- Email: [email protected]
staphylococcal enterotoxin B against fibrosarcoma tumor
Accepted Manuscript Macrophage cell-derived exosomes/staphylococcal enterotoxin B against fibrosarcoma tumor Elham Behzadi, Hamideh Mahmoodzadeh Hosseini, Raheleh Halabian, Abbas Ali Imani Fooladi PII:
S0882-4010(17)30863-X
DOI:
10.1016/j.micpath.2017.08.027
Reference:
YMPAT 2416
To appear in:
Microbial Pathogenesis
Received Date: 17 July 2017 Revised Date:
12 August 2017
Accepted Date: 16 August 2017
Please cite this article as: Behzadi E, Hosseini HM, Halabian R, Fooladi AAI, Macrophage cell-derived exosomes/staphylococcal enterotoxin B against fibrosarcoma tumor, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.08.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Macrophage cell-derived exosomes/staphylococcal enterotoxin B against fibrosarcoma tumor Running title:
Exosomal Macrophage/SEB and fibrosarcoma
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Elham Behzadia, Hamideh Mahmoodzadeh Hosseinia*, Raheleh Halabiana, Abbas Ali Imani Fooladia* Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
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*Corresponding Authors: Abbas Ali Imani Fooladi, PhD
Hamideh Mahmoodzadeh Hosseini, PhD Email: [email protected]
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Email: [email protected]
Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Vanak Sq. Mollasadra St., Tehran - Iran. P.O. Box 19395-5487
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Tel: +98 21 82482568 Fax: +98 21 88068924
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ACCEPTED MANUSCRIPT Abstract Targeted immune therapies are a modern approach to harness the immunity to treat cancer patients. Exosomes (EXOs) are nano-vesicles used for drug delivery in cancer treatment. We aimed to assess the effectiveness of novel designed EXO structures for immunotherapy alone and in combination with other components in animal models.
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EXO derived from untreated macrophage (EXO), WEHI-164 cell lysate treated EXO (EXOLys), HSP70 enriched WEHI-164 cell lysate treated EXO (EXOHSP70), Naloxone (NLX) treated EXO (EXONLX), Propranolol (PRP) treated EXO (EXOPRP) and staphylococcal enterotoxin B (SEB) anchored to three kinds of EXOs designated as
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EXO/SEB, EXOLys/SEB, EXOHSP70/SEB were purified from J774 cell line. To determine the therapeutic effect of these novel constructed nano-vesicles, the animals were immunized with different types of EXOs at weekly intervals for three consecutive weeks and in the fourth week the WEHI-164 tumor cells were injected. Finally,
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the splenocyte proliferation was examined by MTT assay and tumor growth was also determined in each group. We observed that EXOHSP was more effective than EXO and EXOLys to decrease the number of tumor cells and to stimulate immune responses in animal models (P <0.05). In SEB-anchored EXO group, EXOHSP70/SEB has the potency to stimulate immune responses more efficiently than EXO/SEB and EXOLys/SEB and the tumor was not palpable until 28th day which may refer to synergistic effect of HSP70 and SEB on immunity. In EXONLX
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treated mice proliferative response decreased significantly compared to control group (P >0.05) and the tumor number was constant within a period of 28 days and EXOPRP may delay the occurrence of the fibrosarcoma tumor; After development of fibrosarcoma the number of tumors diminished over the studied period of time.
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Our results demonstrate that HSP70 enriched EXO is an effective immunoadjuvant in cancer immunotherapy and causes tumor regression in animal model.
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Key words: Exosome; HSP70; staphylococcal enterotoxin B; Tumor regression; fibrosarcoma
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ACCEPTED MANUSCRIPT 1. Introduction Cancer is the one of the leading causes of death in many parts of the world, but the results of different interventions are poor. The conventional treatments for most cancer patients are a combination of surgery, radiation and/or chemotherapy. In most cases, the primary tumor can be treated with a combination of above
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mentioned therapies, but it is not efficient for the eradication of disseminated tumor cells (DTCs) in the blood circulation and micro-metastases in other organs [1]. Therefore, the main strategy to combat malignancies is cancer immunotherapy by monoclonal antibodies to target tumor specific antigens and cellular therapy using cytotoxic T lymphocytes and dendritic cells [2].
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One type of malignant tumors with mesenchymal cell origin called fibrosarcoma, which arises from fibroblasts predominantly found in the area around bones or in the soft tissues [3]. The primary treatment of fibrosarcoma is
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surgical removal, but recurrence may occur years after treatment [4].
The most innovative strategy for immune cancer therapy is using major histocompatibility complex (MHC) class II-bearing exosomes (EXOs) as drug delivery vehicles in cancer models [5-7]. EXOs are small endogenous vesicles of 40–150 nm in diameter, which are present in nearly all biological fluids and formation of these nanovesicles are through the reverse budding of the late endosomal membrane to develop a multivesicular body
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(MVB). Upon the fusion of MVBs with the plasma membrane, EXOs are released into the extracellular space and can reach and interact with target cells [8-10]. EXOs can contain different kinds of immunostimulatory molecules and antigen-presenting cells (APCs)-derived EXOs can also express MHC II, elicit strong antigenspecific T cell, B cell and NK cell responses and abolish tumors in mouse models [5, 10].
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Normally, professional APCs of the immune system such as dendritic cells (DCs), B cells and macrophages (MΦ), constitutively express MHC class II and have a major role in inducing effective adaptive immune
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response and T cells may recognize antigen complexed with MHC II using T cell receptors (TCRs) on their surface [11]. APCs can also combat tumors via stimulation of B and cytotoxic T cells to respectively provide antibodies against tumor-related antigens and destroy tumor cells [12]. Studies show that many factors can affect APCs activity and improve anti-tumor immunity, including heat shock proteins (HSPs) [13, 14], naloxone (NLX) [15] and propranolol (PRP) [13, 16, 17]. HSPs or stress proteins are conserved proteins that are able to elicit the innate and adaptive immune systems and have essential function in antigen presentation, expression of innate receptors and tumor immunosurveillance [18, 19].
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ACCEPTED MANUSCRIPT Some HSPs derived from cancer cells, such as HSP70, HSP90 and glucose-regulated protein 96 (gp96) have been shown to initiate specific immunity through the transportation of antigenic peptides to APCs to consequently activate tumor-antigen-specific cytotoxic T-lymphocytes (CTL). However, interaction of HSPs with APCs also leads to maturation of DCs as well as release of pro-inflammatory cytokines by MΦs and DCs, hereby providing an efficient induction of immune responses to fight malignant cells [14].
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In this context of cancer therapy, several studies executed on NLX, an opioid receptor antagonist, showed contradictory results, declared both immunosuppressive and immunostimulatory effects of opioid peptides [20]. It is also indicated that NLX enhances cell mediated immunity [15] and can induce a significant increase in the
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lymphocyte proliferation during primary viral infection [21].
Besides, PRP is a beta angiotensin receptor blocker (β-AR blocker), that a number of in vivo and in vitro studies indicated that it has anti-proliferative, anti-migratory, anti-angiogenic and cytotoxic properties. PRP prevents or
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reduces cancer progression through inhibiting cAMP—responsive element–binding protein (CREB), NF-kB, and activator protein (AP-1), inducing apoptosis, or reducing matrix metalloproteinase (MMP)-9 activation and tumor angiogenesis [16, 17].
Furthermore, staphylococcal enterotoxins, particularly type B (SEB), are bacterial superantigens which can form a complex with MHC II on APCs and TCR on CD4+ and CD8+ T cells and detour routine antigen processing
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and presentation pathway [22, 23]. This binding causes hyperactivation of T cells accompanied by an increased release of Th1 cytokines, specifically interleukin 2 (IL-2), tumor necrosis factor β (TNF-β), and interferons. As it is known, T cells have a significant role in the eradication of malignant cells and host cells infected with the
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intracellular pathogens [23, 24]. In addition, SEB combined with MHC class II and CD80 has adjuvant activity against metastatic cancer in animal models [25].
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Furthermore, it has been demonstrated that tumor-derived EXOs are composed of cytosolic and membranous tumor antigens associated with antigen presenting molecules which makes them a good candidate for cancer immunotherapy [26]; but as tumor-derived EXOs carry large quantities of antigens, the possibility of anergy increases. Therefore we hypothesized that MΦ-derived EXOs could solve this problem [10]. In brief, as fibrosarcoma usually escapes from presenting its own antigens to T cells, tumor immunotherapy is the best option to elicit a tumor-specific response that contributes to the elimination of the tumor [27]; Therefore, in this study, we tried to assess the effectiveness of some immunotherapy treatments against fibrosarcoma, alone and in combination with other components, in mouse tumor models.
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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. WEHI-164 Cell Lysate Preparation WEHI-164 mouse BALB/c fibrosarcoma cells were obtained from the Pasteur Institute (Tehran, Iran). WEHI164 cells were grown in T75 flasks (Bioidea, Tehran, IRAN) containing RPMI 1640 medium (Bioidea, Tehran, IRAN) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Inoclon, Tehran, IRAN), 100
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U/ml penicillin and 100 µg/ml streptomycin (Inoclon, Tehran, IRAN) incubated in 5% CO2 and 95% air at 37 °C.
Once the adherent cells reached approximately 80% confluence, the medium was removed and attached cells
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washed three times with phosphate buffered saline (PBS) and detached using trypsin/EDTA (Inoclon, Tehran, IRAN) for further treatment.
Subsequently, WEHI-164 cell lysates were prepared following a previously published method [28] with some
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modifications. Briefly, cell suspensions in PBS were pellet by centrifugation at 2500×g for 5 min and RIPA buffer (phosphate-buffered saline, pH 7.4, 0.5% deoxycholate sodium, 0.1% SDS, 1% Triton X-100) [29] containing protease inhibitors (phenylmethylsulfonyl fluoride, 100 µg/ml; sodium vanadate, 0.4 mM; pepstatin A, 1 µg/ml; leupeptin, 1 µg/ml) was added to pellet following sonication for 30 seconds with 50% pulse to increase the yields. Obtained mixture was shaken gently for 15 min on ice and centrifuged at 14000×g for 15
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min at 4 °C to pellet debris. Supernatant transferred to a new tube for further analysis and stored at -80 °C for later examinations. The protein concentration of total protein was evaluated at 280 nm using a Nanodrop 2000
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spectrophotometer (Thermo-Scientific, US).
2.2. Induction of HSP70 in WEHI-164 Cell Lines
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To increase the expression of Heat Shock Protein 70 (HSP70), in the logarithmic growth phase, the WEHI-164 cells were heated by the incubation of the cells at 42 °C for 60 min [30]; the temperature was controlled within ± 0.1 °C. Then, the cells were collected after 12 h by detachment with trypsin/EDTA (Inoclon, Tehran, IRAN), washed 3 times with phosphate buffered saline (PBS, pH 7.4, centrifuged at 1500 rpm for 5 min). These treated cells were used to prepare HSP70 enriched WEHI-164 cell lysate by above-mentioned method.
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ACCEPTED MANUSCRIPT 2.3. EXO Preparation and Purification J774 cell line from mouse BALB/c monocyte macrophage was obtained from the Pasteur Institute (Tehran, Iran). J774 cell lines were grown in T175 flasks (Bioidea, Tehran, IRAN) containing RPMI 1640 medium (Bioidea, Tehran, IRAN) supplemented with 10% (v/v) fetal bovine serum (FBS) (Inoclon, Tehran, IRAN), 100 U/ml penicillin and 100 µg/ml streptomycin (Inoclon, Tehran, IRAN) incubated in 5% CO2 and 95% air at 37
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°C.
When J774 cell line growth in the T175 flasks (Bioidea, Tehran, IRAN) reached 80% confluence, attached cells were washed three times with PBS to omit FBS and then the fresh medium without FBS was added to each
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flask.
In the next rounds, while adding fresh medium without FBS to each flask, 100 µg/ml of WEHI-164 cell lysate, 100 µg/ml of HSP70 enriched WEHI-164 cell lysate, 10-5M PRP (Tolid Daru Co., Tehran, Iran) [31] and 0.1
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µM NLX (Tolid Daru Co., Tehran, Iran) [32] was added to each flask, separately.
After 24 h, the supernatant of each flask poured to a fresh tube and EXOs were purified using previously published method described by Mahmoodzadeh Hosseini et al. [23]. First, the supernatant was centrifuged at 10000×g for 15 min at 4 ºC to remove debris. Then it was filtered using a 0.22-µm filter (GSV filter technology, USA).
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In the next step, a Centricon Plus-70 centrifugal filter device (Millipore, MA, USA) was used to separate EXOs from supernatant and stored at -80 ºC for future applications. The protein concentration of purified EXOs was evaluated at 280 nm using a Nanodrop 2000 spectrophotometer (Thermo-Scientific, US).
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2.4. Transmission Electron Microscopy
Transmission Electron Microscopy (TEM) was used to assess the morphology and the size of purified EXOs.
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Isolated EXOs suspended in PBS (pH 7.4) fixed with 2.5% glutaraldehyde and then transferred onto a formware/carbon coated grid (Iran University of Medical Sciences, Tehran, IRAN). The grid incubated for 20 min at room temperature, which subsequently transferred to 50 µl of uranyl oxalate (pH 7; Merck, Darmstadt, Germany) for 5 min and then washed with PBS. In the next step, the grid surface was covered with methylcellulose/uranyl acetate (Merck, Darmstadt, Germany) and allowed to stand for 10 min on ice. The excess fluid was blotted off by Whatman filter paper No.1 and the grid was air-dried 5 to 10 min. Ultimately, the morphology and the size of EXOs were observed by Carl Zeiss-Leo 906 TEM (Oberkochen, Germany) at 80 kV and
EXO
sizes
were
determined
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from
the
scale
bar.
ACCEPTED MANUSCRIPT 2.5. Protein Anchorage of SEB on EXOs To anchor SEB on EXO, the protocol described by Mahmoodzadeh Hosseini et al. [23] was utilized. In short, for incorporation of SEB onto the EXO surface, 10 µg SEB (Sigma-Aldrich, St. Louis, USA) was added to 100 µg of purified EXOs (Normal EXO (EXO), HSP70 enriched WEHI-164 cell lysate treated EXO (EXOHSP70) and WEHI-164 cell lysate treated EXO (EXOLys)) in 100 µl PBS. The mixture was shaken at 1000 rpm for 4 h at 37
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°C. Then, the mixture filtered using ultrafree-0.5 biomax100k (Millipore, MA, USA) at 3000×g for 20 min to omit the unbounded SEB. The EXOs, EXOHSP70 and EXOLys attached to SEB were named an EXO/SEB, EXOHSP70/SEB and EXOLys/SEB.
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2.6. Vaccination Protocol and the Tumor Challenge
To examine the effectiveness of prepared vaccines, a total of fifty inbred male Balb/c mice, 3-5 weeks age were purchased from the Pasteur Institute (Tehran, Iran) and kept under recommended conditions according to
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institutional ethical guidelines. The mice were randomly divided into 10 groups of five as follows: 1: PBS (Negative Control), 2: EXO, 3: EXOHSP70, 4: EXOLys, 5: NLX treated EXO (EXONLX), 6: PRP treated EXO (EXOPRP), 7: EXO/SEB, 8: EXOLys/SEB, 9: EXOHSP70/SEB, 10: SEB (Negative Control). Vaccination was performed subcutaneously into the shaved right flank with 10 µg of each component mentioned above at weekly intervals for three consecutive weeks and a week after the last immunization, approximately
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2×106 cells/100 µl of WEHI-164 cells were inoculated to the right flank of the mice to produce fibrosarcoma tumors. Hence, palpable tumors were generated after 7 days and the tumor growth was observed every 7 days. Observation of the tumors was continued until the end of the fourth week after tumor cell injection. The
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behavior of all mice was normal and no sign of pain or any disorder or suffering from inoculations was noticed.
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ACCEPTED MANUSCRIPT 2.7. Proliferation Assay Four weeks after WEHI-164 tumor cell injection, the spleens were removed from sacrificed mice under sterile condition and splenocytes were isolated as described previously [33] The splenocytes from the PBS injected mice were used as a negative control. Briefly, spleens were mashed neatly and passed through 100-µm filters to collect a single-cell suspension and erythrocytes were lysed by ACK lysis buffer (NH4Cl, KHCO3, Na2EDTA)
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at room temperature. The splenocytes were washed and resuspended in RPMI 1640 (Bioidea, Tehran, IRAN), supplemented with 10% FBS (Inoclon, Tehran, IRAN) penicillin, (100 U/ml) and streptomycin (100 mg/ml) (Inoclon, Tehran, IRAN).
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The splenocytes at the concentration of 1×105 cell/well were seeded on each well of a 96-well plate. To stimulate the cells, lysate antigens of the heat shocked and non-heat shocked WEHI-164 cells were exploited in the total volume of 200 µl. The microtiter plates were incubated in a humidified atmosphere containing 5% CO2
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for 3 days at 37°C. The lymphocyte proliferation was determined by adding 20 µl MTT (3, (4, 5dimethylthiazal-2-yl)-2,5-diphenyl tetrazolium bromide) reagent (5 mg/ml) (Sigma-Aldrich, St. Louis, USA) to each well and incubated for 4 h at 37 °C. The supernatants were removed and 100 µl dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St. Louis, USA) were added. Ultimately, the formazan crystals were dissolved and the absorbance of each well was calculated by an ELISA micro plate reader (Bio-Rad, CA, USA) at 570 nm. All
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tests were carried out in triplicate.
The results of the proliferation assay were declared as a stimulation index (SI), which was determined by dividing the mean optical density (OD) of test culture groups by the OD of the control culture group.
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2.8. Statistical analysis
The nonparametric Mann–Whitney test was used to evaluate data obtained from all tests using SPSS.15
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software (SPSS, Chicago, IL, USA). A p-value less than 0.05 was considered statistically significant.
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ACCEPTED MANUSCRIPT 3. Results 3.1. Identification of EXOs Purified from J774 Cells EXOs were negatively stained with uranyl acetate and the morphology and the size of purified EXOs were evaluated by TEM. As it is demonstrated in Fig. 1, the results revealed round-shaped membranous nano-sized with
40–150
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in
diameters
consistent
with
previous
defined
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vesicles
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ACCEPTED MANUSCRIPT 3.2. Tumor Number The tumor numbers on the days 14, 21 and 28 were calculated in ten groups of experimental mice. The results designated that the tumors in some test groups grew more slowly than those in the control groups (PBS and SEB injected groups). The tumor numbers in EXOHSP70 and EXOLys injected groups decreased over the time comparing to EXO
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injected group and control group (Fig. 2a).
The tumor numbers in EXO/SEB, EXOLys/SEB and EXOHSP70/SEB injected groups were compared to two control groups (PBS and SEB). The best response was obtained in EXOHSP70/SEB injected group as it is
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demonstrated in Fig. 2b.
The tumor numbers in EXOPRP injected group decreased over the time comparing to EXONLX injected group and control group. In EXONLX injected group the numbers of tumors were constant over that period of time (Fig. 2c).
results
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Statistics show that there is no difference between EXONLX and various EXO-anchored SEB groups (P>0.05),
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compared
to
the
same
groups
(P>0.05).
ACCEPTED MANUSCRIPT 3.3. Cell proliferation analysis (MTT assay) The proliferation of spleen lymphocytes is indicative of the state of the immune system. Splenocytes obtained from mice test groups were examined for their lymphocyte proliferative response. Cell proliferation was evaluated by MTT assay. Four weeks after tumor cell injection in treated groups (EXO, EXOHSP, EXOLys, EXONLX, EXOPRP, EXO/SEB,
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EXOLys/SEB, EXOHSP/SEB) and control groups (SEB and PBS treated mice), spleen cells were collected, cultured and stimulated with ex vivo tumor antigens.
The results shown in Fig. 3a designate that splenocyte stimulation index in EXOHSP70 treated mice had changed
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prominently compared to the control group and EXO and EXOLys groups; Statistically significant difference in the stimulation index was observed in EXOHSP70 treated group compared to the control group (P <0.05). The splenocyte stimulation index in EXO treated group was also changed notably compared to the control group but
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in EXOLys treated group no significant changes were observed.
The stimulation index (SI) in Fig. 3b indicates that splenocyte proliferative responses in SEB treated mice had changed significantly compared to other groups and statistically significant difference in cell proliferation was observed compared to EXO/SEB, EXOlys/SEB and EXO HSP70/SEB groups (P <0.05). The results shown that SEB anchorage on EXO, EXOLys and EXOHSP has an inhibitory effect on immune responses. Splenocyte
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proliferative responses in EXO/SEB and EXOLys/SEB groups had changed compared to PBS injected but statistically was not significance (P>0.05).
The stimulation index (SI) in Fig. 3c indicate that splenocyte proliferative responses in EXOPRP and EXONLX
4. Discussion
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treated mice did not change significantly and was lower than control group (P <0.05).
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It is believed that an effective vaccine against malignant cells should be potentially strong and have the ability to induce cellular and humoral immunity. Generally the basic mechanism of cancer immunotherapy is to elicit a specific immune response, including the induction of specific immune effector T cells, to suppress target cells. Recently, it has been shown that EXOs as acellular vehicles possess the ability to induce immune responses and therefore can be used as a cancer therapy option [23]. Studies conducted on DC-derived EXOs revealed that these vesicles have the potential to develop anti-tumor immune responses in vivo [36]. But as the MΦs are the most prominent immune cell recruited to the tumor environment and are present in all stages of tumor progression, it may be of great interest to examine the MΦderived EXOs impact on tumor progression. Most of the studies on mouse models demonstrated that MΦs have
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ACCEPTED MANUSCRIPT pro-tumoral role and can stimulate angiogenesis, which consequently enhance tumor invasion [37]. But researchers succeeded to activate MΦs to become tumoricidal [38]. In a recent research a monoclonal antibody targeted at a protein on their cell surface, which stopped the tumors growth and dissemination in vivo [39]. As the tumor cells are poorly immunogenic, a new effective cancer therapy approach may cause a great evolution in the treatment of cancer patients.
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It has been shown that interaction of EXOs with different cell types is not accidental and is dependent on expressed molecules on EXOs and the target cells [34, 35], therefore, we examined the hypothesis that MΦEXOs would exhibit expected function to increase tumor cell antigenicity/immunogenicity.
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Here, we obtained MΦ-derived EXOs to examine the effect of MΦ antigens alone on WEHI-164 tumor progression. We also stimulated J774 cell line with WEHI-164 cell lysate and HSP70 enriched WEHI-164 cell lysate to obtain EXOs containing tumor antigens (EXOLys) and enriched with HSP70 (EXOHSP), respectively.
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Our results show (Fig. 3a) that EXOHSP is more effective than EXO and EXOLys to stimulate immune responses in animal models. This finding may be related to the presence of HSP70 in heat treated WEHI-164 cell lysate, which can stimulate MΦs to have better immunogenic properties. Hence, HSPs are recruited for immunotherapy in cancer models [42, 43]. As it can be seen in Fig. 3a, MΦ-derived EXO (EXO) elicit greater splenocyte proliferative responses in comparison with EXOLys which is in contrast with the result obtained from Cho et al.
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(2005) study [44]; They found that tumor cell-derived EXOs containing tumor antigens, are able of triggering an efficient immune response. They concluded that tumor-derived EXOs can cover the defect of APC-derived EXOs and may be used as a potent cell-free cancer vaccine [43, 44]. Yet, exploiting APC-derived EXOs for
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immunotherapy has some limitations, because the identified tumor antigens are not accessible easily to be pulsed with the APCs [43]. Mahmoodzadeh Hosseini et al. (2014) indicated that tumor-derived EXOs carry
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large amount of antigens, enhances the chance of immune anergy which in turn, suppresses the anti-tumor immunity [23]. This defect may be compensated by the presence of HSP70 as a potent immune stimulator [43]. Here, we believe that MΦ-derived EXOs lack immune anergy and therefore they elicit stronger stimulatory effects compared to EXOlys group. The results also show that the number of tumors in EXOHSP70 vaccinated group diminished in comparison with other tested groups and PBS injected group (Fig. 3b) which can be attributed to the synergistic effect of exosomal components and HSP70 [43]. As tumor cell antigens are usually undetectable for T cells; the main purpose of cancer immunotherapy is to produce tumor-specific T cells to identify and eradicate malignant cells [45]. SAgs such as SEB has the
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ACCEPTED MANUSCRIPT potential to simultaneously bind to the MHC class II on antigen presenting cells and the T cell receptor on T cells, cause massive T-cell activation and results in the secretion of a relatively large amount of cytokines. Hence, they seem a convenient choice in cancer immunotherapy [27]. In brief, SEB is an exotoxin of Staphylococcus aureus with superantigenic properties that may act as a potential agent to promote immune responses against tumor cell and can prevent both tumor growth and metastasis [46].
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In this study, we designed a two-part structure containing MΦ-derived EXO and SEB to study the effect of this structure on WEHI-164 tumor cells progression in mouse models. Three types of SEB-anchored EXOs designated as EXO/SEB, EXOLys/SEB and EXOHSP70/SEB, were synthesized. The conjunction of SEB to all
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types of EXOs was conducted through glycosylphosphatidylinositol (GPI) anchor using protein transfer method. The SEB anchorage depends on the EXOs membrane composition which is rich in lipid content such as sphingomyelin, cholesterol and glycolipid in combination with tetraspanin proteins that make the anchorage
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possible [23, 47]. Our results (Fig. 3b) show that EXOHSP70/SEB has the potency to stimulate immune responses more efficiently than EXO/SEB and EXOLys/SEB. This finding is in accordance with previous results reported by Cho et al (2009). They found that heat treatment increases the HSP70 content in EXOs and consequently enhances the immune stimulating activity of EXOs. The data suggest that EXOHSP70/SEB could activate the immune response to inhibit tumor progression. This consequence may refer to immune stimulating activities and
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Th1 anti-tumor effects of EXOHSP70/SEB structure [43].
Our data also show that SEB induces immune responses more effectively than SEB-anchored EXO, which is in contrast with the findings of the survey conducted by Mahmoodzadeh Hosseini et al. (2014) on breast tumor
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cancer [23]. We suggest that these contradictory results may be referred to the different nature of fibrosarcoma and breast cancer cell lines. Nevertheless, Imani Fooladi et al. (2008) demonstrated that SEB can induce
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cytotoxic T-cell activity and increase in cytokine levels in mice with fibrosarcoma, suggest that SEB may be a suitable choice to treat patients with fibrosarcoma [27]. We also observed that EXOHSP70/SEB vaccinated group did not develop tumors on the 7th and 14th day (Fig. 2b) and on 28th day small tumors were palpable just in two groups of five which may refer to the synergistic effect of HSP70 and SEB on immunity. In this case, enrichment of HSP70 in EXOs may have an adjuvant effect to upregulate Th1 immunity and cause resistance to tumor development [23, 43]. On the other hand, it has been demonstrated that general opioid antagonists like NLX have an inhibitory growth impact on malignant cells and modifies cell proliferative events through interaction with opioid receptors [48]. Some studies indicate that NLX exert this effect through stimulation of Th1 immune responses by increasing the
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ACCEPTED MANUSCRIPT level of IFN-γ compared to IL-2 [49] and inhibit regulatory T cells [15]. Furthermore, it is defined that the NLX has the adjuvant properties which cause lymphocytes to proliferate. It seems that the NLX function at injection site by influencing local APCs, production of pre-inflammatory cytokines and causing inflammatory responses [49] Here, we developed MΦ-derived EXOs containing NLX- designated as EXONLX -through exposing MΦ cell
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culture to 0.1 µM NLX to improve anti-tumor immunity of MΦ-derived EXOs in BALB/c mouse model with fibrosarcoma tumor. The results indicated that splenocyte proliferative responses in that EXONLX treated mice had decreased significantly compared to control group and the tumor number was constant during a period of 28
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days. These findings are in contrast with previous study done by Hassan et al. (2009) [15] which reported a significant increase in the proliferation of splenocytes and IFN-γ production in fibrosarcoma-bearing mice treated with co-administration of gp96 vaccine and naloxone [15, 49]. These conflicting results may be referred
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to different treatment approaches used in both studies.
As the most promising strategy to treat cancer is based on the enhancement of cell mediated immunity, we tried to investigate the effect of PRP, a β-AR drug on fibrosarcoma-bearing mice. Our results show that EXOPRP may delay the occurrence of the fibrosarcoma tumor, which is in accordance with the results obtained by Khalili et al. (2013) [16].
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In addition, our results demonstrate that HSP70 enriched EXO is an effective option in cancer immunotherapy and causes tumor regression in animal model.
Since there is a little information about the effect of EXOs with tested components on tumor growth inhibition
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further investigations are necessary to determine the actual effect of these nano-vesicles for cancer therapy. All in all, based on the obtained data and the study done by Bahreyi Toosi et al. (2004), Balb/c mouse is a good
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animal model for short-term studies on cancer immunotherapy; but for studies longer than 9 days the secretion of TNF may cause tumor reduction. Therefore, for obtaining more reliable results or investigating on human tumor cell lines the nude mice may be the best option to be used [50]. In conclusion, it is proposed to do a more extensive analysis on exosome based vaccine to improve the understanding of its anti-tumor activity and the methodology used here is valuable to other researchers to pursue the clinical and research studies on exosomes.
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ACCEPTED MANUSCRIPT Acknowledgment This study is a part of the thesis research work carried out by Elham Behzadi, PhD student of Applied Microbiology. The work was approved by the Research Vice-chancellor of Baqiyatallah University of Medical Sciences (Record No. 414, dated March 16, 2015). This work was supported by the grant from the Iranian
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National Science Foundation (INSF).
Conflict of interest
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The authors declare no conflicts of interest.
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ACCEPTED MANUSCRIPT References
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1 Schuster M, Nechansky A, Kircheis R: Cancer immunotherapy. Biotechnology journal 2006;1:138-147. 2 Karlitepe A, Ozalp O, Avci CB: New approaches for cancer immunotherapy. Tumor Biology 2015;36:4075-4078. 3 Burningham Z, Hashibe M, Spector L, Schiffman JD: The epidemiology of sarcoma. Clinical sarcoma research 2012;2:1. 4 Wong SL: Diagnosis and management of desmoid tumors and fibrosarcoma. Journal of surgical oncology 2008;97:554-558. 5 Gehrmann U, Näslund TI, Hiltbrunner S, Larssen P, Gabrielsson S: Harnessing the exosomeinduced immune response for cancer immunotherapy: Seminars in cancer biology, Elsevier, 2014, 28, pp 58-67. 6 Greening DW, Gopal SK, Xu R, Simpson RJ, Chen W: Exosomes and their roles in immune regulation and cancer: Seminars in cell & developmental biology, Elsevier, 2015, 40, pp 72-81. 7 Johnsen KB, Gudbergsson JM, Skov MN, Pilgaard L, Moos T, Duroux M: A comprehensive overview of exosomes as drug delivery vehicles—endogenous nanocarriers for targeted cancer therapy. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 2014;1846:75-87. 8 Beninson LA, Fleshner M: Exosomes: An emerging factor in stress-induced immunomodulation: Seminars in immunology, Elsevier, 2014, 26, pp 394-401. 9 Mahmoodzadeh Hosseini H, Ali Imani Fooladi A, Reza Nourani M, Ghanezadeh F: The role of exosomes in infectious diseases. Inflammation & Allergy-Drug Targets (Formerly Current Drug TargetsInflammation & Allergy) 2013;12:29-37. 10 Hosseini HM, Soleimanirad J, Aghdam EM, Amin M, Fooladi AAI: Texosome-anchored superantigen triggers apoptosis in original ovarian cancer cells. Medical Oncology 2015;32:1-8. 11 Kambayashi T, Laufer TM: Atypical mhc class ii-expressing antigen-presenting cells: Can anything replace a dendritic cell? Nature Reviews Immunology 2014;14:719-730. 12 Eggermont LJ, Paulis LE, Tel J, Figdor CG: Towards efficient cancer immunotherapy: Advances in developing artificial antigen-presenting cells. Trends in biotechnology 2014;32:456-465. 13 Khalili A, Shahabi S, Pourfathollah AA, Ostad SN, Noori S, Mahdavi M, Shajiei A, Hassan ZM: Reduced treg and onset of a th1pattern in combined hsp70 and propranolol treatment of fibrosarcomabearing mice. 14 Hashemi SM, Hassan ZM, Soudi S, Shahabi S: The effect of vaccination with the lysate of heatshocked tumor cells on nitric oxide production in balb/c mice with fibrosarcoma tumor. Cell biology international 2008;32:835-840. 15 Hassan ATM, Hassan ZM, Moazzeni SM, Mostafaie A, Shahabi S, Ebtekar M, Hashemi SM: Naloxone can improve the anti-tumor immunity by reducing the cd4+ cd25+ foxp3+ regulatory t cells in balb/c mice. International immunopharmacology 2009;9:1381-1386. 16 Khalili A, Hassan ZM, Shahabi S, Pourfathollah AA, Ostad SN, Noori S, Mahdavi M, Haybar H, Langroudi L: Long acting propranolol and hsp-70 rich tumor lysate reduce tumor growth and enhance immune response against fibrosarcoma in balb/c mice. Iranian Journal of Immunology 2013;10:70. 17 Pasquier E, Ciccolini J, Carre M, Giacometti S, Fanciullino R, Pouchy C, Montero M-P, Serdjebi C, Kavallaris M, André N: Propranolol potentiates the anti-angiogenic effects and anti-tumor efficacy of chemotherapy agents: Implication in breast cancer treatment. Oncotarget 2011;2:797-809. 18 Binder RJ: Functions of heat shock proteins in pathways of the innate and adaptive immune system. The Journal of Immunology 2014;193:5765-5771. 19 Zhang Y, Zheng L: Tumor immunotherapy based on tumor‑derived heat shock proteins (review). Oncology letters 2013;6:1543-1549. 20 Sacerdote P, Manfredi B, Gaspani L, Panerai AE: The opioid antagonist naloxone induces a shift from type 2 to type 1 cytokine pattern in balb/cj mice. Blood 2000;95:2031-2036. 21 Jamali A, Mahdavi M, Shahabi S, Hassan ZM, Sabahi F, Javan M, Farsani MJ, Parsania M, Bamdad T: Naloxone, an opioid receptor antagonist, enhances induction of protective immunity against hsv-1 infection in balb/c mice. Microbial pathogenesis 2007;43:217-223.
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22 Lindsay C, Griffiths G: Addressing bioterrorism concerns options for investigating the mechanism of action of staphylococcus aureus enterotoxin b. Human & experimental toxicology 2013;32:606-619. 23 Hosseini HM, Fooladi AAI, Soleimanirad J, Nourani MR, Davaran S, Mahdavi M: Staphylococcal entorotoxin b anchored exosome induces apoptosis in negative esterogen receptor breast cancer cells. Tumor Biology 2014;35:3699-3707. 24 Fooladi AAI, Halabian R, Mahdavi M, Amin M, Hosseini HM: Staphylococcal enterotoxin b/texosomes as a candidate for breast cancer immunotherapy. Tumor Biology 2016;37:739-748. 25 Pulaski BA, Terman DS, Khan S, Muller E, Ostrand-Rosenberg S: Cooperativity of staphylococcal aureus enterotoxin b superantigen, major histocompatibility complex class ii, and cd80 for immunotherapy of advanced spontaneous metastases in a clinically relevant postoperative mouse breast cancer model. Cancer research 2000;60:2710-2715. 26 Mahmoodzadeh HH, Ali IFA, Soleimanirad J, Reza NM, Mahdavi M: Exosome/staphylococcal enterotoxin b, an anti tumor compound against pancreatic cancer. Journal of BU ON: official journal of the Balkan Union of Oncology 2013;19:440-448. 27 Fooladi AAI, Sattari M, Hassan ZM, Mahdavi M, Azizi T, Horii A: In vivo induction of necrosis in mice fibrosarcoma via intravenous injection of type b staphylococcal enterotoxin. Biotechnology letters 2008;30:2053-2059. 28 Jo M, Stolz DB, Esplen JE, Dorko K, Michalopoulos GK, Strom SC: Cross-talk between epidermal growth factor receptor and c-met signal pathways in transformed cells. Journal of Biological Chemistry 2000;275:8806-8811. 29 Suresh A, Dunaevsky A: Preparation of synaptosomes from the motor cortex of motor skill trained mice. Bio-protocol 2015;5 30 Hashemi SM, Hassan ZM, Soudi S, Ghazanfari T, Kheirandish M, Shahabi S: Evaluation of antitumor effects of tumor cell lysate enriched by hsp-70 against fibrosarcoma tumor in balb/c mice. International immunopharmacology 2007;7:920-927. 31 Dimitrijević M, Pilipović I, Stanojević S, Mitić K, Radojević K, Pešić V, Leposavić G: Chronic propranolol treatment affects expression of adrenoceptors on peritoneal macrophages and their ability to produce hydrogen peroxide and nitric oxide. Journal of neuroimmunology 2009;211:56-65. 32 Alicea C, Belkowski S, Eisenstein TK, Adler MW, Rogers TJ: Inhibition of primary murine macrophage cytokine production in vitro following treatment with the k-opioid agonist u50, 488h. Journal of neuroimmunology 1996;64:83-90. 33 Fantini MC, Dominitzki S, Rizzo A, Neurath MF, Becker C: In vitro generation of cd4+ cd25+ regulatory cells from murine naive t cells. Nature protocols 2007;2:1789-1794. 34 Bhatnagar S, Shinagawa K, Castellino FJ, Schorey JS: Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo. Blood 2007;110:3234-3244. 35 Li XB, Zhang ZR, Schluesener HJ, Xu SQ: Role of exosomes in immune regulation. Journal of cellular and molecular medicine 2006;10:364-375. 36 Romagnoli GG, Zelante BB, Toniolo PA, Migliori IK, Barbuto JAM: Dendritic cell-derived exosomes may be a tool for cancer immunotherapy by converting tumor cells into immunogenic targets. Novel clinical applications of extracellular vesicles 2015;5:62. 37 Noy R, Pollard JW: Tumor-associated macrophages: From mechanisms to therapy. Immunity 2014;41:49-61. 38 Whitworth PW, Pak CC, Esgro J, Kleinerman ES, Fidler IJ: Macrophages and cancer. Cancer and Metastasis Reviews 1990;8:319-351. 39 Georgoudaki A-M, Prokopec KE, Boura VF, Hellqvist E, Sohn S, Östling J, Dahan R, Harris RA, Rantalainen M, Klevebring D: Reprogramming tumor-associated macrophages by antibody targeting inhibits cancer progression and metastasis. Cell reports 2016;15:2000-2011. 40 Yang C, Robbins PD: The roles of tumor-derived exosomes in cancer pathogenesis. Clinical and Developmental Immunology 2011;2011 41 Yu S, Cao H, Shen B, Feng J: Tumor-derived exosomes in cancer progression and treatment failure. Oncotarget 2015;6:37151-37168.
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42 Didelot C, Lanneau D, Brunet M, Joly A-L, Thonel AD, Chiosis G, Garrido C: Anti-cancer therapeutic approaches based on intracellular and extracellular heat shock proteins. Current medicinal chemistry 2007;14:2839-2847. 43 Cho J-a, Lee Y-S, Kim S-H, Ko J-K, Kim C-W: Mhc independent anti-tumor immune responses induced by hsp70-enriched exosomes generate tumor regression in murine models. Cancer letters 2009;275:256-265. 44 Hurdle JG, O'Neill AJ, Mody L, Chopra I, Bradley SF: In vivo transfer of high-level mupirocin resistance from staphylococcus epidermidis to methicillin-resistant staphylococcus aureus associated with failure of mupirocin prophylaxis. J Antimicrob Chemother 2005;56:1166-1168. 45 Restifo NP, Dudley ME, Rosenberg SA: Adoptive immunotherapy for cancer: Harnessing the t cell response. Nature Reviews Immunology 2012;12:269-281. 46 Miao B-P, Zhang R-S, Sun H-J, Yu Y-P, Chen T, Li L-J, Liu J-Q, Liu J, Yu H-Q, Zhang M: Inhibition of squamous cancer growth in a mouse model by staphylococcal enterotoxin b-triggered th9 cell expansion. Cellular & molecular immunology 2015 47 Xiu F, Cai Z, Yang Y, Wang X, Wang J, Cao X: Surface anchorage of superantigen sea promotes induction of specific antitumor immune response by tumor-derived exosomes. Journal of molecular medicine 2007;85:511-521. 48 Zagon I, McLaughlin P: Opioid antagonist (naltrexone) stimulation of cell proliferation in human and animal neuroblastoma and human fibrosarcoma cells in culture. Neuroscience 1990;37:223-226. 49 Jamali A, Mahdavi M, Hassan ZM, Sabahi F, Farsani MJ, Bamdad T, Soleimanjahi H, Motazakker M, Shahabi S: A novel adjuvant, the general opioid antagonist naloxone, elicits a robust cellular immune response for a DNA vaccine. International immunology 2009;21:217-225. 50 Bahreyni TSMH, Sazgarnia A, Mahmoudi M: Establishment and behavioral study of a tumor model in balb/c mice for experimental cancer studies. Iranian Journal Of Basic Medical Sciences 2004;6:9-13.
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ACCEPTED MANUSCRIPT Figure legends: Fig. 1 Transmission electron microscopy image of negative stained purified EXOs. Isolated EXOs were observed by TEM (×10,000). EXOs' diameter ranges from 40 nm to 150 nm
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Fig. 2 Immunization of balb/c mice against fibrosarcoma tumor. (a) Tumor numbers are indicated for three tested group EXO, EXOHSP and EXOLys comparing to control group PBS. (b) Tumor numbers are indicated for three tested groups EXO/SEB, EXOLys/SEB and EXOHSP70/SEB comparing to control groups SEB and PBS (c) Tumor numbers are indicated for two tested groups EXONLX and EXOPRP comparing to control group PBS.
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Fig. 3 Splenocyte stimulation index (SI). (a) SI is indicated for three tested group EXO, EXOLys and EXOHSP comparing to the control group PBS. (b) SI is indicated for three tested groups EXO/SEB, EXOLys/SEB and
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EXOPRP comparing to the control group PBS.
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EXOHSP70/SEB comparing to control groups SEB and PBS. (c) SI is indicated for two tested groups EXONLX and
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EXOHSP70/SEB has the potency to stimulate immune responses more efficiently
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HSP70 enriched EXO is an effective immunoadjuvant in cancer immunotherapy
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Our data also show that SEB induces immune responses more effectively than SEB-anchored EXO
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S0882-4010(17)30863-X
DOI:
10.1016/j.micpath.2017.08.027
Reference:
YMPAT 2416
To appear in:
Microbial Pathogenesis
Received Date: 17 July 2017 Revised Date:
12 August 2017
Accepted Date: 16 August 2017
Please cite this article as: Behzadi E, Hosseini HM, Halabian R, Fooladi AAI, Macrophage cell-derived exosomes/staphylococcal enterotoxin B against fibrosarcoma tumor, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.08.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Macrophage cell-derived exosomes/staphylococcal enterotoxin B against fibrosarcoma tumor Running title:
Exosomal Macrophage/SEB and fibrosarcoma
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Elham Behzadia, Hamideh Mahmoodzadeh Hosseinia*, Raheleh Halabiana, Abbas Ali Imani Fooladia* Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
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*Corresponding Authors: Abbas Ali Imani Fooladi, PhD
Hamideh Mahmoodzadeh Hosseini, PhD Email: [email protected]
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Email: [email protected]
Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Vanak Sq. Mollasadra St., Tehran - Iran. P.O. Box 19395-5487
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Tel: +98 21 82482568 Fax: +98 21 88068924
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ACCEPTED MANUSCRIPT Abstract Targeted immune therapies are a modern approach to harness the immunity to treat cancer patients. Exosomes (EXOs) are nano-vesicles used for drug delivery in cancer treatment. We aimed to assess the effectiveness of novel designed EXO structures for immunotherapy alone and in combination with other components in animal models.
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EXO derived from untreated macrophage (EXO), WEHI-164 cell lysate treated EXO (EXOLys), HSP70 enriched WEHI-164 cell lysate treated EXO (EXOHSP70), Naloxone (NLX) treated EXO (EXONLX), Propranolol (PRP) treated EXO (EXOPRP) and staphylococcal enterotoxin B (SEB) anchored to three kinds of EXOs designated as
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EXO/SEB, EXOLys/SEB, EXOHSP70/SEB were purified from J774 cell line. To determine the therapeutic effect of these novel constructed nano-vesicles, the animals were immunized with different types of EXOs at weekly intervals for three consecutive weeks and in the fourth week the WEHI-164 tumor cells were injected. Finally,
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the splenocyte proliferation was examined by MTT assay and tumor growth was also determined in each group. We observed that EXOHSP was more effective than EXO and EXOLys to decrease the number of tumor cells and to stimulate immune responses in animal models (P <0.05). In SEB-anchored EXO group, EXOHSP70/SEB has the potency to stimulate immune responses more efficiently than EXO/SEB and EXOLys/SEB and the tumor was not palpable until 28th day which may refer to synergistic effect of HSP70 and SEB on immunity. In EXONLX
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treated mice proliferative response decreased significantly compared to control group (P >0.05) and the tumor number was constant within a period of 28 days and EXOPRP may delay the occurrence of the fibrosarcoma tumor; After development of fibrosarcoma the number of tumors diminished over the studied period of time.
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Our results demonstrate that HSP70 enriched EXO is an effective immunoadjuvant in cancer immunotherapy and causes tumor regression in animal model.
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Key words: Exosome; HSP70; staphylococcal enterotoxin B; Tumor regression; fibrosarcoma
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ACCEPTED MANUSCRIPT 1. Introduction Cancer is the one of the leading causes of death in many parts of the world, but the results of different interventions are poor. The conventional treatments for most cancer patients are a combination of surgery, radiation and/or chemotherapy. In most cases, the primary tumor can be treated with a combination of above
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mentioned therapies, but it is not efficient for the eradication of disseminated tumor cells (DTCs) in the blood circulation and micro-metastases in other organs [1]. Therefore, the main strategy to combat malignancies is cancer immunotherapy by monoclonal antibodies to target tumor specific antigens and cellular therapy using cytotoxic T lymphocytes and dendritic cells [2].
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One type of malignant tumors with mesenchymal cell origin called fibrosarcoma, which arises from fibroblasts predominantly found in the area around bones or in the soft tissues [3]. The primary treatment of fibrosarcoma is
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surgical removal, but recurrence may occur years after treatment [4].
The most innovative strategy for immune cancer therapy is using major histocompatibility complex (MHC) class II-bearing exosomes (EXOs) as drug delivery vehicles in cancer models [5-7]. EXOs are small endogenous vesicles of 40–150 nm in diameter, which are present in nearly all biological fluids and formation of these nanovesicles are through the reverse budding of the late endosomal membrane to develop a multivesicular body
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(MVB). Upon the fusion of MVBs with the plasma membrane, EXOs are released into the extracellular space and can reach and interact with target cells [8-10]. EXOs can contain different kinds of immunostimulatory molecules and antigen-presenting cells (APCs)-derived EXOs can also express MHC II, elicit strong antigenspecific T cell, B cell and NK cell responses and abolish tumors in mouse models [5, 10].
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Normally, professional APCs of the immune system such as dendritic cells (DCs), B cells and macrophages (MΦ), constitutively express MHC class II and have a major role in inducing effective adaptive immune
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response and T cells may recognize antigen complexed with MHC II using T cell receptors (TCRs) on their surface [11]. APCs can also combat tumors via stimulation of B and cytotoxic T cells to respectively provide antibodies against tumor-related antigens and destroy tumor cells [12]. Studies show that many factors can affect APCs activity and improve anti-tumor immunity, including heat shock proteins (HSPs) [13, 14], naloxone (NLX) [15] and propranolol (PRP) [13, 16, 17]. HSPs or stress proteins are conserved proteins that are able to elicit the innate and adaptive immune systems and have essential function in antigen presentation, expression of innate receptors and tumor immunosurveillance [18, 19].
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ACCEPTED MANUSCRIPT Some HSPs derived from cancer cells, such as HSP70, HSP90 and glucose-regulated protein 96 (gp96) have been shown to initiate specific immunity through the transportation of antigenic peptides to APCs to consequently activate tumor-antigen-specific cytotoxic T-lymphocytes (CTL). However, interaction of HSPs with APCs also leads to maturation of DCs as well as release of pro-inflammatory cytokines by MΦs and DCs, hereby providing an efficient induction of immune responses to fight malignant cells [14].
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In this context of cancer therapy, several studies executed on NLX, an opioid receptor antagonist, showed contradictory results, declared both immunosuppressive and immunostimulatory effects of opioid peptides [20]. It is also indicated that NLX enhances cell mediated immunity [15] and can induce a significant increase in the
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lymphocyte proliferation during primary viral infection [21].
Besides, PRP is a beta angiotensin receptor blocker (β-AR blocker), that a number of in vivo and in vitro studies indicated that it has anti-proliferative, anti-migratory, anti-angiogenic and cytotoxic properties. PRP prevents or
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reduces cancer progression through inhibiting cAMP—responsive element–binding protein (CREB), NF-kB, and activator protein (AP-1), inducing apoptosis, or reducing matrix metalloproteinase (MMP)-9 activation and tumor angiogenesis [16, 17].
Furthermore, staphylococcal enterotoxins, particularly type B (SEB), are bacterial superantigens which can form a complex with MHC II on APCs and TCR on CD4+ and CD8+ T cells and detour routine antigen processing
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and presentation pathway [22, 23]. This binding causes hyperactivation of T cells accompanied by an increased release of Th1 cytokines, specifically interleukin 2 (IL-2), tumor necrosis factor β (TNF-β), and interferons. As it is known, T cells have a significant role in the eradication of malignant cells and host cells infected with the
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intracellular pathogens [23, 24]. In addition, SEB combined with MHC class II and CD80 has adjuvant activity against metastatic cancer in animal models [25].
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Furthermore, it has been demonstrated that tumor-derived EXOs are composed of cytosolic and membranous tumor antigens associated with antigen presenting molecules which makes them a good candidate for cancer immunotherapy [26]; but as tumor-derived EXOs carry large quantities of antigens, the possibility of anergy increases. Therefore we hypothesized that MΦ-derived EXOs could solve this problem [10]. In brief, as fibrosarcoma usually escapes from presenting its own antigens to T cells, tumor immunotherapy is the best option to elicit a tumor-specific response that contributes to the elimination of the tumor [27]; Therefore, in this study, we tried to assess the effectiveness of some immunotherapy treatments against fibrosarcoma, alone and in combination with other components, in mouse tumor models.
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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. WEHI-164 Cell Lysate Preparation WEHI-164 mouse BALB/c fibrosarcoma cells were obtained from the Pasteur Institute (Tehran, Iran). WEHI164 cells were grown in T75 flasks (Bioidea, Tehran, IRAN) containing RPMI 1640 medium (Bioidea, Tehran, IRAN) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Inoclon, Tehran, IRAN), 100
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U/ml penicillin and 100 µg/ml streptomycin (Inoclon, Tehran, IRAN) incubated in 5% CO2 and 95% air at 37 °C.
Once the adherent cells reached approximately 80% confluence, the medium was removed and attached cells
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washed three times with phosphate buffered saline (PBS) and detached using trypsin/EDTA (Inoclon, Tehran, IRAN) for further treatment.
Subsequently, WEHI-164 cell lysates were prepared following a previously published method [28] with some
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modifications. Briefly, cell suspensions in PBS were pellet by centrifugation at 2500×g for 5 min and RIPA buffer (phosphate-buffered saline, pH 7.4, 0.5% deoxycholate sodium, 0.1% SDS, 1% Triton X-100) [29] containing protease inhibitors (phenylmethylsulfonyl fluoride, 100 µg/ml; sodium vanadate, 0.4 mM; pepstatin A, 1 µg/ml; leupeptin, 1 µg/ml) was added to pellet following sonication for 30 seconds with 50% pulse to increase the yields. Obtained mixture was shaken gently for 15 min on ice and centrifuged at 14000×g for 15
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min at 4 °C to pellet debris. Supernatant transferred to a new tube for further analysis and stored at -80 °C for later examinations. The protein concentration of total protein was evaluated at 280 nm using a Nanodrop 2000
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spectrophotometer (Thermo-Scientific, US).
2.2. Induction of HSP70 in WEHI-164 Cell Lines
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To increase the expression of Heat Shock Protein 70 (HSP70), in the logarithmic growth phase, the WEHI-164 cells were heated by the incubation of the cells at 42 °C for 60 min [30]; the temperature was controlled within ± 0.1 °C. Then, the cells were collected after 12 h by detachment with trypsin/EDTA (Inoclon, Tehran, IRAN), washed 3 times with phosphate buffered saline (PBS, pH 7.4, centrifuged at 1500 rpm for 5 min). These treated cells were used to prepare HSP70 enriched WEHI-164 cell lysate by above-mentioned method.
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ACCEPTED MANUSCRIPT 2.3. EXO Preparation and Purification J774 cell line from mouse BALB/c monocyte macrophage was obtained from the Pasteur Institute (Tehran, Iran). J774 cell lines were grown in T175 flasks (Bioidea, Tehran, IRAN) containing RPMI 1640 medium (Bioidea, Tehran, IRAN) supplemented with 10% (v/v) fetal bovine serum (FBS) (Inoclon, Tehran, IRAN), 100 U/ml penicillin and 100 µg/ml streptomycin (Inoclon, Tehran, IRAN) incubated in 5% CO2 and 95% air at 37
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°C.
When J774 cell line growth in the T175 flasks (Bioidea, Tehran, IRAN) reached 80% confluence, attached cells were washed three times with PBS to omit FBS and then the fresh medium without FBS was added to each
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flask.
In the next rounds, while adding fresh medium without FBS to each flask, 100 µg/ml of WEHI-164 cell lysate, 100 µg/ml of HSP70 enriched WEHI-164 cell lysate, 10-5M PRP (Tolid Daru Co., Tehran, Iran) [31] and 0.1
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µM NLX (Tolid Daru Co., Tehran, Iran) [32] was added to each flask, separately.
After 24 h, the supernatant of each flask poured to a fresh tube and EXOs were purified using previously published method described by Mahmoodzadeh Hosseini et al. [23]. First, the supernatant was centrifuged at 10000×g for 15 min at 4 ºC to remove debris. Then it was filtered using a 0.22-µm filter (GSV filter technology, USA).
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In the next step, a Centricon Plus-70 centrifugal filter device (Millipore, MA, USA) was used to separate EXOs from supernatant and stored at -80 ºC for future applications. The protein concentration of purified EXOs was evaluated at 280 nm using a Nanodrop 2000 spectrophotometer (Thermo-Scientific, US).
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2.4. Transmission Electron Microscopy
Transmission Electron Microscopy (TEM) was used to assess the morphology and the size of purified EXOs.
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Isolated EXOs suspended in PBS (pH 7.4) fixed with 2.5% glutaraldehyde and then transferred onto a formware/carbon coated grid (Iran University of Medical Sciences, Tehran, IRAN). The grid incubated for 20 min at room temperature, which subsequently transferred to 50 µl of uranyl oxalate (pH 7; Merck, Darmstadt, Germany) for 5 min and then washed with PBS. In the next step, the grid surface was covered with methylcellulose/uranyl acetate (Merck, Darmstadt, Germany) and allowed to stand for 10 min on ice. The excess fluid was blotted off by Whatman filter paper No.1 and the grid was air-dried 5 to 10 min. Ultimately, the morphology and the size of EXOs were observed by Carl Zeiss-Leo 906 TEM (Oberkochen, Germany) at 80 kV and
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ACCEPTED MANUSCRIPT 2.5. Protein Anchorage of SEB on EXOs To anchor SEB on EXO, the protocol described by Mahmoodzadeh Hosseini et al. [23] was utilized. In short, for incorporation of SEB onto the EXO surface, 10 µg SEB (Sigma-Aldrich, St. Louis, USA) was added to 100 µg of purified EXOs (Normal EXO (EXO), HSP70 enriched WEHI-164 cell lysate treated EXO (EXOHSP70) and WEHI-164 cell lysate treated EXO (EXOLys)) in 100 µl PBS. The mixture was shaken at 1000 rpm for 4 h at 37
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°C. Then, the mixture filtered using ultrafree-0.5 biomax100k (Millipore, MA, USA) at 3000×g for 20 min to omit the unbounded SEB. The EXOs, EXOHSP70 and EXOLys attached to SEB were named an EXO/SEB, EXOHSP70/SEB and EXOLys/SEB.
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2.6. Vaccination Protocol and the Tumor Challenge
To examine the effectiveness of prepared vaccines, a total of fifty inbred male Balb/c mice, 3-5 weeks age were purchased from the Pasteur Institute (Tehran, Iran) and kept under recommended conditions according to
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institutional ethical guidelines. The mice were randomly divided into 10 groups of five as follows: 1: PBS (Negative Control), 2: EXO, 3: EXOHSP70, 4: EXOLys, 5: NLX treated EXO (EXONLX), 6: PRP treated EXO (EXOPRP), 7: EXO/SEB, 8: EXOLys/SEB, 9: EXOHSP70/SEB, 10: SEB (Negative Control). Vaccination was performed subcutaneously into the shaved right flank with 10 µg of each component mentioned above at weekly intervals for three consecutive weeks and a week after the last immunization, approximately
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2×106 cells/100 µl of WEHI-164 cells were inoculated to the right flank of the mice to produce fibrosarcoma tumors. Hence, palpable tumors were generated after 7 days and the tumor growth was observed every 7 days. Observation of the tumors was continued until the end of the fourth week after tumor cell injection. The
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behavior of all mice was normal and no sign of pain or any disorder or suffering from inoculations was noticed.
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ACCEPTED MANUSCRIPT 2.7. Proliferation Assay Four weeks after WEHI-164 tumor cell injection, the spleens were removed from sacrificed mice under sterile condition and splenocytes were isolated as described previously [33] The splenocytes from the PBS injected mice were used as a negative control. Briefly, spleens were mashed neatly and passed through 100-µm filters to collect a single-cell suspension and erythrocytes were lysed by ACK lysis buffer (NH4Cl, KHCO3, Na2EDTA)
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at room temperature. The splenocytes were washed and resuspended in RPMI 1640 (Bioidea, Tehran, IRAN), supplemented with 10% FBS (Inoclon, Tehran, IRAN) penicillin, (100 U/ml) and streptomycin (100 mg/ml) (Inoclon, Tehran, IRAN).
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The splenocytes at the concentration of 1×105 cell/well were seeded on each well of a 96-well plate. To stimulate the cells, lysate antigens of the heat shocked and non-heat shocked WEHI-164 cells were exploited in the total volume of 200 µl. The microtiter plates were incubated in a humidified atmosphere containing 5% CO2
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for 3 days at 37°C. The lymphocyte proliferation was determined by adding 20 µl MTT (3, (4, 5dimethylthiazal-2-yl)-2,5-diphenyl tetrazolium bromide) reagent (5 mg/ml) (Sigma-Aldrich, St. Louis, USA) to each well and incubated for 4 h at 37 °C. The supernatants were removed and 100 µl dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St. Louis, USA) were added. Ultimately, the formazan crystals were dissolved and the absorbance of each well was calculated by an ELISA micro plate reader (Bio-Rad, CA, USA) at 570 nm. All
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The results of the proliferation assay were declared as a stimulation index (SI), which was determined by dividing the mean optical density (OD) of test culture groups by the OD of the control culture group.
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The nonparametric Mann–Whitney test was used to evaluate data obtained from all tests using SPSS.15
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ACCEPTED MANUSCRIPT 3. Results 3.1. Identification of EXOs Purified from J774 Cells EXOs were negatively stained with uranyl acetate and the morphology and the size of purified EXOs were evaluated by TEM. As it is demonstrated in Fig. 1, the results revealed round-shaped membranous nano-sized with
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vesicles
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ACCEPTED MANUSCRIPT 3.2. Tumor Number The tumor numbers on the days 14, 21 and 28 were calculated in ten groups of experimental mice. The results designated that the tumors in some test groups grew more slowly than those in the control groups (PBS and SEB injected groups). The tumor numbers in EXOHSP70 and EXOLys injected groups decreased over the time comparing to EXO
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injected group and control group (Fig. 2a).
The tumor numbers in EXO/SEB, EXOLys/SEB and EXOHSP70/SEB injected groups were compared to two control groups (PBS and SEB). The best response was obtained in EXOHSP70/SEB injected group as it is
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demonstrated in Fig. 2b.
The tumor numbers in EXOPRP injected group decreased over the time comparing to EXONLX injected group and control group. In EXONLX injected group the numbers of tumors were constant over that period of time (Fig. 2c).
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Statistics show that there is no difference between EXONLX and various EXO-anchored SEB groups (P>0.05),
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ACCEPTED MANUSCRIPT 3.3. Cell proliferation analysis (MTT assay) The proliferation of spleen lymphocytes is indicative of the state of the immune system. Splenocytes obtained from mice test groups were examined for their lymphocyte proliferative response. Cell proliferation was evaluated by MTT assay. Four weeks after tumor cell injection in treated groups (EXO, EXOHSP, EXOLys, EXONLX, EXOPRP, EXO/SEB,
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EXOLys/SEB, EXOHSP/SEB) and control groups (SEB and PBS treated mice), spleen cells were collected, cultured and stimulated with ex vivo tumor antigens.
The results shown in Fig. 3a designate that splenocyte stimulation index in EXOHSP70 treated mice had changed
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prominently compared to the control group and EXO and EXOLys groups; Statistically significant difference in the stimulation index was observed in EXOHSP70 treated group compared to the control group (P <0.05). The splenocyte stimulation index in EXO treated group was also changed notably compared to the control group but
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in EXOLys treated group no significant changes were observed.
The stimulation index (SI) in Fig. 3b indicates that splenocyte proliferative responses in SEB treated mice had changed significantly compared to other groups and statistically significant difference in cell proliferation was observed compared to EXO/SEB, EXOlys/SEB and EXO HSP70/SEB groups (P <0.05). The results shown that SEB anchorage on EXO, EXOLys and EXOHSP has an inhibitory effect on immune responses. Splenocyte
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proliferative responses in EXO/SEB and EXOLys/SEB groups had changed compared to PBS injected but statistically was not significance (P>0.05).
The stimulation index (SI) in Fig. 3c indicate that splenocyte proliferative responses in EXOPRP and EXONLX
4. Discussion
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treated mice did not change significantly and was lower than control group (P <0.05).
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It is believed that an effective vaccine against malignant cells should be potentially strong and have the ability to induce cellular and humoral immunity. Generally the basic mechanism of cancer immunotherapy is to elicit a specific immune response, including the induction of specific immune effector T cells, to suppress target cells. Recently, it has been shown that EXOs as acellular vehicles possess the ability to induce immune responses and therefore can be used as a cancer therapy option [23]. Studies conducted on DC-derived EXOs revealed that these vesicles have the potential to develop anti-tumor immune responses in vivo [36]. But as the MΦs are the most prominent immune cell recruited to the tumor environment and are present in all stages of tumor progression, it may be of great interest to examine the MΦderived EXOs impact on tumor progression. Most of the studies on mouse models demonstrated that MΦs have
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ACCEPTED MANUSCRIPT pro-tumoral role and can stimulate angiogenesis, which consequently enhance tumor invasion [37]. But researchers succeeded to activate MΦs to become tumoricidal [38]. In a recent research a monoclonal antibody targeted at a protein on their cell surface, which stopped the tumors growth and dissemination in vivo [39]. As the tumor cells are poorly immunogenic, a new effective cancer therapy approach may cause a great evolution in the treatment of cancer patients.
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It has been shown that interaction of EXOs with different cell types is not accidental and is dependent on expressed molecules on EXOs and the target cells [34, 35], therefore, we examined the hypothesis that MΦEXOs would exhibit expected function to increase tumor cell antigenicity/immunogenicity.
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Here, we obtained MΦ-derived EXOs to examine the effect of MΦ antigens alone on WEHI-164 tumor progression. We also stimulated J774 cell line with WEHI-164 cell lysate and HSP70 enriched WEHI-164 cell lysate to obtain EXOs containing tumor antigens (EXOLys) and enriched with HSP70 (EXOHSP), respectively.
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Our results show (Fig. 3a) that EXOHSP is more effective than EXO and EXOLys to stimulate immune responses in animal models. This finding may be related to the presence of HSP70 in heat treated WEHI-164 cell lysate, which can stimulate MΦs to have better immunogenic properties. Hence, HSPs are recruited for immunotherapy in cancer models [42, 43]. As it can be seen in Fig. 3a, MΦ-derived EXO (EXO) elicit greater splenocyte proliferative responses in comparison with EXOLys which is in contrast with the result obtained from Cho et al.
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(2005) study [44]; They found that tumor cell-derived EXOs containing tumor antigens, are able of triggering an efficient immune response. They concluded that tumor-derived EXOs can cover the defect of APC-derived EXOs and may be used as a potent cell-free cancer vaccine [43, 44]. Yet, exploiting APC-derived EXOs for
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immunotherapy has some limitations, because the identified tumor antigens are not accessible easily to be pulsed with the APCs [43]. Mahmoodzadeh Hosseini et al. (2014) indicated that tumor-derived EXOs carry
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large amount of antigens, enhances the chance of immune anergy which in turn, suppresses the anti-tumor immunity [23]. This defect may be compensated by the presence of HSP70 as a potent immune stimulator [43]. Here, we believe that MΦ-derived EXOs lack immune anergy and therefore they elicit stronger stimulatory effects compared to EXOlys group. The results also show that the number of tumors in EXOHSP70 vaccinated group diminished in comparison with other tested groups and PBS injected group (Fig. 3b) which can be attributed to the synergistic effect of exosomal components and HSP70 [43]. As tumor cell antigens are usually undetectable for T cells; the main purpose of cancer immunotherapy is to produce tumor-specific T cells to identify and eradicate malignant cells [45]. SAgs such as SEB has the
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ACCEPTED MANUSCRIPT potential to simultaneously bind to the MHC class II on antigen presenting cells and the T cell receptor on T cells, cause massive T-cell activation and results in the secretion of a relatively large amount of cytokines. Hence, they seem a convenient choice in cancer immunotherapy [27]. In brief, SEB is an exotoxin of Staphylococcus aureus with superantigenic properties that may act as a potential agent to promote immune responses against tumor cell and can prevent both tumor growth and metastasis [46].
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In this study, we designed a two-part structure containing MΦ-derived EXO and SEB to study the effect of this structure on WEHI-164 tumor cells progression in mouse models. Three types of SEB-anchored EXOs designated as EXO/SEB, EXOLys/SEB and EXOHSP70/SEB, were synthesized. The conjunction of SEB to all
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types of EXOs was conducted through glycosylphosphatidylinositol (GPI) anchor using protein transfer method. The SEB anchorage depends on the EXOs membrane composition which is rich in lipid content such as sphingomyelin, cholesterol and glycolipid in combination with tetraspanin proteins that make the anchorage
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possible [23, 47]. Our results (Fig. 3b) show that EXOHSP70/SEB has the potency to stimulate immune responses more efficiently than EXO/SEB and EXOLys/SEB. This finding is in accordance with previous results reported by Cho et al (2009). They found that heat treatment increases the HSP70 content in EXOs and consequently enhances the immune stimulating activity of EXOs. The data suggest that EXOHSP70/SEB could activate the immune response to inhibit tumor progression. This consequence may refer to immune stimulating activities and
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Th1 anti-tumor effects of EXOHSP70/SEB structure [43].
Our data also show that SEB induces immune responses more effectively than SEB-anchored EXO, which is in contrast with the findings of the survey conducted by Mahmoodzadeh Hosseini et al. (2014) on breast tumor
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cancer [23]. We suggest that these contradictory results may be referred to the different nature of fibrosarcoma and breast cancer cell lines. Nevertheless, Imani Fooladi et al. (2008) demonstrated that SEB can induce
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cytotoxic T-cell activity and increase in cytokine levels in mice with fibrosarcoma, suggest that SEB may be a suitable choice to treat patients with fibrosarcoma [27]. We also observed that EXOHSP70/SEB vaccinated group did not develop tumors on the 7th and 14th day (Fig. 2b) and on 28th day small tumors were palpable just in two groups of five which may refer to the synergistic effect of HSP70 and SEB on immunity. In this case, enrichment of HSP70 in EXOs may have an adjuvant effect to upregulate Th1 immunity and cause resistance to tumor development [23, 43]. On the other hand, it has been demonstrated that general opioid antagonists like NLX have an inhibitory growth impact on malignant cells and modifies cell proliferative events through interaction with opioid receptors [48]. Some studies indicate that NLX exert this effect through stimulation of Th1 immune responses by increasing the
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ACCEPTED MANUSCRIPT level of IFN-γ compared to IL-2 [49] and inhibit regulatory T cells [15]. Furthermore, it is defined that the NLX has the adjuvant properties which cause lymphocytes to proliferate. It seems that the NLX function at injection site by influencing local APCs, production of pre-inflammatory cytokines and causing inflammatory responses [49] Here, we developed MΦ-derived EXOs containing NLX- designated as EXONLX -through exposing MΦ cell
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culture to 0.1 µM NLX to improve anti-tumor immunity of MΦ-derived EXOs in BALB/c mouse model with fibrosarcoma tumor. The results indicated that splenocyte proliferative responses in that EXONLX treated mice had decreased significantly compared to control group and the tumor number was constant during a period of 28
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days. These findings are in contrast with previous study done by Hassan et al. (2009) [15] which reported a significant increase in the proliferation of splenocytes and IFN-γ production in fibrosarcoma-bearing mice treated with co-administration of gp96 vaccine and naloxone [15, 49]. These conflicting results may be referred
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As the most promising strategy to treat cancer is based on the enhancement of cell mediated immunity, we tried to investigate the effect of PRP, a β-AR drug on fibrosarcoma-bearing mice. Our results show that EXOPRP may delay the occurrence of the fibrosarcoma tumor, which is in accordance with the results obtained by Khalili et al. (2013) [16].
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In addition, our results demonstrate that HSP70 enriched EXO is an effective option in cancer immunotherapy and causes tumor regression in animal model.
Since there is a little information about the effect of EXOs with tested components on tumor growth inhibition
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animal model for short-term studies on cancer immunotherapy; but for studies longer than 9 days the secretion of TNF may cause tumor reduction. Therefore, for obtaining more reliable results or investigating on human tumor cell lines the nude mice may be the best option to be used [50]. In conclusion, it is proposed to do a more extensive analysis on exosome based vaccine to improve the understanding of its anti-tumor activity and the methodology used here is valuable to other researchers to pursue the clinical and research studies on exosomes.
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ACCEPTED MANUSCRIPT Acknowledgment This study is a part of the thesis research work carried out by Elham Behzadi, PhD student of Applied Microbiology. The work was approved by the Research Vice-chancellor of Baqiyatallah University of Medical Sciences (Record No. 414, dated March 16, 2015). This work was supported by the grant from the Iranian
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National Science Foundation (INSF).
Conflict of interest
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The authors declare no conflicts of interest.
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ACCEPTED MANUSCRIPT Figure legends: Fig. 1 Transmission electron microscopy image of negative stained purified EXOs. Isolated EXOs were observed by TEM (×10,000). EXOs' diameter ranges from 40 nm to 150 nm
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Fig. 2 Immunization of balb/c mice against fibrosarcoma tumor. (a) Tumor numbers are indicated for three tested group EXO, EXOHSP and EXOLys comparing to control group PBS. (b) Tumor numbers are indicated for three tested groups EXO/SEB, EXOLys/SEB and EXOHSP70/SEB comparing to control groups SEB and PBS (c) Tumor numbers are indicated for two tested groups EXONLX and EXOPRP comparing to control group PBS.
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Fig. 3 Splenocyte stimulation index (SI). (a) SI is indicated for three tested group EXO, EXOLys and EXOHSP comparing to the control group PBS. (b) SI is indicated for three tested groups EXO/SEB, EXOLys/SEB and
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EXOHSP70/SEB comparing to control groups SEB and PBS. (c) SI is indicated for two tested groups EXONLX and
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EXOHSP70/SEB has the potency to stimulate immune responses more efficiently
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HSP70 enriched EXO is an effective immunoadjuvant in cancer immunotherapy
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