T lymphocyte cell-derived nano-size exosome impedes the proliferation and induces apoptosis of breast cancer MDA-MB231 cells by negative regulation of microRNA27

INTRODUCTION
Breast cancer as the most shared malignanciy in women worldwide could be treated in 70-75 percent of patients more importantly in patients with non-metastatic disease. Unfortunately, developed breast cancer with widespread metastases is mainly noticed incurable with the conventional therapies [1]. Breast cancer is an heterogeneous malignancy and includes several molecular property such as human epidermal growth factor receptor 2 (HER2, encoded by ERBB2), estrogen receptor (ER), and progesterone receptor (PR) activation in association with breast cancer gene 1 (BRCA1) mutations [2]. Based on the recent knowledge, therapeutic methods change respecting the molecular subtypes [3]. The novel treatment option needs to be evolved in a multidisciplinary setting, with respect to the tumor’s molecular subtype and regional load.
Dissimilar microenvironment ingredients of breast cancer, such as soluble mediators, inhibitory immune cells, and modified extracellular matrix (ECM) may provoke breast cancer development and metastasis. These components also could avert efficient antitumor immunity. Various stromal cells which were found in breast cancer microenvironment may complicate in the prediction if clinical outcomes. They frequently are haracterized by molecular modifications and irregular signaling axis [4, 5]. Today, immune cell therapy has attracted evolving attention in the context of breast cancer therapy. Immunotherapy circumvents immune cell tolerance and exhaustion thus inspiring the anti- tumor activity to fight cancer. This therapeutic approach includes but not restricted to immune checkpoint inhibitors (ICIs), cytokines, adoptive T cell therapy (ACT), and cancer vaccines [6, 7]. Meanwhile, growing evidences indicate that immune cell-derived exosomes can arbitrate communication between innate and adaptive immunity and thus moderate cancer development and metastasis.
Exosomes mainly are released by human cell, specifically tumor cell and immune cells. They are 30–150 nm in diameter, and could convey RNA, proteins and other biomolecules to target cells in vivo [8-10]. Exosome generation, release, and uptake of T lymphocyte cell-derived exosomes are tightly adjusted by intracellular proteins and extracellular incentives. T cell derived exosomes was found that could prohibit tumor cell progression [11, 12]. Of course, T cell- exosomes in some case was found that inhibit anti-cancer immunity in vivo [13]. Overall, they could show great capacity tumor diagnosis and immunotherapy and also could possibly be advanced for vaccination and chemotherapy agents transport.
In the current study, we assess the anti-cancer effects of the T cell derived exosomes on human breast cancer cell line MDA-MB-231 in vitro.

MATERIALS AND METHODS
Cell culture
The human blood samples were primed from healthy human donor for efficient isolation of T cell lymphocytes. Human PBMCs were firstly isolated utilizing the centrifugation by ficoll density gradient. After that, T lymphocytes were obtained by MACS concerning the producer instructions. ​Separated T lymphocyte then were cultivated in RPMI-1640 media (Sigma-Aldrich, Germany) enriched with 10% FBS (Sigma-Aldrich, Germany), and 1% pen/strep (PAA, Austria).
The breast cancer cells, MDA-MB-231 (ATCC), were expanded in DMEM enriched with FBS 10% and 1% pen/strep. Then, MDA-MB-231 and also isolated T cells were kept in humidified air with 5% CO2 at 37 °C.

Exosome isolation
T lymphocyte -derived exosomes were attained from the serum-free conditioned media (CM) by the responding kit (MagCapture™ Exosome Isolation Kit) concerning the producer recommendation. For efficient isolation of the exosome, the achieved CM was centrifuged at 10000 × g for 15 min to discard the other vesicles with similar properties. 

Transmission Electron Microscopy (TEM)
Briefly, the procured exosomes’ morphology was analyzed by the transmission electron microscopy (FEI Tecnai 12, Philips, Netherlands).

Western blotting
For evaluation of the CD9, 63 and CD81 expression, the parental T lymphocyte and isolated exosomes were firstly lysed in RIPA buffer (BioLegend, USA) and transported to PVDF membranes. Using primary and secondary anti-bodies, which were acquired from (Abcam, UK), the cited proteins expression was managed. 

MTT assay
The anti-cancer influences T cell- exosome on MDA-MB-231, firstly 5×104 cells were seeded within in each wells on the 96-well plates. MDA-MB231 cells then were treated with 25-400 ng/ml concentrations of exosomes within 12-72 hours of treatment. Following the adding of the 10 µL of 5 mg MTT/ml to wells, the OD of the well was measured at 570 nm by ELISA reader. 

Flow cytometric analysis of apoptosis
Utilizing the FITC Annexin V Apoptosis Detection Kit with PI (Biolegend, USA), MDA-MB-231 cells apoptosis was assessed following treatment with 100 ng/ml T cell-exosome at 24 and 48 hours of treatment. Then, 10 µL of propidium iodide (PI) and 5 µL of fluorescein isothiocyanate (FITC)-conjugated Annexin-V were added. By a FACSCalibur machine (BD Biosciences, USA), the fluorescent emission was recognized. Outcomes were assessed by FlowJo software.

RNA extraction and cDNA synthesis and Quantitative PCR
Exploiting the RNX Plus solution kit (Sinaclon, Iran) total RNA isolation was managed from MDA-MB-231 cells. Then, the cDNA was generated by the high-capacity kit (Bioneer, USA). For evaluation of the expression of candidate genes at mRNA levels, quantitative PCR was conducted. The generated cDNA, forward and reverse primers (Table 1), distilled water, and PrimeTime™ Master Mix (Idtdna, U.S.A) were used. Used primer pairs in Real Time-PCR, have been cited in Table 1.

Statistical Analysis 
Statistical analysis was managed using GraphPad Prism v7. The outcomes were showed as means ± SEM from 3 separate experiments and their differences were estimated using the Student’s t-test. The P-value less than 0.05 were considered as statistically significant.

RESULT AND DISCUSSION
Exosome characterizing
Western blotting results verified the exosome identity based on expression of CD9, CD63 and CD81 on T cell derived exosomes (Fig. 1). Besides, morphological analysis by TEM verified the efficient isolation of the exosomes from T cells. 

Exosome inhibits MDA-MB-231 proliferation
Concerning the MTT assay results, exosomes 25, 50, 100, 200 and 400 ng/ml abrogated the viability of MDA-MB-231 cell lines with 12-72 hours of exposure (P<0.05) (Fig. 2). Based on consequences, the suppressive impacts of exosomes on cell viability were time-dependent and dose-dependent (P<0.05) (Fig. 2). 
Previously, it has been signified that exosomes derived from activated T-cell enhance the growth of other anti-cancer immune cells [14, 15]. Exosomes convey great quantity of microRNAs and affect the biological action of the recipient cells [16, 17]. Immunological synapses stimulate T lymphocyte cell exosome transporting between T lymphocyte and APCs. On the other hand, T cell-derived exosomes may induce ERK and NF-κB signaling axis in some cancers, such as melanoma cells, leading to enhanced metastases of cancerous cells in part by up-regulating matrix metalloproteinase-9 (MMP9) expression [18].

Exosome stimulated MDA-MB-231 cell apoptosis
The apoptosis percentages of MDA-MB-231 cells were treated with exosome 100 ng/ml within 24 and 48 hours of treatment with Annexin-V/PI staining and FACS analysis. Accordingly, exosoms 100 ng/ml resulted in a significant enhancement in the apoptosis percentages of MDA-MB-231 cells within 1 and 2 days of exposure (P<0.05) (Figs. 3A, B). The apoptosis percentages in MDA-MB-231 cells (control) and MDA-MB-231 cells upon exposure with 100 ng/ml  at 1 and 2 days of treatment were 5.31±1.08, 17.78±2.98, 31.07.14±2.06, respectively (Figs. 3A, B).

Exosome inhibited MiR-27a expression in MDA-MB-231 cells
The expression levels of MiR-27a were estimated by real-time PCR in MDA-MB-231 cells upon treatment with exosomes 50 and 100 ng/ml during the 24 and 48 hours of treatment (Fig. 4A). Accordingly, exosomes  at 50 and 100 ng/ml concentrations down-regulated MiR-27a expression in MDA-MB-231 cell at 24 and 48 hours of treatment (P<0.05) (Fig. 4A). The suppressive effects were more evident at 100 ng/ml concentration and 48 hours of treatment.
Culminating evidences indicated the key role for miR-27a in adjusting cancer growth, metastasis along with chemotherapy resistance [19, 20]. The miR-27a is drastically related with the clinical phase and survival breast cancer patients. In this light, the improved levels of the miR-27a sustain breast cancer progression. Mechanistically, various tumor suppressor genes like ZBTB10, FOXO1 and prohibitin could be affected by miR-27a [21]. The miR-27a induces cancer development as well as metastasis through affecting zinc finger and BTB domain containing 10 (ZBTB10) [22]. Also, it reduces the expression of F-Box and WD Repeat Domain Containing 7 (FBXW7) to inspire epithelial-mesenchymal transition (EMT) and human breast tumor cell migration [23]. 

Exosome promoted Bax/Bcl-2 expression ratio in MDA-MB-231 cells 
The expression levels of Bax and Bcl-2 were estimated by real-time PCR in MDA-MB-231 cells upon treatment with exosomes 50 and 100ng/ml during the 24 and 48 hours of treatment (Fig. 4B, C). Accordingly, exosomes at 50 ng/ml concentration did not modify Bcl-2 expression in MDA-MB-231 cell at 24 hours of treatment (P<0.05) (Fig. 4B). However, exosome 50 ng/ml within 48 hours of treatment and exosome 100 ng/ml within 24 and 48 hours of treatment reduced Bcl-2 expression (P<0.05) (Fig. 4B). Besides, exosomes 50 and 100 ng/ml during the 24 and 48 hours of treatment improved Bax expression (P<0.05) (Fig. 4C). This effect was time and dose-dependent. 
It seems that improved expression of Bax in association with down-regulated Bcl-2 expression was ensured as result of miR-27a down-regulation by T cell derived exosome.

CONCLUSION
In this study, T cell derived exosome elicits a robust anti-cancer effect on MDA-MB-231 cells in part by improving Bax/Bcl-2 ratio as result of MiR-27a up-regulation and triggering the apoptosis pathway. Also, study of the detailed underlying mechanism is urgently required.

CONFLICT OF INTEREST
There is no conflict of interest.