Exosomes derived from tumor cells are essential for processes involved in tumor progression, including angiogenesis, tumor cell proliferation and immunoregulation. In addition, exosome secretion may contribute to the mechanisms of hypoxia-induced angiogenesis and metastasis of tumors. In the present study, as it is one of the most common cancers in females, breast cancer, cell lines were cultured under hypoxic (1% O2) and normoxic conditions to evaluate the effects of hypoxia on exosome production. Under hypoxic conditions an increase in the number of exosomes in the medium, determined by CD63 immunoblotting, was observed. Application of these exosomes to T cells revealed that they were able to suppress T cell proliferation. As transforming growth factor-β (TGF-β), interleukin-10, and prostaglandin E2 are important factors in the mediation of T cell suppression, the exosomes were subsequently treated with antibodies against these three factors. The results revealed that anti-TGF-β was capable of ameliorating the immunosuppressive effects of exosomes. These data demonstrate that hypoxia enhances the secretion of exosomes by breast cancer cells, which acts to suppress T cell proliferation via TGF-β. The findings have significant implications for understanding the underlying mechanisms of immunosuppression in tumor microenvironments, and for the potential development of cancer therapies.
The communication dysfunction between tumor cells and stromal cells is essential for tumorigenic processes, including cellular proliferation, differentiation, and apoptosis (
Recently, exosomes have emerged as potent communicators between tumors and their microenvironment, and potential contributors to tumorigenesis and subsequent metastasis (
Accumulating research indicates that exosomes have diverse functions in an array of biological events, such as cellular proliferation and migration (
The essential roles of exosomes have been demonstrated in a number of studies conducted in various tumor types, such as prostate cancer, and melanoma (
Lymphocyte infiltration and activation in tumor microenvironments may have a significant effect on tumor prognosis, growth and metastasis (
Breast cancer is one of the most common cancers in females, and one of the greatest threats to female health. Globally, breast cancer was the cause of 522,000 mortalities (15% of cancer mortalities in females) in 2012 (
The present study explores the immunoregulatory functions of exosomes derived from two different breast cancer cell lines (MDA-MB-231 and BT-474). As transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), the two predominantly considered cytokines, have been demonstrated to suppress the immune response (
BT-474 and MDA-MB-231 breast cancer cells were purchased from American Type Culture Collection (Manassas, VA, USA), and cultured according to the supplier's instructions. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS). When the cells were ~50% confluent, the medium was changed to serum-free medium prior to exosome preparation.
For hypoxic exposure, cells were cultured in an O2/CO2 incubator (Sanyo; Osaka, Japan) in a 1% O2, 5% CO2 humidified atmosphere at 37°C. For certain experiments, cells were treated with dimethyloxalylglycine, a hypoxia-inducible factor hydroxylase inhibitor (Enzo Life Sciences; Farmingdale, NY, USA) at a final concentration of 1 mM. This was added to inhibit the effects of hypoxia.
For normoxic exposure, cells were cultured in an O2/CO2 incubator (Sanyo) in a 20% O2, 5% CO2 humidified atmosphere at 37°C.
BT-474 and MDA-MB-231 cell lines were cultured in serum-free medium for exosome preparation. Exosome preparation was performed as described by Epple
Exosomes were collected and lysed with 1% Nonidet P-40 plus protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Following brief lysis, 5 µg exosome was subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. In this study, CD63 was used as an indicator for exosomes, as CD63 is a exosome marker. After blocking with 5% fat-free milk for 60 min, the membrane was incubated with the appropriate primary antibody (CD63; Santa Cruz Biotech, Santa Cruz, CA, USA; catalog no. sc-15363), followed by a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA; catalog no. 111-035-003). β-actin was used as the loading control. The blotting bands were visualized with enhanced chemiluminescence plus immunoblotting detection reagents (Pierce, Rockford, IL, USA). The membrane was subsequently scanned and the relative densitometry of the bands was determined using ImageJ (Windows version 1.48, NIH, Bethesda, MD, USA).
Splenocytes were isolated from the spleens of female C57BL/6 mice (6–8 weeks of age). The mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China), and all animal experiments were approved by the Institutional Review Board of Nanfang Medical University (Guangzhou, Guangdong, China). Splenocytes were separated and seeded at a density of 5×103 cells/well in DMEM supplemented with 10% FBS. Anti-CD3 (Invitrogen Life Technologies; catalog no. 11452D) and anti-CD28 (Invitrogen Life Technologies; catalog no. 11452D) linked with goat-anti-mouse antibody were added for activation. Splenocytes at a density of 5×104 cells/well in 100 µl expansion medium were added to round-bottom 96-well plates (Nunc; Penfield, NY, USA) and cultured for five days. Activated splenocytes without addition of any exosomes were used as the control group. The immunosuppressive effects of the exosomes were evaluated by incubating 10 µl exosomes per well with the splenocytes for five days. [3H] thymidine (1 µCi ml−1; Perkin Elmer, San Jose, CA, USA) was added 18 h prior to the end of incubation. Cells were subsequently harvested for measurement of [3H] thymidine incorporation to assess their rate of proliferation. To harvest the cells, 1 ml 10% trichloroacetic acid (TCA) was gently added to each well, and incubated at room temperature for 15 min. Following aspiration with 10% TCA and washing with a further 1 ml 10% TCA, the TCA was aspirated and 300 µl 0.2N NaOH was added to each well to dissolve DNA; this was incubated for 15 min at room temperature with agitation. To measure the thymidine incorporation, subsequently, 100 µl of each sample was added to a scintillation vial, and the counts were measured in a scintillation counter (Beckman Coulter LS6500; Brea, CA, USA). In order to assess the effects of TGF-β and IL-10 on splenocyte proliferation, anti-TGF-β and/or anti-IL-10 antibodies (5 µg/ml) were applied at the time of exosome application to block the potential effects of these factors.
The concentrations of TGF-b and IL-10 from exosome lysates were determined by ELISA according to the manufacturer's instructions (R&D, Minneapolis, MN, USA; DB100B and HS100C, respectively). Briefly, 100 µl/well exosome lysate samples were added to 96-well plates and incubated for 2 h at room temperature. Following three washes with washing buffer (R&D), 100 µl/well polyclonal horseradish peroxidase-conjugated anti-TGF-b1 or monoclonal alkaline phosphatase-conjugated anti-IL-10 antibody was added and the samples were incubated at room temperature for 2 h. The samples were then washed three times with wash buffer, 50 µl substrate solution (R&D) was added and incubated for 30 min, followed by the addition of 50 µl stop solution (R&D). Absorbance was measured using a microplate reader (Biotek Synergy 2; Biotek, Winooski, VT, USA) at a wavelength of 490 nm. The concentration of cytokines was quantified, according to a respective standard curve, which was obtained using recombinant human TGF-b1 or IL-10 as the control.
All statistical analyses were performed using SPSS software, version 20 (IBM SPSS Armonk, NY, USA). Student's
Exosomes were isolated from the conditioned media of breast cancer cell lines BT-474 and MDA-MB-231, cultured under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions, using serial centrifugation. The exosomal secretion of these cells was evaluated through immunoblotting using CD63, a well-established exosomal marker. As shown in
The application of exosomes to proliferating splenocytes revealed that exosomes secreted from cells cultured under hypoxic conditions produced significantly stronger immunosuppressive effects when compared with cells cultured under normoxic conditions (P<0.05). Additionally, stronger immunosuppressive effects were observed for exosomes secreted from cells in hypoxia than that from cells in normoxia (P<0.05). (
The concentration of TGF-β1 in exosomes from normoxic conditions in both cell lines was considerably higher compared with that from hypoxic conditions (P<0.05). Additionally, the concentration of IL-10 in exosomes from normoxic conditions in cell line BT-474 was significantly higher compared with hypoxic conditions, however, no considerable difference was observed for MDA-MB-231 cells. Furthermore, in BT-474 cells, the difference for TGF-β is much more prominent than for IL-10 (
As exosomes contain bulky proteins, proteomic analysis has allowed for further investigation into the functions of exosomes. An array of proteins, including vascular endothelial growth factor, epidermal growth factor and interleukin-4, have been shown to be present in the exosomes from cancer cells, and are critical for tumor cell survival, proliferation and migration. TGF-β1, which is produced by regulatory T cells, has potent immunosuppressive effects, which are crucial for protecting the body from excessive immune responses and maintaining immune homeostasis (
In conclusion, these results revealed a novel aspect of exosomes derived from breast cancer cells in immunosuppression, suggesting the essential role of exosomes in tumor microenvironment constitution. These findings could be potentially significant in the development of novel therapeutic strategies.
Exosomes were collected from MDA-MB-231 and BT-474 cells cultured under normoxic or hypoxic conditions and lysed with 1% Nonidet P-40. Following SDS-PAGE, immunoblotting and exposure to detection reagents, relative densitometry was performed, which revealed higher expression of CD63 protein for cells cultured under hypoxic conditions compared with normoxic conditions for both cell lines. (A) Immunoblot showing increased CD63 expression in MDA-MB-231 and BT-474 cells cultured under hypoxic conditions. (B) Relative CD63 expression. For both cell lines, cells cultured in hypoxic conditions exhibited significantly higher CD63 protein levels than those cultured in normoxic conditions. Bars represent mean ± standard error of the mean numbers of transmigrated cells from three independent experiments. *P<0.05.
Splenocytes incubated with breast cancer cell-derived exosomes exhibit lower [3H] thymidine incorporation compared with controls. Additionally, splenocytes incubated with exosomes produced under hypoxic conditions exhibit significantly lower incorporation compared with those produced under normoxic conditions for the cancer cell lines, MDA-MB-231 and BT-474. Bars represent mean ± standard error of the mean numbers of thymidine incorporation (cpm) from three independent experiments. *P<0.05. TdR, radioactive thymidine.
TGF-β and IL-10 in exosomes are responsible for immunosuppression. Concentrations of (A) TGF-β and (B) IL-10 in exosomes from MDA-MB-231 and BT-474, cultured under normoxia or hypoxia was measured by ELISA assay. (C) Addition of anti-TGF-β antibodies was revealed to significantly reduce the effects of exosomes on [3H] thymidine uptake by proliferating splenocytes. Bars represent mean ± standard error of the mean numbers of transmigrated cells from three independent experiments. *P<0.05. TGF-β, transforming growth factor-β; IL-10, interleukin-10; TdR, radioactive thymidine.