Myeloid-derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME). Along with the role of MDSC immunosuppression and antitumor immunity, MDSCs facilitate tumor growth, differentiation, and metastasis in several ways that are yet to be explored. Like any other cell type, MDSCs also release a tremendous number of exosomes, or nanovesicles of endosomal origin, that participate in intercellular communications by dispatching biological macromolecules. There have been no investigational studies conducted to characterize the role of MDSC-derived exosomes (MDSC exo) in modulating the TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant level of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed a higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those in the spleen or bone marrow. Our
Apart from cancer cells, the tumor microenvironment (TME) consists of heterogeneous host cells of the immune system, the tumor vasculature and lymphatics, fibroblasts, pericytes, and sometimes adipocytes (
There is growing evidence that MDSCs harness various immune and nonimmune mechanisms to promote tumor development. MDSCs inhibit adaptive antitumor immunity by inhibiting T-cell activation and function (T-cell receptor downregulation, T-cell cell cycle inhibition, and immune checkpoint blockade) (
Although the role of MDSCs in tumor growth and metastasis is well known, there is a significant knowledge gap for understanding the role of MDSC-derived exosomes (MDSC exo). During the past decade, there has been a huge surge of exosome research and publications that are mostly focused on exosomes derived from tumor cells and immune cells. Exosomes are 30–150 nm lipid bi-layered extracellular bioactive vesicles of endosomal origin that are secreted by all cells and are present in various body fluids. Exosomes have been proposed to act as intercellular communicators as they can transfer their cargo (proteins, lipids, and nucleic acids) to nearby or distant recipient cells. Previously, we observed that MDSC exo carry a significant amount of pro-tumorigenic factors, and a large percentage of MDSC exo injected intravenously was found to be distributed in the primary breast tumor and metastatic sites (
In this study, we characterized the size, yield, and contents of exosomes collected from different MDSC populations and immature myeloid progenitor cells. We now report that, similar to parental MDSCs, exosomes from MDSCs also play a crucial role in inciting the immunosuppressive milieu by way of limiting the functions of cytotoxic T cells and pro-inflammatory M1 macrophages in the TME.
All experiments were performed according to the National Institutes of Health (NIH) guidelines and regulations. The Institutional Animal Care and Use Committee (IACUC) of Augusta University (protocol #2014-0625) approved the experimental procedures. All animals were kept under regular barrier conditions at room temperature with exposure to light for 12 h and dark for 12 h. Food and water were offered
Nanoparticle tracking analysis (NTA) was performed using ZetaView, a second-generation instrument from Particle Metrix for visualizing and counting individual exosome particles as described previously (
For the
Both 4T1 and AT3 cells expressing the luciferase gene were orthotopically implanted in syngeneic BALB/c and C57BL/J6 mice, respectively (The Jackson Laboratory, Bar Harbor, Maine, USA). All mice were between 5–6 weeks of age and weighed 18–20 g. Animals were anesthetized using a mixture of xylazine (20 mg/kg) and ketamine (100 mg/kg) administered intraperitoneally. Hair was removed from the right half of the abdomen using hair removal ointment, and then the abdomen was cleaned by povidone-iodine and alcohol. A small incision was made in the middle of the abdomen, and the skin was separated from the peritoneum using blunt forceps. The separated skin was pulled to the right side to expose the mammary fat pad and either 50,000 4T1 cells or 100,000 AT3 cells in 50 µl Matrigel (Corning Inc.) were injected.
MDSCs were isolated from spleens and tumors of tumor-bearing mice 3 weeks after orthotopic tumor cell implantation. Myeloid progenitor cells were isolated from the bone marrow of normal wild-type mice. We used anti-mouse Ly-6G, and Ly-6C antibody-conjugated magnetic beads (BD Biosciences). The purity of cell populations was >99%. In short, the spleen was disrupted in PBS using the plunger of a 3 ml syringe, and cell aggregates and debris were removed by passing the cell suspension through a sterile 70-µm mesh nylon strainer (Fisherbrand™). Mononuclear cells were separated by lymphocyte separation medium (Corning®) as a white buffy coat layer. Cells were then centrifuged at 1,500 rpm for 10 min followed by a washing step with PBS at 1,200 rpm for 8 min. Then cells were resuspended at 1×108 cells/ml in PBS and antibodies conjugated with magnetic beads were added followed by incubation at 4°C for 30 min. Finally, positive cells were collected using a MACS LS column (Miltenyi Biotec) and a MidiMACS™ magnetic stand followed by a wash step with extra PBS. The purity of isolated MDSCs was checked by flow cytometry using Gr1 FITC and CD11b APC antibodies (purchased from BioLegend). Cell viability was checked with 7-AAD which was less than 0.1–0.2% (dead cells) of the total population. MDSCs were grown in exosome-depleted media consisting of RPMI, 2 mM L-glutamine, 1% MEM non-essential amino acids, 1 mM sodium pyruvate, and 10% FBS, supplemented with 100 ng/ml of GM-CSF.
Exosomes were depleted from the complete media by ultracentrifugation for 70 min at 100,000 × g using an ultracentrifuge (Beckman Coulter) and SW28 swinging-bucket rotor. MDSCs (6×106) were grown in a T175 flask for 72 h under normoxic conditions (5% CO2 and 20% oxygen) at 37°C in a humidified incubator. The cell culture supernatant was centrifuged at 700 × g for 15 min to remove cell debris. To isolate exosomes, we employed a combination of two steps of the size-based method by passing through a 0.20-µm syringe filter and centrifugation with 100k membrane tube at 3,200 × g for 30 min followed by a single step of ultracentrifugation at 100,000 × g for 70 min [as described in our previous publication (
Isolated exosomes resuspended in a minimal amount of PBS were lysed by RIPA buffer with protease and phosphatase inhibitor (100:1 dilution). Exosomal protein was quantified by Bradford assay using Pierce™ BCA Protein Assay Kit (Thermo Scientific™) and serial dilution of BSA standard (Thermo Scientific™; Thermo Fisher Scientific, Inc.).
Proteins were extracted from tumor cells and their corresponding exosomes in both untreated and treated conditions to evaluate the expression profiles of 44 factors in duplicate by mouse cytokine antibody array (AAM-CYT-1000-8; RayBiotech, Inc.). Protein sample (500 µg) was loaded onto the membrane according to the manufacturer's instructions, and the chemiluminescent reaction was detected using a LAS-3000 imaging system (Fuji Film, Japan). All signals (expression intensity) emitted from the membrane were normalized to the average of 6 positive control spots of the corresponding membrane using ImageJ software version 1.53c [National Institutes of Health (NIH)].
A Transwell assay was performed to evaluate the chemotaxis property of the MDSC-derived exosomes. We used 24 Transwell plates with 8-µm inserts in polyethylene terephthalate track-etched membranes (Corning, Inc.). We collected bone marrow cells and splenic mononuclear cells using Ficoll gradient centrifugation, and myeloid cells from bone marrow using CD11b+ magnetic beads from normal Balb/c mice. A total of 1.5×106 cells/insert in serum-free media were added into the upper compartment of the chamber. Inserts were placed in 12-well plates with DMEM containing 0.5% FBS in the presence or absence of exosomes (20 µl containing approximately 3×108 exosomes) isolated from MDSCs. After incubating overnight, we collected suspended immune cells (migrated) from the media of the bottom chamber and loosely adherent immune cells on the surface of the bottom chamber using gentle cell scraping. Then we centrifuged and resuspended the cells in PBS and counted the cells with a hemocytometer. Insert membranes were washed, fixed, and stained with 0.05% crystal violet to detect the migrated/invaded cells. The counting was made with an inverted microscope (Nikon Eclipse E200).
Scratch assay was performed to detect the ability of MDSC-derived exosomes to increase migration and invasion of tumor cells. 4T1 luciferase positive cells were seeded in 6-well plates. After achieving 80–90% confluency, the cells were starved overnight with 0.5% FBS for cell cycle synchronization and a measured wound was inflicted at the center of the culture (from top to bottom). Then, cells were treated with 50 µl of splenic MDSC-derived exosomes in PBS containing 7.5×108 exosomes for 48 h in 2% FBS media. Microphotographs were taken every 24 h using an automated all-in-one microscope (BZ-X710; Keyence). The wound size was measured using Image J software (NIH) by drawing a rectangular region of interest to quantify the visible area of the wound.
MDSC-derived exosomes were injected intravenously (100 µl containing approximately 1.5×109 exosomes) into the wild-type Balb/c and C57Bl/6 mice. The animals were treated for a week with 3 doses (alternate days) of MDSC-derived exosomes. After that, the animals were euthanized and organs were collected for flow cytometric analysis.
Both CD4+ and CD8+ cells were isolated from normal mouse splenocytes by immune-magnetic negative selection kit (Stemcell Technology; catalog #19852 and 19853, respectively). In short, harvested spleens from normal mice were disrupted in cold PBS containing 2% FBS. Clumps and debris were removed by passing the cell suspension through a 70-µm mesh nylon strainer. The single-cell suspension was centrifuged at 300 × g for 10 min and resuspended at 1×108 nucleated cells/ml. Rat serum was added to the sample (50 µl/ml) followed by the addition of isolation cocktail (50 µl/ml). After mixing, the sample mix was incubated at room temperature for 10 min. RapidSpheres™ (75 µl/ml) were added to the sample mix and incubated for 3 min. The tube was placed in EASYSEP™ MAGNETS (catalog #18001; Stemcell Technologies) for 3 min. The enriched cell suspension was collected by decanting into a new tube. Cells were seeded in 24-well plates bound with purified anti-mouse CD3e (5 µl/ml) in T-cell media that consists of RPMI, 10% FBS, 1% MEAM, 2.5% HEPES, 1% penicillin-streptomycin, 0.5% β-mercaptoethanol, and purified anti-mouse CD28 (5 µl/ml).
ROS production from CD8+ T-cells following MDSC-derived exosomes treatment
Following isolation, 20,000 CD8+ cells were seeded in an anti-mouse CD3e-bound 96-well plate and treated with 10 µl of MDSC-derived exosomes or the same volume of PBS (control). After 48 h, 10 µl of WST-1 reagent (Alkali Scientific Inc.) was added to each well and incubated for 4 h. The absorbance of each well was measured at a wavelength of 450 nm by the Perkin Elmer Victor3 V 1420 multilabel plate reader.
Quantitative data are expressed as mean ± standard error of the mean (SEM) unless otherwise stated, and statistical differences between more than two groups were determined by analysis of variance (ANOVA) followed by multiple comparisons using Tukey's multiple comparisons test. A comparison between 2 samples was performed by the Student t-test. GraphPad Prism version 8.2.1 for Windows (GraphPad Software, Inc.) was used to perform the statistical analysis. Differences with P-values <0.05 were considered significant and are indicated in the figures and legends (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
We isolated MDSCs from the bone marrow of normal (non-tumor bearing) wild-type mice, and from spleen and tumors of tumor-bearing mice by magnetic particle separation using Ly6G and Ly6C beads. Tumors were implanted orthotopically in the mammary fat pad and allowed to grow for 3 weeks. Following their separation, by flow cytometry, more than 98% of the cells were estimated to be positive for MDSC markers (CD11b+Gr+) (
Next, we tested if the protein contents of normal BM MDSC exo differ from the spleen MDSC exo and tumor MDSC exo isolated from tumor-bearing mice. We quantified the expression level of cytokines in MDSC exo that are involved in tumor invasion (
MDSCs were demonstrated to promote tumor invasion and metastasis by two mechanisms: i) Increased production of multiple matrix metalloproteinases (MMPs) for extracellular matrix degradation and increased production of chemokines to establish a pre-metastatic milieu (
Tumor-specific endocrine factors systemically stimulate the quiescent immune-compartments (bone marrow, spleen, lymph nodes), resulting in the expansion, mobilization, and recruitment of immunosuppressive cells. Discrete subsets of tumor-instigated immune cells bolster tumor progression and metastasis by governing angiogenesis, inflammation, and immune suppression. Of the immune cells, much focus has been denoted towards the MDSCs (
To determine the chemotaxis capability of MDSC exo, we seeded CD11b+ myeloid cells (from bone marrow) or all bone marrow cells, or all splenic mononuclear cells (following Ficoll separation) isolated from normal mice in the upper chamber and with or without spleen MDSC exo in the bottom chamber of the Transwell insert. After 24 h of incubation, the number of migrated cells in the bottom chamber was significantly higher in the wells treated with spleen MDSC exo compared to untreated control wells (
We further estimated the level of expression of T-cell function-associated and immunomodulatory cytokines in the protein contents of normal BM MDSC exo and exosomes isolated from MDSCs (tumor and splenic) of tumor-bearing mice by protein array. Among the immunomodulatory cytokines, the levels of IL-12, IL-13, IL-1Ra, IL-4, C-X-C motif chemokine 5 (LIX), and tumor necrosis factor (TNF)-α were significantly elevated in both the tumor-MDSC-exo and spleen-MDSC-exo of tumor-bearing mice compared to normal BM-MDSC-exo (
Next, we investigated whether MDSC exo treatment could deplete the CD8+ T-cells in mice. For this
We also explored whether the MDSC exo treatment could change the distribution of the myeloid populations
Since we demonstrated that MDSC exo express a significantly high level of cytokines that facilitate regulatory T-cell or Th2 cell functions and immunosuppression, we wanted to investigate the effect of MDSC exo directly on CD4+ and CD8+ T-cells
MDSCs release ROS molecules as part of a primary mechanism to suppress T-cell responses (
Considering the fact that MDSC exo were able to activate and deplete CD8+ T-cells, we determined the level of FasL in different MDSC exo that could conceivably trigger the apoptosis process in CD8+ T-cells. We detected higher expression of FasL in the exosomes isolated from the MDSCs in tumors compared to that in exosomes of MDSCs from spleen and bone marrow (
It has been perceived that functional differences may exist in myeloid-derived suppressor cells (MDSCs) isolated from different environments within the same host, and that MDSCs from tumors have a stronger immunosuppressive capacity than MDSCs in the peripheral lymphoid organs (spleen, lymph nodes) (
MDSCs are competent in promoting tumor growth through remodeling the TME (
Tumor-associated macrophages (TAMs) are part of the heterogeneous populations of immunosuppressive myeloid cells that produce chemokines for the activation and maintenance of inflammatory processes in the TME (
We observed that MDSC-derived exosomes are able to deplete CD8+ T-cells
In summary, we comprehensively demonstrated that MDSC-derived exosomes inherit pro-tumorigenic factors and functionally resemble parental cells in immunosuppression, tumor growth, angiogenesis, invasion, and metastasis. In addition, MDSC-derived exosomes are capable of increasing ROS production and inciting the Fas/FasL pathway in CD8+ T-cells, which precipitates AICD (
The authors thank Dr Rhea-Beth Markowitz, Director, Office of Grant Development, Georgia Cancer Center for help with the English language editing of the manuscript.
This study was supported by the Georgia Cancer Center Startup Fund and Intramural Grant Program at Augusta University (Augusta, GA, USA) to ASA.
The datasets used during the present study are available from the corresponding author upon reasonable request.
MHR conceived the hypothesis, designed and performed the experiments, and conducted the data collection, data analysis, and interpretation, and wrote the manuscript. TFB conducted acquisition of the
All experiments were performed according to the National Institutes of Health (NIH) guidelines and regulations. The Institutional Animal Care and Use Committee (IACUC) of Augusta University (Augusta, GA, USA) (protocol #2014-0625) approved the experimental procedures.
Not applicable.
The authors have declared that no competing interest exists.
Isolation of MDSC-derived exosomes (exo) from different sources. (A) Flow cytometric analysis of isolated MDSCs from normal bone marrow (BM), spleen of tumor-bearing mice, and tumors, showing that more than 98% of cells were positive for CD11b and Gr1. (B and C) Nanoparticle tracking analysis (NTA) showing no significant differences in the size distribution of exosomes isolated from MDSCs of normal bone marrow (BM), the spleen of tumor-bearing mice, and tumors. (D) NTA analysis showing exosome concentration per ml. Quantitative data are expressed in mean ± SEM. *P<0.05. n=3. MDSCs, myeloid-derived suppressor cells.
The expression level of cytokines in MDSC-derived exosomes (exo) that are involved in tumor invasion, angiogenesis, and myeloid cell activation and function.
Role of MDSC-derived exosomes (exo) in immune cell migration. Isolated mouse myeloid cells, bone marrow cells, and splenic cells were seeded on the top chamber of the Transwell, and splenic MDSC-derived exosomes were added in the bottom chamber with 0.5% FBS. After 24 h, migrated (A) bone marrow cells, (B) splenic cells, and (C) myeloid cells in the bottom chamber were counted with a hemocytometer. In addition, (D) attached myeloid cells on the Transwell membrane were visualized under a light microscope, and (E) quantified. Quantitative data are expressed as mean ± SEM. *P<0.05, ***P<0.001, n=4. MDSCs, myeloid-derived suppressor cells.
Expression levels of cytokines in MDSC-derived exosomes (exo) that are involved in T-cell function and immunomodulation.
Effect of MDSC-derived exosomes on CD4 and CD8-positive T-cells
Schematic diagram showing the process of biogenesis of exosomes from MDSCs and the role of MDSC-derived exosomes in tumor progression and immunosuppression by AICD. Exosomes secreted from the MDSCs contain pro-tumorigenic factors from the parent cells and can play a crucial role in immunosuppression, tumor growth, angiogenesis, invasion, and metastasis by dispensing their contents into the other TME cells or distant cells. MDSC-derived exosomes can activate CD8+ T-cells, and TCR triggering causes activation of DUOX-1 that leads to H2O2 production and eventually generation of ROS in mitochondria. Prolonged TCR stimulation triggers overexpression of both Fas (receptor) and FasL (ligand), which culminates in fratricide (from direct cell contact) or autocrine suicide (interaction of soluble FasL with Fas).