α-tubulin (#T7451), and GAPDH (#98795) were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against α-tubulin (#2125), STAT3 (#9139), pSTAT3 (#9145), VEGF-A (#50661), HIF-1α (#79233) and MMP9 (#13667) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell culture

Human monocytic THP-1 cells and Human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from NCCS, Pune, India. Murine breast cancer 4T1 cells and murine macrophage RAW 264.7 cells were a kind gift from Prof. G.P. Talwar (Talwar Research Foundation, India). THP-1, 4T1 and RAW 264.7 cells were maintained in RPMI-1640 medium while MCF-7 and MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle’s Minimal Essential Medium (with 10 µg/ml insulin) and L-15 medium, respectively. All cell culture media were supplemented with 10% FBS and 1% penicillin-streptomycin and maintained in humidified conditions at 37 °C and 5% CO2 as described previously [31,32,33,34]. THP-1 monocytes were differentiated into M0 macrophages by stimulation with 100 nM PMA for 48 h followed by 24 h of incubation in PMA-free RPMI-1640 medium. For polarization to the M2 phenotype, M0 macrophages were incubated with IL-4 and IL-13 (20 ng/ml each) for 24 h. For polarization to M1 phenotype, M0 macrophages were incubated with LPS and IFN-γ (20 ng/ml each) for 24 h. In other experiments, M0 macrophages were incubated with conditioned medium of MCF-7 cells for 24 h.

MTT assay

Briefly, THP-1 cells and RAW 264.7 cells (5000 cells/well) were differentiated into M0 macrophages and polarized to M1 or M2 in the presence of vehicle, 2ME2 (0 µM to 100 µM) or WP1066 (0 µM to 100 µM). After treatment, MTT was added, and the cells were incubated for 4 h at 37 °C. DMSO was used to dissolve the formazan crystals, and the absorbance was measured at 570 nm.

Collection of conditioned medium

M0 macrophages were polarized to M2 macrophages in the presence of either 0.01% DMSO (vehicle), 2ME2 or WP1066 for 24 h. Then, the drug-containing medium was removed, and the cells were incubated in FBS-free RPMI medium for 6 h. For collection of breast cancer CM, MCF-7 cells were washed with PBS and cultured in 2% FBS for 24 h. The obtained conditioned medium (CM) was centrifuged at 5000 rpm, and the supernatants were collected and stored at -80 °C until further use.

Nuclear-cytoplasmic protein extraction for Western blotting

The control and treated cells were harvested by scraping and centrifuged at 3000 rpm for 5 min. Cells were lysed in 200 µl ice-cold cell fractionation buffer (20 mM HEPES, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA with freshly prepared DTT (1 mM), protease inhibitors, and phosphatase inhibitors) by passing through a 27 G needle, incubated for 15 min and centrifuged again at 3000 rpm for 15 min. The supernatant was collected as cytoplasmic extract, and the pellet was resuspended in 200 µl cell fractionation buffer. The sample was lysed by passing through a 25 G needle and centrifuged at 3000 rpm for 15 min. The pellet was resuspended in TBS with 0.1% SDS and sonicated to prepare the nuclear extract. Western blotting of each fraction was performed using antibodies specific for pSTAT3, α-tubulin, and histone H3 [33].

Western blotting

Briefly, after the indicated treatments, cells were harvested by centrifugation and washed with ice-cold PBS, and lysis of the cells was performed with RIPA buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton X) containing protease (P1860) and phosphatase (P2850) inhibitors. Protein was harvested from homogenized tumor tissues similarly. Bradford assay (Bio-Rad) was used to determine the protein concentrations. Proteins were separated based on size by using SDS‒PAGE and were subsequently transferred to PVDF membranes. Wherever necessary, membranes were cut prior to incubation with primary antibody. The membrane was blotted with primary antibodies against α-tubulin, acetylated α-tubulin, GAPDH (1:1000), STAT3 (1:1000), pSTAT3Y705 (1:1000), MMP9 (1:1000), VEGF-A (1:1000) or HIF-1α (1:1000), and histone H3 (1:60,000) as per the manufacturer’s protocol. The membranes were further incubated with secondary antibodies (1:3000) for 1 h. The signals were developed using the ECL reagent, captured by the gel documentation system (GE Biosciences), and quantified by densitometry using ImageJ software. The original, full-length Western Blot images are provided in Supplementary material. The cropped region of the blot shown in respective figures is indicated by red dashed line.

Immunofluorescence assay

Cells were seeded on coverslips in 24-well plates. After the indicated treatments, M0 or M2 macrophages were fixed in 3.7% paraformaldehyde (PFA) for 15 min, and permeabilization was performed with ice-cold methanol (100% v/v) for 20 min at -20 ℃. For surface markers (CD68 and CD206), cells were fixed in 3.7% PFA but were not permeabilized with methanol. After blocking (2% BSA-PBS), incubation with primary antibodies was performed for 2 h using the following dilutions: α-tubulin (1:500), acetylated α-tubulin (1:500), STAT3 (1:300), pSTAT3 (1:500), CD68 (1:200) and CD206 (1:200) [28]. Samples were further incubated with secondary antibodies (Alexa Fluor 488 or Alexa Fluor 594) for 1 h in the dark. Cells were incubated with DAPI for 15 min. Coverslips were then mounted on glass slides using 10 µl of mounting solution (80% glycerol in PBS) and visualized under a confocal microscope (Nikon, Tokyo, Japan). For image quantification, individual channels of the image were selected using ImageJ software. To determine the nuclear translocation of STAT3 under different conditions, the percentage of cells with nuclear-translocated STAT3 was scored, and total cells (n = 200) were counted using the DAPI channel [34]. Data from three independent experiments were averaged.

Gelatin zymography

Matrix metalloproteinase activity was determined by gelatin zymography. M2 macrophages were serum starved and treated as indicated. The conditioned medium was collected and concentrated 10X (Eppendorf centrifugal vacuum concentrator). Then, MCF-7 cells were incubated with CM (50% v/v) from vehicle- or drug-treated macrophages for 12 h and incubated with FBS-free medium for 6 h to obtain the sample for gelatin zymography. Samples were separated by SDS‒PAGE (containing 1 mg/ml gelatin) at 4 °C. Furthermore, the gel was incubated with zymogram washing buffer (2.5% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl2, 1 µM ZnCl2, pH 7.5), followed by incubation with zymogram incubation buffer (1% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl2, 1 µM ZnCl2, pH 7.5) at 37 °C for 16 h. The gel was then stained with 0.25% Coomassie Blue for 6 h and destained in 7% (v/v) acetic acid and 40% (v/v) methanol solution. The gelatinolytic activity of MMPs appeared as clear bands against the blue background. The original, full-length zymogram images are provided in Supplementary Material. The cropped region of the zymogram shown in respective figures is indicated by red dashed line.

Real-time RT‒PCR

RNA (1 µg) (isolated from TRIzol reagent) was reverse transcribed to synthesize complementary DNA by using a RevertAid first-strand cDNA kit (Thermo Scientific). Real-time PCR was performed using SYBR® Premix Ex Taq™ II (Takara Bio, Inc.) on a Step one (Applied Biosystems) using the primers (Genei, Banglore) (Supplementary Table S1).

Colony formation assay

MCF-7, MDA-MB-231 and 4T1 breast cancer cells (500 cells/well) were seeded, and drug-free conditioned medium from control and 2ME2-treated macrophages was collected and added to the attached breast cancer cells (50% v/v). RPMI medium with vehicle was used as a control. Colonies were allowed to develop for 5 days, fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet solution. The resultant colonies were counted in at least three random fields under an inverted microscope [35].

Scratch wound healing assay

MDA-MB-231, MCF-7 and 4T1 breast cancer cells (0.5 ml of 3 × 105 cells/ml) were seeded in 24-well plates for 24 h. The resultant monolayer of cells was mechanically scratched using a sterile 20 µl pipette tip, followed by PBS washes. The cells were then incubated with DMEM containing RPMI or conditioned medium (50% v/v) from vehicle-treated, 2ME2-treated (5 µM and 20 µM), and 20 µM WP1066-treated M2 macrophages. Wound images were captured under an inverted microscope at 0 and 24 h. Wound distances were measured using ImageJ software, and the percent wound closed was calculated [32].

Transwell assay

For the Transwell assay, MCF-7, MDA-MB-231 or 4T1 breast cancer cells were serum-starved by incubating in FBS-free medium and then seeded in the Transwell chamber (24-well, 8 μm pore size). RPMI medium (with 10% FBS) or the indicated CM was added to the lower chambers. After incubation for 24 h, the cells on the top of the upper chamber were gently scraped off (non-migrated cells), and the inserts were fixed with methanol, followed by staining with 0.1% Coomassie blue solution. The number of migrated cells was counted in at least three random fields.

Apoptosis assay

THP-1 cells were differentiated with PMA and polarized to M2 with 2ME2 for 24 h. Cells were stained with an Annexin V-FITC/PI apoptosis kit (Invitrogen), and the manufacturer’s protocol was followed. An Accuri C6 flow cytometer (BD Biosciences) was used for analysis, and data were analyzed by BD Accuri C6 Software.

Flow cytometry

THP-1 macrophages were harvested and washed with PBS. Fc receptors were blocked by incubating cells with an Fc blocker (Sigma Aldrich, USA) for 15 min. Cells were incubated with a mouse monoclonal CD163 antibody (1:500) or isotype control antibodies for 2 h. Cells were further incubated with Alexa Fluor 488-labeled secondary antibodies for 1 h in the dark. The analysis was performed using BD Aria III. Analysis was performed using BD-FACS Diva software, version 8.8.4.

ELISA assay

THP-1 macrophages were cultured in 6-well plates, polarized in presence or absence of 2ME2. The supernatants derived from the cell culture medium were collected for ELISA experiments. The amounts of MMP-9, TGF-β, VEGF-A, and interleukin-10 (IL-10) in the supernatant were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits obtained from Thermo-Fisher Scientific (BMS249-4) and Krishgen BioSystems (KBH0936, KBH0050, KB1072). The methods provided by the manufacturer were followed for each respective assay. Subsequently, absorbance was measured at a wavelength of 450 nm.

Animal experiments

Female BALB/c mice were purchased from the Rodent Research Center (Jind, Haryana, India). All experiments were conducted strictly as per guidelines by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The experiments described were approved by the Institutional Animal Ethical Committee, Amity University, Uttar Pradesh, India (Approval No: CPCSEA/IAEC/AIP/2022/12/22). Briefly, 4T1 cells (4 × 105 cells in 100 µl ice-cold PBS) were injected into the mammary fat pad of 4- to 6-week-old mice. After 7 days, when the tumors became palpable (4–5 mm diameter), the mice were randomly divided into control and treatment groups (n = 5 in each group). Doses of 75 or 125 mg/kg 2ME2 were administered intraperitoneally every day for 2.5 weeks [36, 37]. The control group received an equal volume of ethanol as vehicle. Tumor sizes were measured regularly using calipers, and tumor volumes were calculated as width2 × length × 0.52. At the end of the dosing regimen, animals were sacrificed by carbon dioxide asphyxiation with secondary cervical dislocation and tumor mass was measured. The tumor sections were stored in 4% paraformaldehyde and TRIzol for immunohistochemistry staining and RNA isolation, respectively. Lungs from mice were harvested and fixed in Bouin’s solution. Histological analysis of lungs was performed by staining with hematoxylin and eosin (H&E), and the sections were further observed under a microscope to determine nodule formation.

Immunohistochemistry

The tumor tissues were formalin fixed, embedded in paraffin, and sectioned into 3 μm sections followed by staining with primary antibodies against CD163 and CD86. Briefly, the sections were deparaffinized and rehydrated by ethanol. Antigen retrieval was performed by immersing and heating the samples in 10 mM sodium citrate buffer for antigen retrieval. The sections were incubated with 3% hydrogen peroxide for 15 min. The sections were incubated with blocking solution and incubated with primary antibody against CD163 and CD86 overnight. Subsequently, incubation was performed with HRP-conjugated secondary antibodies, and antigen detection was performed by DAB staining and hematoxylin was used for counterstaining. The sections were further observed under a microscope.

Statistical analysis

Statistical analysis was performed with student's t-tests and one-way ANOVA using GraphPad Prism software, Inc., version 8.2.1. Data are expressed as the mean ± SD (standard deviation).

Results

Polarization of THP-1 cells into alternatively activated M2 macrophages is associated with reorganization of the microtubule cytoskeleton

Initially, we differentiated THP-1 monocytes into M0 macrophages using 100 nM PMA [38, 39] and co-cultured THP-1 derived M0 macrophages with conditioned medium (CM) from MCF-7 cells. Consistent with previous reports [40,41,42], our results showed a significant increase in M2 macrophage markers (CD206, CD209, CD163, TGF-β, VEGF-A, CCL18). However, we did not observe significant increase in the expression of the M1 macrophage markers (CD80, CD86, iNOS, TNF-α) upon co-culturing of M0 macrophages with breast cancer cell CM (Supplementary Fig. S1a, b). Furthermore, IHC staining in 4T1-induced breast tumors from mice also showed more pronounced staining for CD163+ cells as compared to CD80+ cells, thus confirming a predominant M2 macrophage population in breast cancer (Supplementary Fig. S1c). We then polarized M0 macrophages to the alternatively activated M2 phenotype by incubation with IL-4 and IL-13 (20 ng/ml each) [38]. Immunofluorescence analysis confirmed strong expression of CD68 (a classical pan macrophage marker) on the surface of the differentiated cells compared to THP-1 monocytes (Supplementary data, Fig. S2a). Furthermore, the quantification of immunofluorescence images showed that CD68 was expressed in 79.66 ± 3.21% and 81.3 ± 3.78% of M0 and M2 macrophages, respectively, compared with 46.3 ± 5.67% of monocytes (Supplementary data, Fig. S2b). Quantitative RT‒PCR confirmed that CD68 transcripts were significantly upregulated in M0 and M2 macrophages compared with monocytes (Supplementary data, Fig. S2c). Next, we determined the expression of a panel of cytokines and growth factors associated with the M2 polarized phenotype, including the surface markers CD206 (mannose receptor C-type 1) and CD163 (a scavenger receptor). Immunofluorescence analysis showed increased expression of CD206 in M2 macrophages (78.6 ± 4.04%) compared to that in M0 macrophages (29 ± 2%) (Supplementary data, Fig. S2d, e). Furthermore qRT‒PCR results showed an increase in the expression of both CD206 and CD163 in M2-polarized macrophages in comparison to M0 macrophages (Supplementary data, Fig. S2f, g). Flow cytometry also confirmed increased expression of CD163 in M2 macrophages compared with that in M0 macrophages (Supplementary data, Fig. S2h). M2 TAMs modulate the tumor microenvironment for tumor growth and metastasis by secreting various tumorigenic factors, including proteases, cytokines, and chemokines, such as matrix metalloproteases (MMPs), VEGF-A, IL-10, and TGF-β [6, 42]. As expected, qRT‒PCR analysis showed an increase in the cytokines CCL18, CCL22, TGF-β, MMP9, MMP2, fibronectin (FNT), IL-10, and arginase in M2 macrophages compared to M0 macrophages (Supplementary data, Fig. S2i). Gelatin zymography showed an increase in the activity of secreted MMP9 in M2 macrophages compared to M0 macrophages (Supplementary data, Fig. S2j). These results suggest that THP-1 cells were polarized into M2 macrophages, which exhibit intrinsic traits of tumor-associated macrophages.

Reportedly, LPS-mediated classical activation of macrophages is associated with an increase in acetylated microtubules in the cytoplasm [43]. Hence, we analyzed the status of microtubules in M0 and M2 macrophages. Our immunofluorescence data demonstrated that M0 and M2 macrophages exhibit a well-developed and spread network of microtubule filaments (Fig. 1a), with a microtubule network more elaborately organized in M2 macrophages. However, monocytes, which are mainly spherical, exhibit peripheral α-tubulin staining with essentially no microtubule network. We also examined the acetylation level of microtubules (Lys 40 of α-tubulin) as a marker of microtubule stability [44]. Interestingly, we found that acetylation of α-tubulin at lysine 40 residue was significantly higher in M2 macrophages than in resting M0 macrophages (Fig. 1b, c).

Fig. 1
figure 1

Microtubule network of macrophages changes with their activation to the M2 phenotype (a) THP-1 monocytes and macrophages (M0 and M2) were immunostained with antibodies against acetylated α-tubulin, α-tubulin and DAPI. Scale bar: 50 μm. b Western blotting of α-tubulin and acetylated tubulin in M0 and M2 macrophages. c Acetylated α-tubulin and total α-tubulin were measured by densitometry using ImageJ software. The results are expressed as the mean ± SD, n = 3. **p < 0.01

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2-Methoxyestradiol (2ME2) suppresses M2 polarization of macrophages

To elucidate the effect of 2ME2 on M2 macrophages, we first evaluated the effect of 2ME2 on cell viability. The results of the MTT assay showed that 2ME2 inhibited the viability of M2 TAMs in a concentration-dependent manner. The cell viability was not discernibly affected up to a concentration of 20 µM 2ME2, while cell viability was significantly reduced at higher concentrations (50 and 100 µM) (Fig. 2a). 2-Methoxyestradiol showed a similar concentration-dependent effect on the viability of M0 and M1 macrophages (Supplementary Fig. S3a, b). Annexin-V/PI staining confirmed that 2ME2 treatment did not induce significant cell death of M2 macrophages at low concentrations (Fig. 2b, c) as the percentage of alive M2 macrophage cells were not found to be significantly different in vehicle (81.25 ± 2.1%) and 5 µM (78.28 ± 2.74%, p = 0.248) or 20 µM (72.87 ± 1.8%, p = 0.14) 2ME2 treated samples. Subsequent experiments were carried out with 5 and 20 µM 2ME2 to determine its cell death-independent effects.

Fig. 2
figure 2

Effect of 2ME2 on M2 macrophages: a Bar graph representing the effect of 2ME2 on the viability of M2 macrophages. b Annexin V/PI assay shows the effect of 2ME2 on apoptosis in M2 macrophages. c Bar graph representing the effect of 2-methoxyestradiol (5 µM and 20 µM) on apoptosis in M2 macrophages (d) Immunostaining images of M2 macrophages treated with vehicle or 2-methoxyestradiol (5 µM and 20 µM). Scale bar: 50 μm. e Western blotting of acetylated tubulin in vehicle- and 2ME2 (5 µM and 20 µM)-treated M2 macrophages; alpha tubulin was used as a loading control. f Acetylated α-tubulin and total α-tubulin were measured by densitometry using ImageJ software. g Western blotting of HIF-1α in vehicle- and 2ME2 (5 µM and 20 µM)-treated M2 macrophages; α-tubulin was used as a loading control. h HIF-1α and total α-tubulin were measured by densitometry using ImageJ software. The results are expressed as the mean ± SD, n = 3. **p < 0.01

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Since 2ME2 is a well-known microtubule-depolymerizing agent, we analyzed the status of microtubules in 2ME2-treated M2 TAMs. Our data revealed that 5 µM 2ME2 partially depolymerized the microtubule network, whereas 20 µM 2ME2 led to a complete disruption of the microtubule network, concomitantly reducing microtubule acetylation (Fig. 2d). Similarly, Western blotting results also showed a decrease in microtubule acetylation after treatment with 5 and 20 µM 2ME2 (Fig. 2e, f). We also examined the effect of 2ME2 on HIF-1α in M2 macrophages [45]. As shown, Western blotting results showed a decrease in the expression of HIF-1α in 2ME2-treated M2 macrophages (Fig. 2g, h). Taken together, our results indicated that 2ME2 is taken up by THP1 cells and that even non-cytotoxic concentrations of 2ME2 affected its well-known cellular targets (α-tubulin and HIF-1α) in M2 TAMs.

Next, we investigated the effect of 2ME2 on the polarization of M2 macrophages. As shown by qPCR analysis, the expression of the M2 surface markers CD206, CD209, and CD163 decreased after treatment with 2ME2 (Fig. 3a). In line with the gene expression data, flow cytometry analysis also demonstrated a decrease in the expression of the M2 macrophage surface marker CD163 in 2ME2-treated macrophages (Fig. 3b). 2ME2 showed a decrease in expression of CD68 (pan macrophage marker), however, at these concentrations no significant effect was observed on M1 macrophage markers (Supplementary Fig. S4a, b). The data showed that 2ME2 notably reduced the expression of typical macrophage surface markers associated with the M2 phenotype.

Fig. 3
figure 3

2-Methoxyestradiol inhibits M2 polarization of macrophages: a mRNA expression of CD206, CD209 and CD163 in control and 2ME2 (5 µM and 20 µM)-treated macrophages. b Flow cytometry shows the expression of the CD163 surface marker in control M2 and 2ME2-treated macrophages and bar graph representing the percentage of CD163 + cells in control M2 and 2ME2-treated macrophages (c) mRNA expression levels of cytokines, including CCL18, TGF-β, MMP9, IL-10, fibronectin (FNT), arginase and CXCL12, were analyzed. d Western blotting of MMP9 in the whole cell lysate of control and 2ME2 (5 µM and 20 µM)-treated macrophages and densitometry analysis of Western blots of MMP9. e Western blotting of VEGF-A in whole cell lysates of control and 2ME2 (5 µM and 20 µM)-treated macrophages and densitometry analysis of Western blots of VEGF-A. f Concentration of TGF-β, IL-10, VEGF-A, and MMP9 in cell culture supernatant of M2 macrophages and 2ME2 (5 µM and 20 µM)-treated M2 macrophages was determined by ELISA. g Gelatin zymography of conditioned medium of M2 macrophages and 2ME2 (5 µM and 20 µM)-treated macrophages. Gelatinolytic activity of purified recombinant MMP9 (R-MMP9) is shown. The results are expressed as the mean ± SD, n = 3. *p < 0.05, **p < 0.01 and ***p < 0.001

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We then hypothesized that 2ME2-mediated suppression of M2 polarization may reduce the abundance of pro-tumoral factors in the conditioned medium (CM) from M2 TAMs. Our results also showed a decrease in the mRNA expression of anti-inflammatory factors, including CCL18, TGF-β, MMP9, interleukin-10 (IL-10), fibronectin (FNT), arginase and CXCL12 (Fig. 3c). Furthermore, Western blotting revealed reduced expression of MMP9 and VEGF-A, a key angiogenic molecule secreted by TAMs, in 2ME2-treated M2 macrophages (Fig. 3d, e). ELISA showed that the concentration of TGFβ, IL-10, VEGF-A, and MMP-9 decreased significantly in the cell culture media of M2 macrophages upon 2ME2 treatment (Fig. 3f). In accordance, gelatin zymography of the CM also showed a concentration-dependent decrease in MMP9 activity in 2ME2-treated M2 macrophages (Fig. 3g). In addition, we polarized murine RAW 264.7 cells to M2 macrophages in absence or presence of sub-cytotoxic concentrations of 2ME2. Our results showed a decrease in CCL22, CCL2, VEGF-A, IL-10 and FNT1 in RAW 264.7 cells indicating a reduction in M2 cytokines when polarized along with 2ME2 (Supplementary Fig. S5).

2-Methoxyestradiol impairs the protumor functions of alternatively activated M2 macrophages in breast cancer cells

We next hypothesized that, in addition to M2 macrophage polarization, 2ME2 could impair the pro-tumoral functions of M2 macrophages. To examine this, MDA-MB-231 and MCF-7 cells were separately incubated with drug-free conditioned medium (CM) from 2ME2-treated M2 macrophages. THP-1 cells were polarized to the M2 phenotype in the presence of either vehicle or 2ME2 (5 and 20 µM) for 24 h, 2ME2-containing medium was removed, and the cells were cultured in FBS-free RPMI medium for 6 h. As shown in Fig. 4a-c, M2 macrophage CM resulted in increased proliferation of breast cancer cells, which was markedly reduced upon incubation with 2ME2-treated CM. We then determined the effect of CM from M2 macrophages on the migration of breast cancer cells. The percent wound closure at 24 h in scratch-wounded MDA-MB-231 cells decreased by 31.29 ± 1.18% (p < 0.0001) and 40.03 ± 3.74% (p < 0.001) and percent wound closure in MCF-7 cells decreased by 40.93 ± 5.17% (p < 0.01) and 48.95 ± 1.63 (p < 0.001) when cells were grown in CM from M2 macrophages treated with 5 µM and 20 µM 2ME2, respectively, compared to CM from vehicle-treated M2 macrophages (Fig. 4d-f). Moreover, the transwell assay also showed that 2ME2 significantly reduced the migration-inducing ability of M2 macrophages in both MDA-MB-231 and MCF-7 cells (Fig. 4g-i). Furthermore, gelatin zymography results showed that conditioned medium from control M2 macrophages stimulated the secretion of MMP9 from MCF-7 cells. A drastic reduction in secreted MMP9 was observed in MCF-7 cells induced by M2 macrophages polarized with 2ME2 in comparison with control M2 macrophages (Fig. 4j). Similarly, we found that the conditioned medium of RAW 264.7 macrophages polarized in presence of 2ME2 showed a significant reduction in the proliferative and migration ability of 4T1 cells (Supplementary Fig. S6) as compared to CM from untreated RAW264.7 M2 macrophages. Overall, these observations showed that treatment with 2ME2 impaired the pro-tumoral functions of M2 macrophages.

Fig. 4
figure 4

2-Methoxyestradiol impairs the protumor functions of alternatively activated M2 macrophages in breast cancer cells. a Representative images of the colony formation assay in MDA-MB-231 and MCF-7 cells after incubation with RPMI medium (control), vehicle-treated CM and 2ME2-treated CM. b, c The number of colonies per field was counted and is presented as the mean ± SD. d Wound healing assay showing migration in MDA-MB-231 and MCF-7 cells after incubation with RPMI medium (control), vehicle-treated CM and 2ME2-treated CM. Scale bar: 150 μm. e, f Histogram representing the percentage of wound closure after 24 h. g MDA-MB-231 and MCF-7 cells were seeded in the upper chamber of a Transwell, and RPMI medium (control), vehicle-treated CM and 2ME2-treated CM were kept in the lower well of a 24-well plate. The cells that migrated to the other side of the Transwells after 24 h were collected, stained, and counted. h, i The cell number per field was counted under an inverted microscope and is presented as number of migrated cells. j MCF-7 cells were incubated with RPMI medium (control), vehicle treated CM and 2ME2 treated CM for 24 h. The medium was then replaced with FBS-free DMEM for 6 h, which was collected to perform Gelatin Zymography. The results are expressed as the mean ± SD, n = 3. **p < 0.01, ***p < 0.001, ****p < 0.0001 

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2ME2 decreased M2 macrophages and inhibited breast tumor metastasis in vivo

To confirm the effects of 2ME2 on tumor-associated macrophages and breast cancer metastasis in vivo, we developed a 4T1 syngeneic breast tumor model in immunocompetent BALB/c mice and administered vehicle or 75 or 125 mg/kg 2ME2 to these mice. The tumors were excised after 25 days. Immunohistochemistry analysis showed reduced expression of the M2 macrophage marker CD163 in tumors isolated from 2ME2-treated mice (Fig. 5a). Concomitantly, in comparison with vehicle-treated mice, 2ME2-treated mouse tumors showed a significant decrease in the mRNA expression of CD206, CD209 and CD163 as well as the cytokines and growth factors, including VEGF-A, CCL22, FNT1 and IL-10 (Fig. 5b, c).

Fig. 5
figure 5

2ME2 decreased M2 macrophages and inhibited breast tumor metastasis in vivo. a IHC staining was performed on vehicle- and 2ME2-treated mouse tumor tissue sections using CD163 antibody (400X magnification). b mRNA expression level of CD163, CD209 and CD206 in vehicle- and 2ME2-treated mouse tissue. c mRNA expression levels of VEGF-A, CCL22, FNT-1 and IL-10 in vehicle- and 2ME2-treated mouse tissue. d Images of tumors excised from vehicle- and 2ME2-treated mice. e Tumor volumes were measured at various time points. f Weights of the tumors excised at the end were measured. g Body weight of vehicle- and 2ME2-treated mice was measured at various time points. h Representative images of lungs (arrows showing metastatic lung nodules) isolated from vehicle- and 2ME2-treated mice. i Bar graph showing the number of metastatic lung nodules. j H&E staining of lungs in vehicle- and 2ME2-treated mice. Data are presented as the mean ± SD. *, **, *** represent significant differences between samples with p < 0.05, p < 0.01, and p < 0.001, respectively

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Next, we analyzed the effect of 2ME2 on tumor metastasis. Our results showed that 2ME2-treated mice showed reduced tumor volume and tumor weight in comparison with vehicle-treated mice (Fig. 5d-f). However, irrespective of the treatment provided, no significant reduction in mouse body weight was observed (Fig. 5g). Interestingly, our results showed fewer metastatic lung nodules after 2ME2 treatment (Fig. 5h, i) which was confirmed by H&E staining of the lungs sections (Fig. 5j). Together, these results suggest that, consistent with our in vitro results, 2ME2 inhibits M2 macrophage polarization, and metastasis to the lungs in vivo.

2-Methoxyestradiol inhibits STAT3 in alternatively activated M2 macrophages

To further investigate the mechanism by which 2ME2 dampens the polarization and pro-tumoral functions of M2 macrophages, we determined its effect on STAT3 signaling, which is implicated in the polarization and functions of M2 macrophages. Compared to that in M0 macrophages, the level of p-STAT3 (pY705) was found to be nearly two-fold higher in M2 TAMs (Fig. 6a, b). Immunofluorescence imaging further showed that M0 macrophages had a uniform punctate distribution of STAT3 in the cytoplasm, while in M2 macrophages, activated STAT3 accumulated in the nucleus (Fig. 6c, d). After analyzing the kinetics of activated STAT3 nuclear translocation, our results revealed that almost all M2 cells exhibited accumulation of pSTAT3 in the nucleus within 2 h of IL-4/13 polarization (data not shown). Western blotting showed that 2ME2 decreased pSTAT3 in M2 TAMs (Fig. 6e, f). Consistently, western blotting results showed decrease in pSTAT3 in tumor sections of mice treated with 2ME2 (Fig. 6g, h). Furthermore, immunofluorescence images showed diffuse localization of STAT3 primarily in the cytoplasm of M2 macrophages polarized with 2ME2. The inhibitory effect of 2ME2 on the phosphorylation and nuclear transfer of pSTAT3 was diminished in the presence of colivelin, a STAT3 activator (0.5 µM) (Fig. 6i, j) [46]. Western blotting also showed the inhibition of the nuclear translocation of STAT3 after 2ME2 treatment, which was partly reversed by colivelin (Fig. 6k-m). The data show that 2ME2 inhibits the phosphorylation and nuclear translocation of STAT3 in M2 TAMs, suggesting that the functional impairment of TAMs could be mediated by the inhibitory effect of 2ME2 on STAT3 signaling.

Fig. 6
figure 6

2-Methoxyestradiol inhibits STAT3 in alternatively activated M2 macrophages. a Western blot of pSTAT3 and STAT3 in M0 and M2 macrophages, respectively. b The densitometry analysis of Western blot of pSTAT3 and STAT3 is shown in the graph. c Immunostaining of STAT3 in M0 and M2 macrophages showing nuclear translocation of STAT3. Scale bar: 50 μm (d) Histogram representing the percentage of cells with nuclear accumulation of STAT3 in M0 macrophages and M2 macrophages. e Western blot of pSTAT3 and STAT3 in control and 2ME2 (5 µM and 20 µM)-treated M2 macrophages. f Densitometry analysis of Western blots of pSTAT3 and STAT3 is shown in the graph. g Western blot of pSTAT3 and STAT3 in tumors isolated from mice treated with vehicle and 2ME2 (75 mg/kg and 125 mg/kg) (h) Densitometry analysis of Western blots of pSTAT3 and STAT3 is shown in the graph (i) Immunostaining of STAT3 in control, 2ME2 (5 µM and 20 µM) and colivelin-treated macrophages. Scale bar: 50 μm. j Bar graph shows the percentage of cells with accumulation of STAT3 in the nucleus. Total cells were counted using DAPI staining. k-l Western blotting of STAT3 in (k) cytoplasmic and (l) nuclear fractions of M2 macrophages. m Bar graph showing densitometric analysis of Western blotting. The results are expressed as the mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

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To confirm that STAT3 is essential for M2 functions and that its pharmacological inhibition can impair their functions and target M2 TAMs, we polarized TAMs in the presence of WP1066, a specific inhibitor of STAT3 phosphorylation in clinical trials, and examined the polarization and pro-tumoral functions of M2 TAMs. Using the MTT assay, we first determined the sub-cytotoxic concentrations of WP1066. We then examined whether inhibition of STAT3 activation could indeed inhibit the polarization and protumor functions of M2 macrophages. Gene expression analysis by qPCR revealed significantly decreased expression of CD206 and CD163 after treatment with sub-cytotoxic concentrations of WP1066 (Supplementary data, Fig. S7a, b). Flow cytometry analysis also showed a decrease in the CD163+ cell population after treatment with WP1066, suggesting that STAT3 inhibition affected M2 polarization (Supplementary data, Fig. S7c). Similarly, we found reduced expression of M2 macrophage-secreted cytokines, including FNT-1, MMP9, VEGF-A, IL-10 and TGF-β, in WP1066-treated macrophages (Supplementary data, Fig. S7d). Gelatin zymography also showed decreased gelatinolytic activity of MMP9 in conditioned medium from WP1066-treated M2 macrophages (Supplementary data, Fig. S7e). We found that WP1066-mediated inhibition of STAT3 also reduced the migration-augmenting effects of M2 macrophages in breast cancer cells (Supplementary data, Fig. S7f-h). Overall, these results show that STAT3 activity is required for M2 polarization and pro-tumoral functions and that 2ME2 dampens the functions of STAT3 in M2 macrophages and phenocopies the effects of STAT3 inhibition.

Discussion

As the understanding of the tumor microenvironment has advanced, it has become evident that TAMs are critical in growth, angiogenesis, and immune escape in tumors. In stark contrast to the proinflammatory “classically activated” M1 macrophages, which are considered antitumor in nature, the “alternatively activated” M2 macrophages secrete cytokines such as TGF-β, IL-10, CCL18, fibronectin, MMP2, MMP9 and CCL22, which promote tumor angiogenesis and metastasis. Macrophage-secreted TGF-β is known to promote epithelial-mesenchymal transition (EMT), while CCL18, an important chemokine secreted by M2 TAMs, stimulates tumor vascularization in breast cancer [10, 11]. Proangiogenic factors secreted by M2 TAMs induce the formation of hypo-perfused abnormal blood vessels, which are also responsible for limiting the delivery of drugs to the tumor, thus contributing to chemoresistance. An increased infiltration of macrophages (CD68+ macrophages) is an indicator of reduced overall survival (OS) and reduced relapse-free survival (RFS) in breast cancer patients [47, 48]. In this study, we found that, as expected, IL-4 and IL-13 induced macrophages to differentiate into M2 TAMs (as revealed by the high expression of well-known M2 TAM-specific markers) and promoted the expression of anti-inflammatory cytokines (Supplementary Fig. 2). Furthermore, incubation with the conditioned medium of M2 TAMs increased the proliferation and migration of breast cancer cells. These findings suggest that M2 TAMs are crucial for the enhanced tumorigenesis of breast cancer cells. Interestingly, TAM-targeting therapies are gaining significant attention as anticancer strategies. In this regard, we focused on 2-methoxyestradiol and its effect on the polarization and functioning of M2 TAMs.

2ME2 inhibits mitosis in cancer cells by binding to tubulin at the colchicine binding site, affecting microtubule assembly dynamics and mitotic spindle assembly, and thus interfering with cell cycle progression [18]. Recently, a study has identified the inhibitory effect of 2ME2 on nNOS and heat shock protein through DNA damage in glioblastoma cells [49]. Also, Zhang et al. (2023) have identified new perspective on effects of 2ME2 in progression of non-small cell lung cancer through inhibition of circ_0010235/miR-34a-5p/NFAT5 axis [50]. The effects of 2ME2 have been extensively studied in the preclinical and clinical setting in multiple tumor models, including liver, prostate, multiple myeloma, and breast cancer [19,20,21,22,23]. However, the effect of 2ME2 on stromal cells in breast cancer has not been well explored. Here, we show that 2ME2 suppresses the polarization of macrophages to the M2 phenotype. Treatment of monocytes with 2ME2 decreased the expression of CD206, CD209, and CD163 and reduced the percentage of CD206-expressing cells, indicating that it represses the polarization of TAMs. Furthermore, this was confirmed by in vivo experiments, and IHC of breast tumor sections showed marked reduction in the staining of CD163 upon treatment with 2ME2.

Several reports suggested that due to its anti-inflammatory properties, 2ME2 has a beneficial role in autoimmune conditions, including rheumatoid arthritis and autoimmune encephalomyelitis [51,52,53]. In a recent study, 2ME2 was found to ameliorate high-fat diet-induced obesity by increasing M2 phenotypic macrophages [53]. However, another study showed that 2ME2 inhibited the infiltration and M2 activation of macrophages (as evidenced by a reduction in Ym1, an M2 macrophage-specific marker, in CCl4-induced liver fibrosis [54]. Similarly, 2ME2 treatment in melanoma was noticed to increase the infiltration of CD8+ T cells. Interestingly, this study observed that 2ME2 resulted in the synergistic anticancer efficacy of PD-1 blockade immunotherapy [28], which is well known to be suppressed and inhibited by M2 TAMs in the tumor microenvironment. These studies highlight that the therapeutic effects of 2ME2 on immune modulation are very disease specific and warrant thorough investigation.

In this context, ours is the first study to show that 2ME2 can inhibit M2 TAMs in breast cancer. In addition, treatment with 2ME2 reduced pro-tumoral cytokines and growth factors, including MMP9, VEGF-A, CCL18, TGF-β, arginase, CXCL12, IL-10 and fibronectin (Fig. 3). Gelatin zymography, Western blotting, and quantitative RT‒PCR revealed that TAM-derived proangiogenic and pro-metastatic factors were significantly reduced by 2ME2, suggesting that 2ME2-mediated suppression of TAM polarization alters their secretome. Consequently, unlike the conditioned medium of vehicle-treated TAMs, the conditioned medium of 2ME2-treated TAMs could not promote the proliferation and migration of breast cancer cells, indicating that treatment with 2ME2 impedes the pro-tumoral functions of TAMs (Figs. 3 and 4). Overall, the data showed that 2ME2 affected M2 polarization and clearly affected tumor metastasis in vivo. Thus, our study suggests that modulation of TAM polarization and inhibition of VEGF-A secretion by TAMs could mediate the well-known antiangiogenic activity of 2ME2.

It is well established that 2-methoxyestradiol, an estradiol metabolite, does not stimulate estrogen receptors and exerts its antiproliferative, anticancer and anti-inflammatory activities independent of estrogen receptors [55]. We next investigated the mechanism through which 2ME2 may lead to functional impairment of M2 macrophages. The functional consequences of M2 macrophages are intricately orchestrated by the activation of STAT3. Several factors released by breast cancer cells, including IL-6 and LIF, activate STAT3 and drive M2 polarization of macrophages in a feed-forward loop-type manner [56]. In addition, JAK2/STAT3 signaling pathway activation promotes an increase in the expression of PD-L1 in macrophages, thus facilitating tumor immune escape and an immunosuppressive environment [57, 58]. Ablating STAT3 in hematopoietic cells has been shown to alter the functional activity of macrophages and can trigger the immune system to inhibit tumor growth and metastasis [59]. Due to its central role in the functional regulation of TAMs, STAT3 signaling is increasingly being recognized as an attractive cellular target for improving conventional, targeted and immunotherapy, and several STAT modulators are being identified to inhibit TAM functions [12, 60]. We also found that inhibition of STAT3 activation by WP1066, a STAT3 inhibitor in clinical trials, diminished M2 macrophage polarization and reduced TAM-derived pro-metastatic factors, thereby affecting TAM functions in breast cancer. Our data show that STAT3 is phosphorylated (pY705) and translocates to the nucleus during M2 polarization in a time-dependent manner (data not shown). Based on previous reports, we hypothesized that 2-methoxyestradiol may suppress the protumoral polarization of tumor-associated M2 macrophages through STAT3 inhibition and found that 2-methoxyestradiol inhibits Y705 phosphorylation and the nuclear translocation of STAT3 in a concentration-dependent manner (Fig. 6). Interestingly, at these concentrations, 2ME2 depolymerizes the microtubules in TAMs as well. It is possible that the activation and nuclear translocation of STAT3 in TAMs might be dependent on microtubules. Walker et al. (2010) identified the interaction between STAT3 and microtubules, and their study showed that microtubule-targeted drugs affect the phosphorylation and signaling of STAT3 in cancer cells [61]. STAT3 is known to interact with microtubules through sequestration of the microtubule depolymerizing protein stathmin [62]. Accordingly, several microtubule inhibitors have been shown to suppress STAT3 activity [63, 64]. STAT3 is also known to modulate HIF-1α and activate HIF-1α target genes, including VEGF-A [65]. Pharmacological inhibition of STAT3 blocks HIF-1α in vitro and inhibits tumor growth in vivo [66]. Together, these studies indicate an intricate relationship between STAT3, microtubules, and HIF-1α.

We hypothesized that 2ME2 may suppress pro-tumoral polarization of tumor-associated M2 macrophages through STAT3 inhibition and found that 2ME2 inhibited Y705 phosphorylation and nuclear translocation of STAT3 without affecting total STAT3 levels. The 2ME2-induced functional impairment of TAMs was similar to that produced by WP1066, which does not affect microtubules but inhibits HIF-1α in macrophages. Previous studies have shown that 2ME2 may disrupt a variety of signaling cascades in various types of cells. 2ME2 triggers p53-dependent apoptosis in breast cancer cells by phosphorylating Bcl-2 and Bxl through activation of P38 and NF-κB as well as activation of JNK and AP-1 [67]. 2ME2 affects ERK and p38 MAPK signaling in breast cancer and impairs the PI3/Akt signaling pathway in gastric cancer to limit gastric cancer metastasis [68, 69]. In a recent report, swept-source optical coherence tomography was used to identify the inhibitory effect of 2ME2 on ovarian multicellular tumor spheroids [70]. In addition to cancer cells, 2ME2 treatment inhibits the proliferation of stromal cells, thus exhibiting immunomodulatory effects [28, 29]. Batth et al. showed that 2ME2 can inhibit macrophage stimulatory protein 1 receptor and RON signaling, thereby inhibiting prostate cancer growth [30]. It has been shown that 2ME2 markedly lowers the expression of chitinase-like molecules, CHI3L3/YM1, another typical alternatively activated M2 marker upregulated by STAT3/6 signaling, and limits CCl4-induced liver fibrosis in mice [54]. In this study, we provide evidence that 2ME2 diminished the phosphorylation and nuclear translocation of STAT3. Abrogation of STAT3 signaling reduced the skewing of TAMs to the M2 phenotype and limited the metastasis-promoting characteristics of TAMs.

Conclusions

Collectively, our results demonstrated that 2ME2 affects the functioning of M2 macrophages, which may contribute to its antiangiogenic and antitumor effects. This may have important considerations in the clinical application of 2ME2 in chemotherapeutics either as a monotherapy or in combination with existing chemotherapies in future. This study provides a basis for further investigation of the immune modulation potential of recently developed derivatives of 2-methoxyestardiol in cancer.

Availability of data and materials

The data used to support the findings of this study are included within the article and the supplementary information. The data and materials in the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors thank the Confocal Microscopy Facility, AIMT and the Department of Science and Technology (DST) FIST sponsored Flow Cytometry Facility (SR/FST/LS-II/2017/115) at AIMMSCR, Amity University, Uttar Pradesh.

Funding

This research was funded by SERB-Department of Science and Technology (project number CRG/2022/008699 and ECR/001173/2016) and Department of Biotechnology (project number BT/PR18562/BIC/101/424/2016), Govt. of India, awarded to SK.

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Authors and Affiliations

  1. Amity Institute of Molecular Medicine and Stem Cell Research, Amity University Uttar Pradesh, Noida, Uttar Pradesh, 201313, India

    Bhawna Deswal, Urmi Bagchi, Manoj Garg & Sonia Kapoor

  2. National Centre for Cell Science Complex, Savitribai Phule Pune University, Campus Ganeshkhind Road, Pune, Maharashtra, 411007, India

    Manas Kumar Santra

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  1. Bhawna Deswal
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  2. Urmi Bagchi
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Contributions

BD conducted experiments, analyzed data, and wrote the manuscript along with SK. UB conducted experiments and analyzed data; MKS analyzed data and helped improve the manuscript; MG analyzed data and reviewed the manuscript, SK designed the concept, acquired funding, supervised the work, analyzed data, and reviewed the manuscript.

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Correspondence to Manoj Garg or Sonia Kapoor.

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Ethics approval and consent to participate

The animal experiments were approved by the Institutional Animal Ethical Committee, Amity University, Uttar Pradesh, India (Approval No: CPCSEA/IAEC/AIP/2022/12/22). All methods of this study were carried out in accordance with the guidelines by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The study is reported in accordance with ARRIVE guidelines.

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Deswal, B., Bagchi, U., Santra, M.K. et al. Inhibition of STAT3 by 2-Methoxyestradiol suppresses M2 polarization and protumoral functions of macrophages in breast cancer. BMC Cancer 24, 1129 (2024). https://doi.org/10.1186/s12885-024-12871-w

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Keywords

BMC Cancer

ISSN: 1471-2407

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