Document Type : Original Research Article
1 Department of Surgery, Alborz University of Medical Sciences, Karaj, Alborz, Iran.
2 Department of Oral and Maxillofacial Pathology,Dentistry Faculty,Tabriz University of Medical Sciences,Tabriz,Iran.
3 Department of Obstetrics and Gynecology, Medical college of Iran university, Tehran, Iran.
4 Anesthesia technology department, Al-Turath University College , Al Mansour, Baghdad, Iraq.
5 Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
6 Dietary Supplements and Probiotic Research Center, Alborz University of Medical Sciences, Karaj, Iran.
7 Department of surgery, afzalipour hospital, kerman university of medical sciences, kerman, iran.
Different microenvironment parts of human tumors, like soluble mediators, tumor suppressive cells, and amended extracellular matrix (ECM) inspire tumor development and invasion [1, 2]. These mechanisms also may prevent effective antitumor effects of the immune cells. Numerous stromal cells usually are detected in tumor microenvironment (TME) and participate in the estimating clinical outcomes . These cells are largely described by altered molecular mechanism and deregulated signaling pathways [4, 5]. Now, tumor immunotherapy has become an efficient approach for targeting transformed cells. Immunotherapy avoids immune cell dysfunctions and consequently supports the anti-cancer impacts of immune cells [6, 7]. Meanwhile, using immune cells or derivate exosomes has attracted increasing attention. Accumulating proofs have showed that immune cell-derived exosomes facilitate connections between innate and adaptive immunity and thereby inhibit tumor progression .
Exosomes are typically secreted by a variety of human cells with 30–150 nm in diameter [9, 10]. They convey biological molecules such as protein and microRNAs. Through targeting a wide spectrum of procedures, immune cell-exosome prohibit cancer cell progression [11, 12]. Nonetheless, T cell- exosomes acts a dual role sometimes and may target anti-tumor functions of the other immune cells . Reports have exhibited that human immune cells can secret exosomes with either stimulatory and tolerogenic possessions into the TME, conferring the importance of their future application in tumor treatment. In sum, they offer unique strategy for cancer diagnosis and therapy.
Herein, we evaluate the anti-tumor influences of the T cell derived exosomes on human breast cancer cell line MCF-7, lung cancer A549 and liver carcinoma HepG2 cells.
MATERIALS AND METHODS
We used healthy human donor derived blood samples to attain T cell. In brief, PBMCs were primarily procured by employing the centrifugation using the ficoll density gradient. Then, T-cells were procured by magnetic cell sorting (MACS) based on the producer instructions. Isolate T cell then were expanded in RPMI-1640 media containing the 10% FBS, and 1% pen/strep all acquired from Sigma-Aldrich, Germany.
The MCF-7, A549 and HepG2 cells (ATCC), were expanded in DMEM containing the FBS 10% and 1% pen/strep. Then, cancer cells along with the procured T cells were kept in special conditions (5% CO2 at 37 °C).
T cell-exosome was acquired from the CM using the MagCapture™ Exosome Isolation Kit with respect to the producer recommendation. The CM was centrifuged by UC at 100,000 × g for 90 min. Then, suspension of the achieved pellet was conducted in PBS and RPMI 1640 medium.
The CD81 and CD9 expression was assessed in T cells and also their exosomes. Cells were lysed using the RIPA buffer (BioLegend, USA) and directed to PVDF. By specific primary and secondary antibody obtained from “Abcam, UK”, CD81 and CD9 expression was assessed.
To evaluate the anti-tumor impacts of exosome isolated from T cells on MCF-7, A549 and HepG2 cells, firstly 1×105 cells (100 µL) were cultured within 96-well plates. Upon, treatment with 100 ng/ml concentrations of exosomes at 12- 72 hours of exposure, 5 µL of 5-10 mg MTT/ml was added to each well. Finally, the wells OD then estimated at 570 nm using ELISA reader.
Flow cytometric analysis of apoptosis
Annexin V Apoptosis Detection Kit with PI (Biolegend, USA) was applied to evaluate the cytotoxicity of exosome on tumor cells apoptosis upon cell incubation with T cell-exosome (100 ng/ml) at 48 hours of exposure. Then, 5 µL of PI and FITC- Annexin-V were used. Finally, the fluorescent emission was detected.
Transmission electron microscopy (TEM)
To analyze the exosome’ morphology, the procured exosomes were evaluated by TEM. For negative staining, the exosomes suspension in PBS accomplished and then exosomes put on carbon-coated grids to conduct electron microscopy.
Statistical analysis was performed by GraphPad Prism. The results were presented as means ± SEM from 3 or 4 separate experiments. The statistical differences determined by Student’s t-test.
RESULT AND DISCUSSION
Characterizing of Exosome
Western blotting examination was executed to evaluate the expression of CD9 and CD81 on T cell-exosomes and also the morphology of exosomes (Fig. 1 A, B).
T cell-exosome inhibits cancer cells proliferation
Concerning the MTT assay results, exosomes 100 ng/ml decreased the viability of MCF-7, A549 and HepG2 cell lines during 12-72 hours of incubation (P<0.05) (Fig. 2). Respecting to outcomes, the exosomes-exerted inhibitory influences on cell viability were more evident within 72 hours of treatment and higher concentration (P<0.05) (Fig. 2). Further, the inhibitory effect on HepG2 cells were more evident than other cells, MCF-7 and A549.
The exosomes isolated from induced T-cell may improve the proliferation neighboring immune cells, thus inspiring the anti-tumor effects [14, 15]. Exosomes through the transporting of the microRNAs and other molecules modify the biological process in target cells [16, 17]. Immunological synapses could evoke the conveyance of exosome among immune cells such as T-cell and APCs . Further, T cell-exosomes can trigger ERK and NF-κB axes in malignant cells thus inspiring tumorigenesis expression . Furthermore, tumor-derived exosome mainly suppress CD8+ T cell activation and induces its apoptosis and exhaustion [20, 21].
T cell-exosome induces MCF-7, HepG2 and A549 cells apoptosis
The apoptosis percentages of all cell lines were assessed following exposure with exosome (100 ng/ml) within 48 h of treatment with by flowcytometry. In this light, exosoms therapy caused a marked increase in the apoptosis percentages of MCF-7, HepG2 and A549 cell within 48 hours of exposure (P<0.05) (Figs. 3A, B). The apoptosis percentages in MCF-7 was 19.38±1.88, in HepG2 was 18.84±2.04 and in A549 cell was 21.17±2.06 (Figs. 3A, B).
Rendering the achieved outcomes, human T cell derived exosome is capable of exerting anti-tumor effects in vitro. However, study of the proteome of T cell-exosome is required to elucidate the underlying mechanism. Also, evolving the novel potency test is of paramount importance to enable its translation to clinic.
CONFLICT OF INTEREST
The authors declare no conflict of interest.