Bone marrow cells are differentiated into MDSCs by BCC‐Ex through down‐regulating the expression of CXCR4 and activating STAT3 signalling pathway

Abstract Studies showed that the increase of myeloid‐derived suppressor cells (MDSCs) in tumour microenvironment is closely related to the resistant treatment and poor prognosis of metastatic breast cancer. However, the effect of tumour‐derived exosomes on MDSCs and its mechanism are not clear. Here, we reported that breast cancer cells (4T1)‐secreted exosomes (BCC‐Ex) were able to differentiate bone marrow cells into MDSCs and significantly inhibited the proliferation of T lymphocytes to provide an immunosuppressive microenvironment for cancer cells in vivo and in vitro. The number of MDSCs in bone marrow and spleen of 4T1 tumour‐bearing mice and BCC‐Ex infused mice was significantly higher than that of normal mice, whereas the number of T lymphocytes in spleen was significantly decreased. In addition, BCC‐Ex markedly promoted the differentiation of MDSCs from bone marrow cells or bone marrow cells derived macrophages, seen as the increased expressions of MDSCs‐related functional proteins Arginase‐1 (Arg‐1) and inducible nitric oxide synthase (iNOS). Furthermore, BCC‐Ex significantly down‐regulated the expressions of chemokine receptor CXCR4 and markedly up‐regulated the levels of inflammatory cytokines IL‐6 and IL‐10 in bone marrow cells and macrophages and remarkably inhibited the division and proliferation of T cells. Importantly, CXCR4 agonist, CXCL12, could reverse the function of BCC‐Ex, indicating that BCC‐Ex‐induced MDSCs might be dependent on the down‐regulation of CXCR4. Western blot showed that BCC‐Ex significantly promoted the phosphorylation of STAT3 in bone marrow cells, resulting in the inhibitions of the proliferation and apoptosis of bone marrow cells, and the aggravation of the differentiation of bone marrow cells into MDSCs.


| INTRODUC TI ON
Breast cancer is one of the most common malignant tumours in women with a incidence about 1/8-1/10. 1,2 At present, the treatments of breast cancer include surgery, chemotherapy, radiotherapy, endocrine therapy and molecular targeted therapy. 3,4 In the case of highly invasive tumours such as triple-negative breast cancer which is easy to relapse and metastasize in the later stage, the general treatment could not achieve a good result. 5 Numerous clinical studies confirmed that there was an important relationship between the insensitivity of late metastatic patients to immunotherapy and the complex tumour microenvironment. 6 Myeloid-derived suppressor cells (MDSCs) are important components of the immunosuppressive microenvironment in tumours. The increase in MDSCs is closely associated with the resistant treatment and poor prognosis of metastatic breast cancer. 7 Normally, myeloid hematopoietic stem cells differentiate into immature bone marrow cells, which migrate to different peripheral organs and then further differentiate into macrophages, dendritic cells or neutrophils. 8 However, in the case of acute or chronic inflammation, infectious trauma and tumour, an accumulation of the immature bone marrow cells will occur, thus resulting in immunosuppression, locally. 8,9 In mice, MDSCs express both myeloid differentiation antigens Gr-1 and CD11b 10 and the cells are further defined as monocytic MDSCs (mMDSCs) and granulocytic MDSCs (gMDSCs), in which the mMDSCs and gMDACs are characterized with CD11b+Ly6G-Ly6C high and CD11b+Ly6G+Ly6C low phenotypes, respectively. 11,12 In general, MDSCs produce immunosuppression by directly or indirectly influencing a variety of immune cells such as inhibiting the proliferation of T cells, destroying the function of NK cells, and inducing macrophages to differentiate into M2 types and so on. [13][14][15] Exosome, a lipid bilayer structure with a diameter of about 50-200 nm, plays an important role in the intercellular communication and tumour microenvironment reprogramming. 16 As a potential biomarker and therapeutic target of tumour, exosome has received extensive attention in recent years. 17,18 Studies showed that tumour cells-derived exosomes could be secreted into the microenvironment of tumour, which is crucial to the invasion and metastasis of tumour cells and the proliferation and survival of immune cells. 19 There were studies showing that tumour exosomes promoted the production of IL-6 and inhibited the differentiation of the CD11b+ myeloid progenitor cells into dendritic cells and macrophages in bone marrow, thus promoting tumour growth. 20 In addition, exosomes derived from tumour cells could be able to make MDSCs secrete inflammatory cytokines such as IL-6 and TNFα, which further promoted the expansion of MDSCs.
The continuous expansion of MDSCs around the breast cancers could further lead to tumour deterioration and immune escape. 21 However, the molecular mechanism of the tumour-derived exosomes in induction of inflammatory factors and the production of MDSCs is not clear.
Signal transduction and activator of transcription 3 (STAT3) is a transcription factor of the STAT family, which is highly expressed in many malignant tumours. The activation of STAT3 led to abnormal cell proliferation and malignant transformation. 22 Studies showed that breast cancer cell-derived exosomes (BCC-Ex) promoted the production of MDSCs in bone marrow to induce immunosuppression through activating STAT3 signalling pathway, 23 thus promoting the invasion and metastasis of breast cancer. 24 The chemokine receptor CXCR4 and its ligand CXCL12 play an important role in the immune and inflammatory response, invasion and metastasis of malignant tumours, and stem cell migration and homing.

| Animal models
BALB/c mice were obtained from Changsha SLAC Laboratory Animal Company (http://www.hnsja.com/). All animal experiments were performed according to institutional guidelines and approved by the Animal Care and Use Committee of Nanchang University.

| Isolation of mouse bone marrow cells
Kill Balb/c mice by cervical dislocation and spray whole body thoroughly with 70% ethanol solution. Carefully take out the femur and tibia and place them in a tube of ice-cold, sterile PBS. Prepare a syringe full of 37°C medium (RPMI 1640 (Gibco), supplemented with 10% FBS (Gibco)), with a 27-gauge needle and a sterile 50 mL tube (Sorfa). Cut the top and bottom ends of the femur and tibia with sterile scissors.
Insert the needle through the cut end and flush bone marrow with medium into a sterile tube. Blow the bone marrow into single cell suspension. The single cell suspension was filtered through a 70μm cell strainer (BD Labware) and centrifuge at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended with RPMI medium supplemented with 10% FBS. Bone marrow cells were placed in cell culture dishes at 37°C, with 5% CO 2 atmosphere.

| Isolation of mouse spleen cells
Aseptically remove spleen and place it in a tube of ice-cold, sterile PBS. Remove any remaining fat and connective tissue using two needles and transfer spleen to a small petri dish containing 7 mL enzyme digestion mix (RPMI 1640, supplemented with collagenase type III at 1 μg/mL and Dnase I at 20 μg/mL). Cut the tissue into very small fragments, digesting the tissue for 20-25 minutes at room temperature. Add 600 μL of EDTA solution to the digestion mix and continue the incubation for a further 5 minutes. Run the digestion mix through a 70μm cell strainer to remove any remaining undigested tissue. The cells were treated with aseptic erythrocyte lysate for 5 minutes at room temperature and then add 10 mL precooled PBS to terminate the lysis. The single cell suspension was centrifuged at 1000 rpm for 5 minutes and discarded the supernatant, and the cells were resuspended with RPMI medium supplemented with 10% FBS.

| Isolation of exosomes
When the breast cancer cells (4T1) were reached to 80%-90% confluent the cells were washed with PBS and cultured for an additional 2 days in 4T1 cell medium with exosome-free FBS. The conditional medium was collected and centrifuged at 500 g for 5 minutes at 4°C to remove the dead cells, cell fragments and apoptotic bodies. After the centrifugation, the supernatant was transferred to an Ultra-clear tube (Millipore) and centrifuged at

| Transmission electron microscopy
The morphology and size distribution of exosomes were identified by using transmission electron microscopy. Briefly, the exosomes were fixed in 3% glutaraldehyde and 2% paraformaldehyde in cacodylate buffer and then loaded to formvar-carbon coated copper grids. After washing, the grids were contrasted in 2% uranyl acetate, dried and then examined by TEM (JEM-1400PLUS).

| Western blot analysis
Exosomes and total protein were run on 10% denaturing SDS-PAGE gels, then transferred to nitrocellulose membranes (BioRad), which (1:1000, mouse monoclonal, Abcam) at 4°C overnight. Blots were detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibody (Invitrogen) for 1 hour at room temperature. Images were quantified using the Super Signal West Pico or Femto chemiluminescent detection system (Pierce).

| Maturation, polarization and BCC-Ex treatment of bone marrow-derived macrophages
Maturation of macrophages: On the first day, primary bone marrow cells were cultured in cell culture dishes, and M-CSF (25 ng/mL) were added into the medium at the same time. At day 3, half of the medium was changed, 25 ng/mL M-CSF were added. At day 5, the medium was fully changed, 25 ng/mL M-CSF were added. The bone marrow cells differentiated into M0 type macrophages.
Macrophage polarization: At day 6, add 10 ng/mL IL-4 to stimulate macrophages to M2a polarization. At day 7, add 10 ng/mL LPS and 10 ng/mL IFNγ to stimulate macrophages to M1 polarization, and add 10 ng/mL IL-10L to stimulate macrophages to M2C polarization.

| Microarray
After M0 macrophages were treated with PBS or BCC-Ex for

| Quantitative real-time PCR
Total RNA from the cells was extracted using the RNA isolation kit (Agilent technologies) per the manufacturer's instructions. After removing the genomic DNA using DNase I (Ambion), 1 μg RNA was reverse-transcribed into cDNA using a commercially available kit (Applied Biosystems). Quantitative real-time PCR was performed with iCycler Thermal Cycler (Bio-Rad) using 2X SYBR Green master mix (Bio-Rad). Forty cycles were conducted as follows: 95°C for 30 seconds, 60°C for 30 seconds, preceded by 1 minute at 95°C for polymerase activation. Primer sequences for all genes we measured in this report are shown in Table 1. Quantification was performed by the delta cycle time method, with GAPDH used for normalization.

| CFSE labelling CD3 positive T cells
CD3-positive T cells were suspended with 1 mL RPMI medium without serum, and CFSE (5 μmol/L) was added. After incubating 20 minutes in 37°C incubator, the complete culture medium was added and placed it on ice for 5 minutes to stop the reaction and then, the sample was centrifugated at 4°C, 225 g for 5 minutes, and the supernatant was discarded. After washing twice with 5 mL complete culture medium, the CSFE-labelled CD3+ T cells were placed in cell culture dishes at 37°C, with 5% CO 2 atmosphere.

| Statistical analysis
The results are presented as average value ± standard deviation (SD). Student's t test was used for analysis between two groups.
One-way analysis of variance (ANOVA) was used to compare data among three or more groups. Differences with a P-value of < .05 were considered statistically significant.

TA B L E 1 Primers for qPCR
Genes Sequence Abbreviations: F, Forward primer; R, Reverse primer.

| The polarizations of macrophages were altered by breast cancer cell-derived exosomes (BBC-Ex)
Various

| BCC-Ex differentiated bone marrow cells into MDSCs
After stimulated with M-CSF (50 ng/mL) for 24 hours, the primary bone marrow cells were treated with exosomes derived from 4T1 cells F I G U R E 2 Effect of 4T1 breast cancer cells derived exosomes on the polarization of bone marrow-derived macrophages. A, The morphology of exosomes was detected by transmission electron microscope. B, Size distribution analysis of BCC-Ex was detected by NTA. C, The expression of exosomes associated surface marker protein TSG101, RAB5 and CD9 was detected by Western blot. D, The changes of gene expression in MO type macrophages and BCC-Ex treated MO type macrophages were detected by microarray method. Left: all gene changes. Right: differentially expressed gene heat map. E-I, The results of microarray were verified by qPCR, and the genes detected were CXCR4, CX3CR1, IL-6, IL-10, Arg-1 (n = 3). J, The results of microarray were verified by Western blot, and the genes detected were CXCR4, IL-6, IL-10 and Arg-1. Significance was measured using a two-way ANOVA. *P < .05, **P < .01, ***P < .001 vs control  After 72 hours, T cells were collected and the fluorescence intensity of CFSE was detected by flow cytometry. The results showed the BCC-Ex decreased the numbers of T cells ( Figure 5A) and increased the CFSE positive cells ( Figure 5B). In addition, the CD3+ cells in the proliferative phase were decreased from 15.5% to 13.6%

| Bone marrow cells treated with BCC-Ex inhibited the proliferation of T lymphocytes
( Figure 5C). The above results indicated that the bone marrow cells treated by 4T1-Ex were able to inhibit T-cell proliferation, in which the inhibition was consistent with MDSCs.

| BCC-Ex promoted MDSCs induction and inhibited T lymphocyte proliferation in vivo
The healthy Balb/c mice were injected with 200 μL PBS containing  Figure 6G).
QPCR analysis shown that BCC-Ex increased the expressions of IL-6 and IL-10 in bone marrow cells, whereas CXCL12 reversed this effect to some extent, suggesting that BCC-Ex-induced the differentiation of bone marrow cells into MDSCs was partially related to the downregulation of CXCR4.

| BCC-Ex inhibited proliferation and apoptosis of bone marrow cells by activating STAT3 signalling pathway
STAT3 is a main regulator of MDSCs proliferation and activation.
The primary bone marrow cells were treated with PBS or BCC-Ex (30 μg/mL) for 24 hours. As shown in Figure 7A

| D ISCUSS I ON
In recent years, the treatment of breast cancer has been continuously developed and updated, from traditional surgery, ra-

| CON CLUS IONS
In summary, we demonstrated that BCC-Ex differentiated the bone

CO N FLI C T O F I NTE R E S T
The authors have declared that no competing interests exist.

AUTH O R CO NTR I B UTI O N S
Quan-Wen Liu: Conceptualization-Equal, Data curation-Equal,

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the finding of this study are available from the corresponding author upon reasonable request.