Tumor-associated macrophage derived IL-6 enriches cancer stem cell population and promotes breast tumor progression via Stat-3 pathway

Background Cancer stem cells (CSCs) play crucial role in tumor progression, drug resistance and relapse in various cancers. CSC niche is comprised of various stromal cell types including Tumor-associated macrophages (TAMs). Extrinsic ques derived from these cells help in maintenance of CSC phenotype. TAMs have versatile roles in tumor progression however their function in enrichment of CSC is poorly explored. Methods Mouse macrophages (RAW264.7) cells were activated by interaction with conditioned media (CM) of murine breast cancer cells (4T1) into TAMs and the effect of activated macrophage (TAM) derived factors was examined on enrichment of cancer stem cells (CSCs) and tumor growth using in vitro and in vivo models. Results In this study, we report that macrophages upon interaction with breast cancer cells activate tumor promoting function and exhibit differential expression of various proteins as shown by secretome analysis using proteomics studies. Based on secretome data, we found that Interleukin-6 (IL-6) is one of the up-regulated genes expressed in activated macrophages. Further, we confirm that TAMs produce high levels of IL-6 and breast cancer cell derived factors induce IL-6 production in activated macrophages via p38-MAPK pathway. Furthermore, we demonstrate that tumor activated macrophages induce enrichment of CSCs and expression of CSC specific transcription factors such as Sox-2, Oct-3/4 and Nanog in breast cancer cells. We further prove that TAM derived IL-6 plays a key role in TAM mediated CSC enrichment through activation of Signal transducer and activator of transcription 3 (STAT-3) signaling. TAM derived IL-6 influences breast cancer cell migration and angiogenesis. Moreover, our in vivo findings indicated that TAM derived IL-6 induces CSC population and resulting tumor growth in breast cancer. Conclusion These finding provide evidence that TAM derived IL-6 plays a major role in CSC enrichment and tumor progression in breast cancer and IL-6 and its regulated signalling network may act as potential therapeutic target for management of breast cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-022-02527-9.


Secretomic analysis
To examine the status of cytokine profile in macrophages in response to 4T1 cells, quantitative secretomics was performed using label free conditions. In brief, RAW264.7 cells were either cultured alone or treated with CM of 4T1 for 12 h and conditioned media were collected. The concentrated conditioned media were further replaced using 0.5 M Tri ethyl ammonium bicarbonate (TEAB) by performing centrifugation at 16000 rpm at 15 o C. Further, secretome protein estimation was performed using 2D quantification kit (Thermofisher) and label free quantification was performed for secretome analysis.
Fifty micro gram protein was used either from control or treated sample for enzymatic protein digestion. Briefly, control and treatment samples were first reduced using 10 mM DTT, followed by 50 mM Iodoacetamide (IAA) alkylation and samples were digested by trypsin (1:50; enzyme: protein). Further the peptides were desalted using zip-tips and suspended in 0.1% formic acid (Sigma Aldrich, USA) containing LC-MS grade water (JT Baker, USA). For LFQ data acquisition, Orbitrap fusion mass spectrometer (ThermoScientific™, USA) coupled with EASY-nLC 1200 nanoflow liquid chromatography system (ThermoScientific™, USA) equipped with EASY-Spray column (50 cm × 75 µm ID, PepMap C18) was used. One microgram of desalted tryptic peptide samples with three biological replicates were used from each sample and injected to Orbitrap Fusion mass spectrometer. Further, peptides were separated using 5 to 95% phase B (0.1% formic acid in 80% acetonitrile) and 300 nL/min flow rate for 140 min through gradient process. MS scan analysis was performed by analysing MS1 spectrum within 375-1500 m/z mass range at 60,000 resolution using Orbitrap analyser with 50 ms injection time. Further the identified MS1 precursors had undergone fragmentation by high energy collision-induced dissociation (HCD) and analysed by Orbitrap mass analyser.
Further proteins were identified and quantified from the mass spectrometry data using proteome Discoverer software (PD) (version 2.2, Thermo Scientific) by using Sequest HT database. Database search includes all entries from the Mus musculus UniProt reference proteome database. Fold change values were obtained based on the mean area from all triplicate samples.

Isolation of TAMs from 4T1 breast tumors
To isolate TAMs from tumors, 4T1 cells (2x10 5 ) were implanted into the mammary fat pads of female BALB/c mice and tumors were allowed to grow up to standard size. Mice were

Flow cytometry
For FACS analysis, RAW264.7 cells were treated with either CM of 4T1 or pre-treated with 10 µM of SB203580 for 1 h, followed by treatment with CM of 4T1 for 24 h and further treated with Brefaldin for 4 h and levels of expression of IL-6 were analysed. For intracellular staining of IL-6, RAW264.7 cells were scraped after treatments, suspended in 1X PBS and centrifuged at 1000 rpm for 10 mins. Cells were fixed using 2% Formaldehyde for 20 mins in ice followed by permeabilization with chilled methanol for 30 mins at 4 °C. Cells were washed with 1X PBS until methanol was completely removed followed by suspension in specific antibody. In separate studies, RAW264.7 cells were co-cultured with CM of 4T1 for 24 h and CD206 expression was analysed. In brief, cells were trypsinized and re-suspended in 100 µl of 1X PBS containing 3% FBS and anti-CD206 or anti-IL-6 antibody was added, incubated for 45 mins on ice in dark along with their respective matched unstained control and then washed twice with 1X PBS. Stained cells were analysed on FACSCanto (BD-Biosciences) and data was analysed using FACS DIVA software (Version 6.1.3).
To study the role of TAM derived IL-6 in enrichment of CSCs through STAT-3 pathway in breast cancer cells, 4T1 cells were treated with either CM of activated macrophages or IL-6 neutralized CM of activated macrophages or pre-treated with Stattic for 3 h followed by treatment with CM of activated macrophages for 24 h and processed for FACS analysis. In brief, cells were trypsinized and re-suspended in 100 µl of 1X PBS containing 3% FBS and anti-Sca-1 antibody was added, incubated for 45 mins along with their respective matched unstained control and then washed twice with 1X PBS. Stained cells were examined on FACSCanto (BD-Biosciences) and data was analysed using FACS DIVA software (Version 6.1.3).

Chromatin immunoprecipitation (ChIP) assay
The IL-6 promoter sequence and putative AP-1 binding site was obtained from The Eukaryotic Promoter Database (EPD) and primers were designed for binding site (Table S2).
The untreated or 4T1 CM-treated RAW264.7 cells were fixed with 0.75% formaldehyde for 10 min followed by quenching with 125 mM glycine for 5 min at RT. After centrifugation, cell pellets were resuspended in FA lysis buffer followed by sonication. 10% lysate removed for Input. Primary antibody (anti-cJun antibody) was used for immunoprecipitation at 4 o C. The immuno-precipitate was then pulled down with SureBeads Protein G magnetic beads and eluted with elution buffer. The immuno-precipitates were then reverse cross-linked using RNase A and Proteinase K and purified. Semi-quantitative PCR was used to quantify DNA levels of specific loci by analyzing PCR end-product using agarose gel electrophoresis.

Aldefluor assay
4T1 single-cell suspensions were used for Aldefluor assay (Stem Cell Technologies) as per the manufacturer's directions. In brief, 4T1 cells (1x10 6) were re-suspended in 1 ml Aldefluor buffer containing activated Aldefluor substrate. As a negative control, for each sample ALDH inhibitor, DEAB (diethylaminobenzaldehyde) was used. Stained cells were examined on FACSCanto (BD-Biosciences) and data was analysed using FACS DIVA software (Version 6.1.3).

Transwell migration assay:
4T1 cell (1 x 10 4) either untreated or treated with CM of Act RAW264.7 were seeded in the upper chamber. In separate experiments, 4T1 cells were treated with IL-6 neutralized CM of activated macrophages or pre-treated with Stattic for 3 h followed by CM of activated macrophages seeded in the upper chamber. 10% FBS was used in lower chambers as a chemoattractant. The migrated cells were stained with crystal violet, counted in five highpower fields (hpfs) and analysed.

Wound healing assay
The wound healing assay was performed using 4T1 cells under different experimental conditions. Briefly, 4T1 cells were grown in monolayer until formed cobblestone morphology.
Uniform sized wounds were made using sterile tip and cells were co-cultured with either CM of activated macrophages or IL-6 neutralized CM of activated macrophages or 4T1 cells pretreated with Stattic for 3 h followed by CM of activated macrophages. Untreated 4T1 cells were used as control. Photographs of wounds were taken at t = 0 h and t = 18 h using phase contrast microscope (Nikon). Area migrated was analysed by using Image-Pro plus software and displayed in the form of bar graph. Figure   Oct 3/4 and Nanog were analysed by q-PCR. Bar graphs represent relative fold change in IL-6 expression of Sox-2, Oct-3/4 and Nanog respectively. (E-G) 4T1 cells were treated with CM of activated RAW264.7 cells for 24 h and expressions of Sox-2, Oct 3/4 and Nanog were analysed by confocal microscopy. Bar graphs represent corrected total cell fluorescence (CTCF) of Sox-2, Oct-3/4 and Nanog respectively as quantified by ImageJ software, mean ± SEM, *denotes p < 0.05, ***denotes p < 0.001, n=3 independent experiments. Figure S5. Comparison of IL-6 level in 4T1 and Activated RAW conditioned media. Bar graph represents IL-6 concentration in CM of 4T1 cells and CM of Activated RAW cells estimated by ELISA, mean ± SEM, ***denotes p < 0.001, n=3 independent experiments. Figure S6. TAM derived IL-6 enriches CSCs in breast cancer. 4T1 cells were treated with either CM of activated RAW or CM of activated RAW neutralized with IL-6 antibody (20 µg/ml) and co-expression ALDH1 as well as Sca-1 was examined by flow cytometry using their specific antibody. Figure S7. IL-6 alone is sufficient to enhance cancer stem cell phenotype in breast cancer cells. (A, B) 4T1 cells were treated with recombinant IL-6 (50 ng/ml) for 24 h and CSC phenotype was examined by analysing Sca-1 expression and ALDH1 activity using flow cytometry. (C) 4T1 cells were treated with recombinant mouse IL-6 (50 ng/ml) for 24 h and expressions of Sox-2, Oct 3/4 and Nanog were analysed by western blot. (D-F) 4T1 cells were treated with recombinant mouse IL-6 (50 ng/ml) for 24 h and expressions of Sox-2, Oct 3/4 and Nanog were analysed by confocal microscopy. Bar graphs represent corrected total cell fluorescence (CTCF) of Nanog, Oct-3/4 and Sox-2 respectively as quantified by ImageJ software, mean ± SEM, *denotes p < 0.05, **denotes p< 0.01, n=3 independent experiments).

Figure S8. IL-6 enhances stemness in breast cancer through STAT-3 pathways. (A, B)
4T1 cells were treated with Stattic (5 µM), a STAT-3 inhibitor, prior to the treatment with recombinant IL-6 and CSC phenotype was examined by analysing Sca-1 expression and Aldefluor activity using flow cytometry. Figure S9. TAM derived IL-6 augments migration in breast cancer cells. 4T1 cells were grown in confluent monolayer with cobblestone morphology and wounds with constant width were given. Cells were treated with CM of activated RAW264.7 cells or CM neutralised with IL-6 antibody (20 µg/ml). In separate experiments, 4T1 cells were pre-treated with Stattic (5 µM) followed by treatment with CM of activated RAW264.7 cells. Wounds were imaged at 0 h and 16 h and area migrated was analysed using Image-Pro Plus software and represented in the form of bar graph. The error bar represents mean ± SEM, *denotes p < 0.05, n=3 independent experiments.