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Myeloid-derived suppressor cells (MDSCs) promote tumor progression. The mechanisms of MDSC development during tumor growth remain unknown. Tumor exosomes (T-exosomes) have been implicated to play a role in immune regulation, however the role of exosomes in the induction of MDSCs is unclear. Our previous work demonstrated that exosomes isolated from tumor cells are taken up by bone marrow myeloid cells. Here, we extend those findings showing that exosomes isolated from T-exosomes switch the differentiation pathway of these myeloid cells to the MDSC pathway (CD11b+Gr-1+). The resulting cells exhibit MDSC phenotypic and functional characteristics including promotion of tumor growth. Furthermore, we demonstrated that in vivo MDSC mediated promotion of tumor progression is dependent on T-exosome prostaglandin E2 (PGE2) and TGF-β molecules. T-exosomes can induce the accumulation of MDSCs expressing Cox2, IL-6, VEGF, and arginase-1. Antibodies against exosomal PGE2 and TGF-β block the activity of these exosomes on MDSC induction and therefore attenuate MDSC-mediated tumor-promoting ability. Exosomal PGE2 and TGF-β are enriched in T-exosomes when compared with exosomes isolated from the supernatants of cultured tumor cells (C-exosomes). The tumor microenvironment has an effect on the potency of T-exosome mediated induction of MDSCs by regulating the sorting and the amount of exosomal PGE2 and TGF-β available. Together, these findings lend themselves to developing specific targetable therapeutic strategies to reduce or eliminate MDSC-induced immunosuppression and hence enhance host antitumor immunotherapy efficacy.
Myeloid-derived suppressor cells (MDSCs) are expanded in large numbers during tumor growth, resulting in accumulation in secondary lymphoid organs, blood, and tumor tissue. These expanded myeloid cells contribute to tumor progression, providing supporting stroma and immune evasion1–4. In mice, removal of the primary tumor results in the reduction of the number of systemic MDSCs, revealing a causal role for MDSCs in tumor growth 5. Purified MDSCs have been shown to inhibit both CD4+ and CD8+ T cell responses in vitro6–8. Furthermore, studies imply that these cells can down-regulate T cell functions in vivo8 and promote tumor metastasis by releasing a number of chemokines9.
Expansion of MDSCs is likely dependent on tumor ability to secrete myeloid-influencing factors (ie, colony-stimulating factor-1, IL-6, vascular endothelial growth factor (VEGF), PGE2, and granulocyte-macrophage colony stimulating factor [GM-CSF])3, 10–12. However, which of these tumor-associated factors are critical for MDSC accumulation and how these tumor factors affect MDSC development have not been fully evaluated.
Many cells, including tumor cells, have the capacity to release exosomes. Recently, increased evidence has suggested that T-exosomes might act as a vehicle for transmitting signals for suppression thus having negative effects on antitumor immune responses. T-exosomes interfere directly with T cell effector functions to induce apoptosis in activated tumor-specific T cells 13–15 and suppress NK cell tumor cytotoxicity through the downmodulation of perforin expression 16. In addition T-exosomes also impair the capacity of bone marrow derived CD11b+ myeloid cells and CD14+ monocytes to differentiate into functional dendritic cells (DCs) 12, 15.
In the present study, we show that T-exosomes can induce the accumulation of MDSCs. Once T-exosomes are taken up, the MDSCs markedly increase production of inflammatory cytokines, including IL-6 and VEGF, and promote tumor growth. Blocking tumor exosomal PGE2 and TGF-β leads to the attenuation of T-exosome mediated induction of accumulation of MDSCs. PGE2 and TGF-β have been shown to suppress immune response by induction of MDSCs 17–20. We further show that tumor microenvironmental factors producing exosomal PGE2 and TGF-β are enriched or increased as the tumor progresses; further highlighting the importance of the tumor microenvironment in regulating T-exosome-mediated immune suppression and tumor growth.
BALB/c mice were purchased from the Jackson Laboratory. Seven- to 10-week old female mice were used in this study. All animal studies were conducted within the guidelines established by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.
The TS/A cell line, 4T-1 cell line, murine mammary adenocarcinomas of spontaneous BALB/c origin, were maintained in vitro at 37°C in a humidified 5% CO2 atmosphere in air in complete medium (DMEM with 5% FBS) as described previously16. FBS used in cell cultures was exosome depleted by differential centrifugation using a method described previously 16.
TS/A or 4T-1 tumor cells from 80% confluent cultures were injected subcutaneously into mice. Mice received 1.2 × 105 tumor cells in 50 μl of PBS per site at three sites in mammary fat pads. Tumor tissue was removed from BALB/c mice at day 7, 14, and 21 post injection, weighed and subsequently dissociated enzymatically for 1 h with 1 mg/ml type I collagenase (Sigma-Aldrich) in the presence of 50 units/ml of RNase and DNase (Sigma-Aldrich) ., Single cell suspensions of tumor tissue in PBS were washed by centrifugation at 600×g for 5 min. The cell supernatants were collected and used for exosome purification by differential centrifugation using a previously described method 16. The cell pellets were washed with PBS and total leukocytes were isolated using Percoll gradients as described 16. Total numbers of leukocytes isolated from tumor tissue were determined using a hemocytometer method. Isolated leukocytes (10,000/sample) were analyzed for the presence of CD11b, and Gr-1 molecules by flow cytometry. Percentages of double-positive CD11b+Gr-1+ myeloid cells were determined by FACS. Purity and integrity of sucrose gradient purified exosomes was analyzed using a Hitachi H7000 electron microscope (Electronic Instruments) as previously described 16. Care was taken to minimize potential contamination during T-exosome purification. The precautions taken included: 1) only using tumor tissues that had no necrosis; 2) bacterial cultures of collagenase digested tumor tissue were negative; 3) rerunning the exosomes isolated from the first run on another sucrose gradient; and 4) determining the presence of common exosome markers before using the T- and C-exosomes.
The TS/A and 4T-1 tumor cell derived exosomes (C-exosomes) were isolated from the supernatants of 36 h cell cultures using minor modifications of the method described above . In brief, the TS/A and 4T-1 cells were cultured in vitro at 37°C in a humidified 5% CO2 atmosphere in air in complete medium (DMEM with 5% exosome-depleted FCS). When the cells reached 60% confluence, the cells were washed two times with CD293 media (Invitrogen, sera free media originally developed for culturing 293 cells without FBS) and returned to culture in CD293 media until 90% confluency was achieved. The cells were cultured in fresh CD293 medium for an additional 36 h before the supernatants were harvested for exosome purification. The concentration of exosomes was determined by analyzing protein concentration using the Biorad protein quantitation assay kit (Biorad) with BSA as a standard.
Sucrose purified exosomes for western blots were prepared by lysing exosomes in radioimmunoprecipitation assay (RIPA) buffer containing 1 mM EDTA. Lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes. After transfer, blots were analyzed using an Odyssey infrared imaging system (LI-COR, Biosciences, NE, USA). Briefly, membranes were rinsed with PBS for several minutes and blocked with Odyssey blocking buffer for 1 h at 22°C. The membrane was then incubated for 1 h at 22°C in a dilution of a primary antibodies in blocking solution containing 0.1% Tween-20, after which the membrane was washed in PBS + 0.1% Tween-20. Subsequently the membrane was incubated in the dark with fluorescent secondary antibody at a dilution of 1:10,000 in blocking solution containing 0.1% Tween-20. Secondary antibodies were IRDye 800 anti-mouse (Molecular Probes, Rockland Immunochemicals, PA), and Alexa Fluor 700 anti-rabbit (Molecular Probes, OR) probes. Images were acquired with an Odyssey infrared imaging system and analyzed using software specified by Odyssey systems.
C-exosomes and T-exosomes were lysed in protein lysis buffer and 100 μg of proteins were electrophoresed on 10% SDS-polyacrylamide gels. Coomassie-stained SDS-polyacrylamide gels were cut into 10 strips to correlate with the gel lanes and trypsinized. The digested peptides were loaded on a 100 nm × 10 cm capillary column packed in-house with C18 Monitor 100 A-spherical silica beads and eluted by a 1 h gradient of 10–100% acetonitrile, 0.1% TFA. Mass spectrometric analysis was performed and analyzed using an LTQ XL spectrometer (Thermo Finnigan) in the UAB Proteomic Core Facility.
UniProt protein IDs were enriched for GO terms using the Protein Information and Property Explorer (http://pipe.systemsbiology.net/pipe/#summary). Sorting of data on proteins based on cellular location and biological process were done using a spreadsheet.
Analysis of the production of T-exosomes revealed that ~100 ± 4.2 μg of T-exosomes are released from a tumor (1.1 ± 0.27 g of weight per tumor at 21 day after the injection). Based on the assumption that 100 μg of T-exosomes are released to the peripheral tissues continuously, we used 100 μg of exosomes for injection experiments. T-exosomes (100 μg) isolated from TS/A or 4T-1 tumor removed at day 21 post tumor cell injection were injected into BALB/c mice via the tail vein twice weekly for three weeks. Five mice received PBS and served as controls. One day after the final injection, mice were sacrificed and each organ was removed for isolation of CD11b+Gr-1+ cells using a protocol described below. The percentage of CD11b+Gr-1+ myeloid cells or others analyzed in the total number of leukocytes isolated from each organ were determined by FACS12.
To determine whether T-exosomes play a role in the accumulation of CD11b+Gr-1+ cells in the tumor, tumor cells (5×105) were co-injected with T-exosomes (50 μg). On day 5 and 10 post injection the tumors were removed. Tumor tissue was dissociated enzymatically for 1 h. with 1 mg/ml of type I collagenase. The resulting single cell suspensions of tumor tissue in PBS were washed by centrifugation at 1,000xg for 5 min. Cells (50,000/sample) were analyzed for the presence of CD11b and Gr-1+ by flow cytometry. The percentage of double positive myeloid cells, i.e., CD11b+Gr-1+, was determined by FACS analysis 12.
Bone marrow was isolated and cultured after RBC lysis as described previously12. Spleen cells were positively selected for CD11b using MACS beads (Miltenyi Biotec) and then stained with anti-Gr-1 Ab. CD11b+Gr-1+ populations were isolated by cell sorting. Purified spleen myeloid cells were used for co-injection with tumor cells. RBC-depleted bone marrow cells were cultured in RPMI 1640 medium containing 10% FBS, with the addition of glutamine, 2-ME, sodium pyruvate, nonessential amino acid, antibiotics (Invitrogen), and GM-CSF (20 ng/ml).
C-exosomes and T-exosomes were labeled with the PKH67 green and PKH26 red fluorescent dyes, respectively, using a commercially available kit (Sigma-Aldrich) according to the manufacturer's instructions. The efficiency of labeling of the exosomes (>92%) with PKH dyes was determined by FACS analysis as described previously 12. Fifty μg mixtures of labeled C- and T- exosomes at a ratio of 1:1 were injected i.v. into BALB/c mice (5 mice). Twenty-four h post injection, the spleen, liver, and lung were resected and retained and the BM was collected. Single-cell suspensions of each tissue were prepared in RPMI 1640 medium and subjected to FACS analysis.
For cell surface marker staining, isolated cells were blocked at 4°C for 5 min with 10 μg/ml Mouse Fc Block (BD), and then reacted for 30 min at 4°C with various fluochrome-labeled Abs including appropriate isotype controls. After washing twice, cells were analyzed using a FACSCalibur (BD Biosciences).
The following antibodies were used for flow cytometry: fluorescein isothiocyanate (FITC) anti-mouse CD19, PE anti-mouse CD11c, PE anti-mouse Ly-6G and Ly-6C (Gr-1), and APC anti-mouse Mac-1 (CD11b) and PE anti-mouse F4/80 from e-Bioscience (San Diego, CA). Anti-mouse CD3, CD19, NK cell surface expression of DX5 and isotype-matched antibodies (all from BD Biosciences) were used as controls for nonspecific binding.
The following antibodies were used for western blotting: rabbit polyclonal anti-CD81, calnexin, and mouse monoclonal antibodies anti-CD9, anti-TSG101, and anti-CD63 all obtained from Santa Cruz Biotechnology (CA). Mouse monoclonal Ab anti-TGF-β was purchased from R&D Systems (Minneapolis, MN). Mouse monoclonal anti-PGE2 antibody was purchased from Cayman Chemical (Ann Arbor, MI). One μg of anti-TGF-β or 2 μg of anti-PGE2 antibody was used for blocking the effects of 100 μg TS/A T- exosomes on induction of CD11b+Gr-1+ bone marrow derived cells.
Tumor cells for injection were prepared from cultures grown to near confluency with 95% viability. Cells were enumerated, adjusted to the proper number, and mixed with sorted CD11b+Gr-1+ cells at the ratio of 3:1 (tumor cell:CD11b+Gr-1+). Tumor cells alone or mixed with CD11b+Gr-1+ cells were injected subcutaneously into the mammary fat pads of mice in 0.2ml injection volumes. Additional groups of mice were injected with tumor cells and tumor sized was measured once a week using calipers. Two independent measurements (length and width) were taken for each tumor weekly. Tumor size was calculated according to the formula V = L × W (L: length, mm. W: width, mm). Animals were sacrificed when the maximal allowable tumor size was reached or after observation for 50 days.
Total RNA from the CD11b+Gr-1+ cells prepusled for 12 h with T-exosomes (1 μg/ml) was extracted using TRIzol reagent (Invitrogen) and reverse transcription-PCR (RT-PCR) analysis was done as previously described 21. Specific primers used in the RT-PCR were mouse Cox2, Arginase-1, IL-10 and GAPDH. 21. Three replicates from each cDNA were analyzed. The primers for mouse Cox2 were: forward primer 5'--TGAGTACCGCAAACGCTT CT -3' and reverse primer 5'-CTCCCCAAAGATAGCATCTGG -3'. The primers for mouse arginase-1 were: forward primer 5'- CAGAAGAATGGAAGAGTCAG-3' and reverse primer 5'-CAGATATGCAGGGAGTCACC-3'. The primers for mouse IL-10 were: forward primer 5'- CCAGTTTTACCTGGTAGAAGTGATG-3'. The primers for mouse GAPDH were: forward primer 5'-AGGTCATCCCAGAGCTGAACG-3' and reverse primer 5'- ACC CTGTTGCTGTAGCCGTAT-3'.
An ABI PRISM 7700 sequence detection system (Applied Biosystems) was used for amplification and detection of the gene transcripts of interest. In each TaqMan run, serial 5-fold dilutions of a single-stranded cDNA derived from a commercially available mouse positive control RNA (Applied Biosystems) were amplified to create a standard curve, and values of unknown samples were estimated relative to this standard curve. Standard curves showed a linear relationship between the copy number (defined as 1 ng of 1000-bp DNA = 9.1 × 1011 molecules) of the original internal standard and the number of PCR cycles that were required to exceed a preset threshold, according to the method described previously 21. PCRs for each sample were run in duplicate in three 5-fold serial dilutions. From these standard curves, the relative amount of cDNA for the 18S ribosomal RNA, GAPDH, and each gene was determined and expressed as a ratio of the amount of this material in cells treated or untreated with TS/A exosomes after samples were standardized to the relative expression of the 18S ribosomal RNA and GAPDH.
Mouse VEGF produced in cultures was determined using a VEGF ELISA (R&D Systems, Minneapolis, MN). Mouse TNF-α and IL-6 produced in culture supernatants was also quantified using an ELISA (e-Bioscience). PGE2 levels were determined using the prostaglandin E2 monoclonal EIA kit (Cayman Chemical). In each case the assay was run according to the manufacturer's instructions.
One hundred μg of exosomes (based on protein content) were incubated for 1 h at 22°C with mouse anti-TGF-β antibody (1μg per ml, R&D System), mouse anti-PGE2 antibody (Cayman Chemical), or mouse IgG at 2μg per ml final concentration. (This concentration was chosen because more than 1 μg/ml of anti-TGF-β antibody or 2 μg/ml of anti-PGE2 antibody did not further reverse T-exosome mediated induction of CD11b+Gr-1+ cells in an in vitro assay 12). The exosomes were then washed once at 100,000 × g for 1 h, and resuspended in 200 ul of PBS for use in induction assays on bone marrow derived CD11b+Gr-1+ cells.
All data represents the mean ± SEM, and the error bars in figures also represent SEM. Unpaired two-tailed t tests were used to compare significant differences between two groups. One-way ANOVA followed by Bonferroni tests were used to analyze data for more than two groups.
Our previous data suggest that T-exosomes are taken up by bone marrow precursor cells (Gr-1+CD11b+) in vivo 12. Experiments were conducted in a mouse model using intravenously injected exosomes isolated from TS/A tumor removed at day 21 post tumor cell injection to examine the effects of T-exosomes or C-exosomes on the induction of accumulation of Gr-1+CD11b+ populations. After twice weekly injections over a 3 week period, FACS analysis of the splenocytes of mice (Figure 1A) demonstrated that the apparent splenomegaly (data not shown) was associated with marked accumulation of cells expressing Gr-1 and CD11b markers, but did not reveal an increase in cells expressing CD3, CD19, DX5 or CD11c (data not shown). A less dramatic increase in the percentage of CD11b+Gr-1+ cells occurred when mice were treated with C-exosomes (Figure 1A, right panel). This result suggested that tumor derived factors enhance T-exosome mediated induction of CD11b+Gr-1+ cells. We also looked for CD11b+Gr-1+cells in other tissues and secondary lymphoid organs. No significant increases in CD11b+Gr-1+cells were observed in the mesenteric lymph nodes or bone marrow (data not shown). However, in lung tissue a marked increase in the percent of CD11b+Gr-1+ cells was noted 21 d post injection where C-exosome and T-exosome injected mice had 8.2% and 14.2% CD11b+Gr-1+ cells, respectively, and PBS injected mice had 3.2% CD11b+Gr-1+ cells. Similar results were observed when exosomes isolated from 4T-1 tumor bearing mice were used for the injection. The limited induction in CD11b+Gr-1+cells in the spleen of mice treated with C-exosomes is most likely not due to preferential up take of T-exosomes because CD11b+Gr-1+cells took up the co-injected PKH67 labeled C-exosomes and PKH26 labeled T-exosomes with equal efficacy as determined by FACS analysis (data not shown). The data published by other groups indicate that the majority of MDSCs accumulate in both spleen and tumor, and we further determined whether T-exosomes played a role in the accumulation of CD11b+Gr-1+ cells in the tumor. Co-injection of tumor and T-exosomes resulted in an increase of CD11b+Gr-1+cell accumulation in the tumor when evaluated over a 10 day period (Figure 1B). Collectively, these results suggest that T-exosomes play a role in MDSCs accumulation in the spleen and tumor.
To examine whether CD11b+Gr-1+ cells induced by T-exosomes affect tumor growth directly, we sorted CD11b+Gr-1+ cells from the spleen of naïve mice or mice having been treated with C-exosomes or T-exosomes and coinjected BALB/c mice subcutaneously with TS/A cells (1.2 × 105) and the isolated CD11b+Gr-1+ cells (0.4 × 105). Tumor size was measured using calipers. We found that tumor growth occurred significantly faster when TS/A cells were coinjected with CD11b+Gr-1+ cells from mice treated with T-exosomes than tumor cells coinjected with naïve mouse CD11b+Gr-1+ cells (p < 0.01) (Figure 1C). Results of tumor growth experiments also indicated that spleen CD11b+Gr-1+ cells isolated from mice pretreated with C-exosomes promote tumor growth but to a slower degree than CD11b+Gr-1+ cells from T-exosome treated mice (p < 0.05). However, this difference in tumor growth was reduced approximately 42 days after tumor inoculation (Figure 1C). This is likely due to the significant induction of endogenous CD11b+Gr-1+ cells in host mice bearing large tumors and having higher amounts of exosomes released. The co-injection of T-exosome or C-exosomes –pretreated CD11b+Gr-1+ cells also led to early deaths in increased numbers of mice (Figure 1D). These data support the idea that T-exosomes induce the accumulation of CD11b+Gr-1+ cells which directly promotes tumor growth.
To examine whether the tumor microenvironment has an effect on tumor derived exosome mediated induction of CD11b+Gr-1+ population, bone marrow derived cells were cultured ex vivo in 24-well plates in the presence of C-exosomes or T-exosomes isolated from TS/A tumor removed from day 21 post tumor cell injected mice. In the absence of T-exosomes the majority of these cells differentiate into myeloid dendritic cells in the presence of GM-CSF 12. In contrast, culturing bone marrow derived cells for 7 d with C-exosomes or T-exosomes led to ~19% and ~29%, respectively, of these cells retaining CD11b+Gr-1+ cells phenotypically compared with ~3% of PBS control treated cells (Figure 2A). To determine the potential effects of T-exosomes on induction of genes/cytokines associated with promoting tumor growth, cytokine profiles in the 7-d cultured supernatants were determined using an ELISA and the expression of genes associated with tumor growth was quantified by RT-PCR and TaqMan PCR analysis of RNA isolated from the 7-d cultured CD11b+Gr-1+ cells. Treatment with T-exosomes most effectively up-regulated Cox2 and arginase-1 expression in 7 day cultured bone marrow cells than did C-exosome treatment (Figure 2B, top panel). Similar results were obtained from additional T-exosomes (n=5) isolated from different tumor-bearing mice when the expression of Cox2 and arginase-1 mRNA was quantified by TaqMan PCR analysis. Cox2 and arginase-1 mRNA was most up-regulated after bone marrow-derived CD11b+Gr-1+ cells were treated with T-exosomes (Fig. 2B, bottom panel). This result was not due to the amount of mRNA used for the TaqMan PCR analysis as there were no differences in the amount of IL-10 gene amplified (Fig. 2B, bottom panel).
The quantity of VEGF and IL-6 in the culture supernatants was significantly higher when T-exosomes were added (P < 0.01, Figure 2C). The response appeared selective as the induction of IL-10 was not observed (Figure 2C). Interestingly, when C-exosomes were added to bone marrow cells, there was no induction of VEGF (Figure 2C). Consistent with the above findings, the coinjection of T-exosome pretreated CD11b+Gr-1+ cells with TS/A tumor cells resulted in the most rapid tumor growth among the three groups of mice (Figure 2D). Taken together, these results suggest that in taking up T-exosomes, bone marrow derived immature myeloid cells gain an ability to induce inflammatory cytokine IL-6 and tumor growth factors that promote tumor growth. Although both C- and T- exosomes can induce myeloid-derived suppressor cells, T-exosomes are far more potent than C-exosomes at inducing MDSCs.
Given the results showing the majority of products induced by T-exosomes are found to be proinflammatory associated, in particular Cox2, we speculated that T-exosomes may contain sufficient quantities of PGE2 to induce a number of proinflammatory cytokines including Cox2 that facilitates tumor growth. The ELISA results showed that PGE2 is highly enriched in the T-exosomes when compared with C-exosomes (Figure 3A). In addition, we also found that higher percentages of CD11b+Gr-1+ cells infiltrating a tumor are correlated with increased amounts of PGE2 (Figure 3B). To determine if T-exosomes with increased quantities of PGE2 are capable of inducing more CD11b+Gr-1+ cells, the induction of bone marrow derived CD11b+Gr-1+ cells was determined in the presence of T-exosomes purified at different times after tumor cell injection. FACS analysis of 7-day bone marrow T-exosome cultures indicated that higher percentages of CD11b+Gr-1+ cells were induced with T-exosomes isolated from later stages of tumor growth (Figure 3C, left panel) where the T-exosomes contained higher quantities of PGE2 (Figure 3C, right panel). To further confirm the role of exosomal PGE2 in the induction of CD11b+Gr-1+ myeloid cells and proinflammatory cytokine expression, anti-PGE2 antibody was used to block exosomal PGE2 effects. Pre-incubation of T-exosomes with anti-PGE2 antibody led to a partial inhibition of tumor exosomal mediated induction of MDSCs (Figure 3D, left panel) with a concomitant reduction of proinflammatory cytokines (Figure 3D, right panel) when 7-day cultures of bone marrow derived cells were analyzed.
Since neutralization of exosomal PGE2 only partially reverses the induction of CD11b+Gr-1+ myeloid cells, we searched for additional molecules that might promote the induction of MDSCs. Another group has reported that TGF-β, which causes immune suppression, is packed into exosomes13. Using western blots our data also shows that TGF-β is packed into both C- and T- exosomes and is enriched as a tumor progresses (Figure 4A). The results of analyzing other exosomal protein markers, including CD63, CD81, and Tsg101, (Figure 4A) suggests that the tumor microenvironment regulates enrichment of TGF-β in a selective fashion as the increase of other proteins was not observed. Calnexin was also detected in the lysates of tumor tissue, but not in the purified exosome preparations (Figure 4A), indicating that our exosomal preparations were free of contamination with non-exosomal membrane proteins. To further determine if the tumor microenvironment only regulates sorting of TGF-β into exosomes, we did a comparison of protein profiles of T-exosomes isolated from TS/A tumor with C-exosomes. Specific proteins were at undetectable levels in C-exosomes when compared with protein amounts in T-exosomes (Table 1). This analysis indicated a protein composition of T-exosomes typical of exosomes derived from other cell types 22–27. The proteins are not detected in the C-exosomes (Table 1) but in both cases the exosomes contained proteins known to be involved in cell metabolism, signal transduction, cell endocytosis, protein transport, and several class E vacuolar protein-sorting (VPS) proteins (associated with MVB biogenesis) (Table 2), with 36 of the proteins identified in this study having been described previously in exosomes derived from other cell types (Table 3) 22–27.
We determined the effect of anti-PGE2 and anti-TGF-β antibodies on induction of bone marrow CD11b+Gr-1+ cells. and found that neutralization of tumor exosomal PGE2 and TGF-β led to a more significant reduction in the induction CD11b+Gr-1+ cells (P < 0.01, Figure 4B) than anti-PGE2 or anti-TGF-β alone (Figure 4B). In addition, neutralization of PGE2 and TGF-β together also resulted in a decreased induction of expression of IL-6 and VEGF (Figure 4C). More importantly, blocking exosomal PGE2 and TGF-β by pre-incubation of T-exosomes with anti-PGE2 and anti-TGF-β antibodies reduced tumor growth when tumor cells were co-injected with bone marrow derived CD11b+Gr-1+ cells pulsed with T-exosomes (Figure 4D).
The present study revealed that T-exosomes can cause the accumulation of myeloid-derived suppressor cells in a tumor. This is supported by the finding that 1) mice injected with T-exosomes developed enlarged spleens containing accumulations of CD11b+Gr-1+ MDSCs; 2) these exosome-induced CD11b+Gr-1+ cells also released proinflammatory cytokine IL-6 and tumor growth factor VEGF; and 3) the co-injection of T-exosomes taken up by bone marrow derived CD11b+Gr-1+ cells with tumor cells led to the promotion of tumor growth. We further identified that tumor exosomal PGE2 and TGF-β play a role in the induction of the accumulation of MDSCs. Together, these data provide evidence of the immune suppression properties of exosomal PGE2 and TGF-β in cancer.
PGE2, a product of inflammation, is thought to promote tumor growth by inducing neoangiogenesis 28–33 and by inducing the accumulation of MDSCs 19. In addition to PGE2 regulating production of a number of cytokines, including IL-6 34–38 and VEGF 29, 31, 39–41, PGE2 also regulates arginase 1 42–44. In the late stages of cancer, TGF-β promotes tumor spreading by enhancing the expression of VEGF 17, 20, 45–50. The current study shows that both PGE2 and TGF-β are enriched in exosomes isolated from late stages of tumor growth. Blocking exosomal PGE2 and TGF-β pathways (with anti-PGE2 and TGF-β antibodies) results in the attenuation of tumor exosomal induced expression of VEGF, and delays tumor growth in an in vivo transplantation tumor model.
Further characterization of the T-exosomes was performed by examining the effects of the tumor microenvironment on T-exosome mediated induction of MDSCs. We found that the tumor microenvironment can dramatically effect the recruitment of PGE2 and TGF-β into the exosomes. The more advanced the tumor stage, the more enriched T-exosomes are with PGE2 and TGF-β. It is conceivable that the tumor microenvironment during the early stages of tumor growth or establishment may regulate the sorting of exosomal proteins in/out differently from during later stages of tumor growth. During the early stages of tumor development, the host immune response against tumor growth may be dominant in the tumor microenvironment when compared to tumor derived immune suppression factors. The exosomes released during the early stages of tumor growth and establishment may actually stimulate host immune responses 51. However, as the tumor becomes established new factors regulated by the tumor microenvironment are expressed 52–57 and these factors could drive the sorting of tumor exosomal molecules toward those molecules that tend to cause immune suppression. This study identifies potential markers for further identification of the tumor microenvironment derived master regulator(s) that preferentially sort immune suppressor molecules such as exosomal PGE2 and TGF-β into exosomes.
Exosomes packed with multiple immune suppression molecules, including PGE2 and TGF-β, could act on a variety of levels and pathways to suppress immune function in multiple targets simultaneously. Therefore, given the broad array of bioactive molecules incorporated into tumor-produced exosomes, the possibility exists that other factors might be involved in the induction of MDSCs (besides exosomal PGE2 and TGF-β). As a matter of fact, our mass spectrometry analysis results suggest that other proteins might be selectively sorted in or out of T-exosomes. These proteins could also play a role in exosome-mediated induction of MDSCs. This assumption is supported with the evidence that neutralization of the activities of exosomal PGE2 and TGF-β does not lead to complete inhibition of T-exosome mediated induction of MDSCs. Therefore, the higher amounts of exosomal PGE2 or TGF-β may be one reason why these molecules are more effective but not necessarily the master regulators of induction of MDSCs and tumor promoting activity of these cells. One important factor to explore is the regulation of protein sorting in and out of exosomes. Data published by other groups indicate that ceramid may play a role in exosomal protein sorting58. Ceramide regulates cell apoptosis. Our MS/LC data suggest that a relatively large group of proteins regulating cell apoptosis is preferentially sorted into T-exosomes. Whether these proteins are regulated by ceramide warrants further investigated.
Regulators within cells are tightly controlled, but may be affected by microenvironment changes. Therefore, difference in the induction of MDSCs and the tumor promoting activity of cells induced by C-exosomes and T-exosomes might be due to tumor microenvironment factors. Tumor microenvironment factors could have effects on the T-exosome mediated accumulation of MDSCs in the tumor and spleen through multiple steps, from controlling quality and quantity of the exosomes released from the tumor to in vivo trafficking routs and targeted cells of the exosomes. These issues are important and require further study.
Finally, caution should be exercised so as to not over interpret our results in regard to the resource of T-exosome producer cells, although our data indicate that the exosomes released by tumor cells can induce MDSCs. Other types of cells rather than tumor cells including infiltrating MDSCs, tumor stroma cells and regulatory T cells should not be excluded for their potential release of exosomes. Future experiments of this nature will be fundamental to understanding how the tumor cells and stroma cells in the tumor microenvironment communicate with infiltrating immune cells via exosomes to regulate immune suppression in a host.
This work was supported by National Institutes of Health (NIH) grant no. RO1CA116092, RO1CA107181, R01AT004294; Birmingham Veterans Administration Medical Center (VAMC) Merit Review Grants (H.-G.Z.); and a grant from the Susan G. Komen Breast Cancer Foundation. We thank Dr. Jerald Ainsworth for editorial assistance.