Background
Exosomes are extracellular nano-sized (< 150 nm) membrane vesicles released by almost all cell types, including cancer cells, into almost all bodily fluids. They are spherical bilayered proteolipids harbouring specific proteins [
1], RNA [
2] and DNA [
3]. Non-coding RNA (microRNA, siRNA and piRNA) and mRNA are key cargos of exosomes [
2]. Their key function being intercellular communication with both neighbouring as well as distant cells [
2]. It has been suggested that tumour cells exploit this intercellular communication mechanism to confer target cell reprogramming that leads to cancer-associated pathologies such as angiogenesis, immune evasion/modulation, cell fate alteration and metastasis. Emerging evidence suggests that tumour viruses also exploit the exosomal message delivery system to induce pathogenesis. Identification of oncogenic exosomal RNA is prerequisite to the understanding of tumour pathophysiology.
Protein composition of exosomes is informative of any existing pathology as they can carry tumour antigens and inflammatory mediators. They also carry customary proteins including HSC70, TSG101 and tetraspanins [
1], in addition they carry specific proteins which are involved in vesicle formation and trafficking such as ALIX (Apoptosis linked gene 2-interacting protein X) [
4]. Exosomes are enriched in tetraspanins, a family of proteins that organizes membrane microdomains called tertraspanin enriched microdomains, by forming clusters and interacting with transmembrane and cytosolic signalling proteins [
5]. Among tetraspanin CD9, CD63, CD81, CD82 and CD151 have a broad tissue distribution. They are involved in biological processes including cell adhesion, motility, membrane fusion, signalling and protein trafficking [
6].
Biogenesis of intraluminal vesicle (ILV, which later become exosomes when excreted) involves endosomal sorting complex required for transport (ESCRT). ESCRT consist of approximately 20 proteins that assemble into four complexes ESCRT-0, I, II and III with associated proteins VPS4 (Vacuolar protein sorting- associated protein 4), VTA1 (vesicle trafficking 1) and ALIX forming ESCRT accessory complex [
7]. ESCRT-0 complex recognizes and segregates ubiquitylated proteins in endosomal membrane. ESCRT I and II deform the membrane into buds with sequestered cargo. ESCRT III is responsible for cleavage into free vesicles [
8]. The mechanism by which ESCRT III complex detaches ILV into multi-vesicular body is similar to final cut between two dividing daughter cells [
9]. Recent studies have shown formation of a helix with a centrosomal protein (CEP55), which translocates to the mid-body during the late phase of cell division and functions as a scaffold for components of the abscission machinery. CEP55 interacts with ESCRT and ALIX-binding region (EABR) [
10]. Previously we have shown that CEP55 is a downstream target of FOXM1, an oncogene that regulates cell cycle, DNA repair and maintenance of genomic stability [
11,
12]. This study investigated the presence of CEP55 protein in normal and cancer exosomes.
The presence of exosomes in bodily fluids (eg., saliva) represents a promising surrogate approach to investigate tumour exosomal RNA biomarkers which has important clinical implications for developing non-invasive salivary diagnostics and therapeutics [
13]. Human saliva is an ideal fluid for developing non-invasive diagnostics and salivary biomarkers have been demonstrated in clinical studies showing promising diagnostic potentials but lacking in sensitivity mostly due to complexity of saliva [
13]. Hence, the ability to purify the highly stable (RNA cargo within exosomes are resistant to RNase [
2]) and protected biomolecules within exosomes helps in reducing background noise in a highly complex and heterogeneous environment such as saliva [
13]. Most of the salivary exosome studies to date have been restricted to characterization of normal healthy samples [
13]. Emerging studies began looking at biochemical properties of disease-derived saliva exosomes but most of these studies focused on proteomics analysis [
1,
13].
HNSCC is diagnosed in over half a million individuals worldwide each year, with an expected global incidence of 750,000 by 2015 [
14]. According to the 8th national annual head and neck cancer audit report published by UK Health and Social Care Information Centre, there were 8272 cases within England and Wales in 2012. Survival rates are poor (10–30% at 5 years) among patients presenting with advanced disease. Early detection of precancer lesions coupled with early intervention could significantly improve patient outcome, reduce mortality and alleviate healthcare costs. Unfortunately, conventional histopathology is currently unable to accurately identify which individual lesions from the oral potentially malignant disorders spectrum will transform to squamous cell carcinoma (SCC). Given similar pathogenesis of other epithelial SCCs, the same clinical dilemmas apply to the management of vulva and skin premalignancies. Current screening methods for HNSCC in otherwise symptom-free persons include the use of oral cytology (brush biopsy), toludine blue staining and various light-based detection systems. More advanced screening methods such as salivary proteomics and antibody-based detection are under investigation. However, the effectiveness of these oral screening adjuncts in detecting early cancer remains unproven [
15]. Hence, there is an urgent clinical need to explore novel cancer biomarkers with better sensitivity and specificity. Salivary exosomal RNA represents a promising new avenue for developing a non-invasive HNSCC screening tool [
13]. In an attempt to identify novel biomarkers for early detection of HNSCC, this study characterised and investigated normal and cancer-derived exosomes and their transcriptome modulating profiles on recipient primary human normal oral keratinocyte cells.
Methods
Cell culture
Normal primary human oral keratinocytes were cultured in serum free medium (SFM) containing 15 ng/ml of human recombinant epidermal growth factor cat no. 10450–013), 62.5 μg/ml bovine pituitary extract (cat no. 13028–014) and 1% penicillin/streptomycin (cat no. 15070–063 from Life technologies UK). HNSCC and transformed cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal bovine serum (FBS) (cat no. 02–00-850, from First Link Ltd. UK) and 1% penicillin/streptomycin. All cells were cultured in a humidified incubator with 5% CO2/95% atmospheric air at 37 °C. Due to the abundance of bovine exosomes in FBS, we have found that human exosomes, although still detectable (by qPCR against specific human markers), were partially masked by the much larger quantity of bovine exosomes (data not shown). Hence, for cells grown in FBS-containing DMEM, to prevent contamination from bovine exosomes, once cells reached ~ 90% confluent (~ 2 × 10
7 cells in a 175 cm
2 flask), we switched to culturing cells in SFM. All cells were left to grow in SFM for 3 days prior to exosome isolation. Additional information regarding each cell line used in this study can be found in (Additional file
1).
Isolation of exosomes by ultracentrifugation
Exosomes were isolated from cell culture supernatant according to well-established ultracentrifugation method [
5,
17,
18] with minor modification. Briefly, the conditioned SFM supernatant was collected and centrifuged in 50 mL tubes at the speed of 500×g for 10 min to remove cellular debris and apoptotic bodies. The supernatant was then centrifuged (SORVALL® Discovery™ SE ultracentrifuge with a SORVALL® T-865 Fixed 23.5° Angle Rotor, k-factor = 51.7) at the speed of 16,500×g for 20 mins to collect microvesicles. Special polycarbonate high speed centrifuge tubes from Thermo Scientific (cat. no. 314348) with screw cap lids (cat no. 314347) were used for high speed ultracentrifugation. The supernatant was filtered through a 0.22 μm filter to remove protein and debris prior to ultracentrifugation at 118,000×g for 70 mins to pellet exosomes. The last ultracentrifugation step was repeated to wash exosomes pellet in PBS.
Scanning electron microscopy
Exosome pellet was re-suspended and fixed in PBS containing 2.5% glutaraldehyde for 1 h at room temperature. Fixed exosomes were washed by adding 20 mL PBS followed by ultracentrifugation at 118000×g for 70 mins. The exosome pallet was resuspended in 100 μl of PBS and incubated on fibronectin-precoated 13 mm round coverslips, without allowing the coverslips to dry, the samples were incubated overnight at 37 °C. Coverslips were then dehydrated in ascending series of ethanol concentrations from 30, 50, 70, 80, 90 to 95% for 5 mins each, followed by two 5 mins incubations in 100% ethanol. The samples were then chemically dried in 100% HMDS (Hexamethyldisilazane) for 3 mins and allowed to air dry at 37 °C for 30 mins. Double-sided adhesive carbon coated conductive discs were used to secure the coverslips onto the SEM stubs. A carbon conducting cement was used to aid a conducting pathway between the stub and the coverslips. The cement was allowed to dry for 24 h prior to coating with gold or carbon particles. Scanning electron micrographs (at 0.5 to 30 kV) were obtained using an FEI Inspect F system. The xT microscope control software was used to control the operation of the microscope while the image capturing software was used to obtain images. SEM was supervised by Dr. Russell Bailey at the Nano Vision centre, QMUL.
Transmission electron microscopy
Exosome pellet was resuspended and fixed in 100 μl of 4% (
w/
v) paraformaldehyde for 10 mins and 5 μl of the fixed exosomes were placed on carbon coated EM grids (Catalogue no. S160–4 carbon film, 400 Mesh Cu by Agar scientific) for 20 mins. A drop of 100 μl of PBS on parafilm sheet was used to wash the grid (membrane side down). The grids were transferred to a 50 μl drop of 2.5% (w/v) gluteraldehyde for 10 mins, followed by eight washes in 100 μl of molecular-grade DNase/RNase/Protease-free water (W4502, Sigma-Aldrich) [
16]. The samples were stained by 0.4% (w/v) lead citrate for 1 min. Stained grids were washed twenty times with distilled water and air dried on filter paper. TEM was supervised by Dr. Russell Bailey from Nano Vision centre QMUL.
Immuno-gold TEM
Exosomes were fixed in 100 μl of 4% (w/v) paraformaldehyde from which 5 μl was placed on carbon coated electron microscopy grids. The grids were covered and left for 20 mins to facilitate adsorption. Further, the grids were washed in 100 μL drops of PBS and transferred in PBS/50 mM ammonium chloride (NH
4CL) for 3 mins. The grids were transferred to blocking buffer (10% foetal calf serum) for 10 mins followed by a transfer to 5 μl drops of CEP55 antibody in the dilution of 1:50 in blocking buffer for 30 mins. The grids were washed multiple times in washing buffer for 3 mins. The grids were incubated with secondary (bridging) antibody diluted in blocking buffer for 30 mins and transferred to 100 μl drops of PBS/0.5% BSA and washed 3 times. Further, the exosomes were incubated in 5 μl drops of protein A-gold conjugates diluted in blocking buffer for 20 mins followed by 7 washes in PBS. The grids were transferred to 50 μL drops of 1% glutaraldehyde for 5 mins to stabilize immunoreaction. The grids were washed 7 times in 100 μl drops of double distilled water, each time for 2 mins. The samples were contrasted using uranyl oxalate at pH 7 for 5 mins. Immuno-gold labelling was done by Dr. Giulia Mastroianni (TEM Facility Manager) at QMUL, following published protocol [
16].
Dynamic light scattering
Fractions of apoptotic cell debris (1st pellet following 500×g centrifugation), microvesicles (2nd pellet following 16,500×g) and exosomes (3rd pellet following 118,000×g) were resuspended in 1 mL molecular grade water for use in a Zetasizer Nano ZS instrument (Model: ZEN3600, Serial no.: MAL500457, Malvern Instruments) to measure the Brownian motion of particles in a sample using Dynamic Light Scattering providing 3 fundamental parameters of nano-sized particles or molecules in a liquid medium: particle size; zeta potential; and molecular weight. The Zetasizer Nano ZS can measure: particle size for the size range 0.6 nm to 6 μm; Zeta potential for a size range 5 nm to 10 μm; Molecular weight in the size range 1000 to 2 × 107 Da. All samples were read at 4 °C and particle size was measured as intensity percent with respect to diameter in nanometre (nm).
Nanoparticle tracking analysis
Particle size verification of exosomes was carried out in School of Pharmacy, University College London using NanoSight Nanoparticle Tracking Analysis (NTA NanoSight, Malvern Inc., United Kingdom) [
17,
18]. Samples were prepared in 500 μl molecular-grade DNase/RNase/Protease-free water (W4502, Sigma-Aldrich) and stored at − 80 °C until use. Using a 1-mL syringe, samples were loaded into the assembled sample chamber of the NanoSight LM10. One minute video images were captured with manual shutter and gain adjustments (Hamamatsu C11440 ORCA-Flash 2.8 digital camera) and analysed using the NanoSight NTA 2.0 software.
RNase protection assays and RNA isolation and quality control
RNase, detergent and protease protection assays were performed to investigate the origin of RNA whether it was protected by exosomes and/or protein complexes. Following ultracentrifugation, the supernatant was gently aspirated and the exosome pellet was resuspended in 260 μL molecular grade water and subdivided into equal fractions (50 μL) which received either: 1) vehicle (dH20), 2) RNase A digestion (0.6 mg/mL final concentration, #R6513, SIGMA) (30 min at 37 °C), 3) detergent incubation (2% Triton-X, 10 min at 55 °C) followed by RNase digestion, 4) proteinase K digestion (PK, #03115828001, ~ 0.4 mg/mL final concentration, Roche Diagnostics) (10 min at 55 °C prior to 5 min heat inactivation at 95 °C) followed by RNase digestion, 5) Triton-X and PK treatment followed by RNase digestion. RNase activity was then inactivated by adding RNase inhibitor (03335399001, 1 U/μl final, Roche, 5 mins at RT). Total RNA was purified using RNeasy Micro Kit (#74004, Qiagen) and quantified using Quan-iTTM RiboGreen® RNA Assay kit (R11490, Molecular Probes, Life Technologies). RNA size, quality and relative quantity were assessed by Agilent BioAnalyzer RNA 6000 Pico chip (#5067–1513, Agilent Technologies, Germany). Our typical exosomal RNA yield from each cell line sample (grown in 2× T157 flasks with a total of 80 mL culture supernatant) was 1–4 ng (10–50 pg RNA/mL supernatant). Given that on average each mL of supernatant contains 1-5 × 107 particles (determined by NTA), we estimated each exosome to contain approximately 0.2-5 × 10−18g (attogram) RNA.
Reverse transcription-quantitative PCR (RT-qPCR)
Exosomal RNA were converted to cDNA using qPCRBIO cDNA Synthesis kit (#PB30.11–10, PCRBIO Systems, UK) and the cDNA was diluted 1:4 with RNase/DNase free water and stored at − 20 °C until used for qPCR. Relative quantitative PCR were performed using qPCRBIO SyGreen Blue Mix Lo Rox (#PB20.11–50, PCRBIO Systems, UK) in the 384-well LightCycler 480 qPCR system (Roche) according to our well-established protocols [
11,
12,
19,
20] which are MIQE compliant [
21]. Briefly, thermocycling begins with 95 °C for 30s prior to 45 cycles of amplification at 95 °C for 6 s, 60 °C for 6 s, 72 °C for 6 s, 76 °C for 1 s (data acquisition). A ‘touch-down’ annealing temperature intervention (66 °C starting temperature with a step-wise reduction of 0.6 °C/cycle; 8 cycles) was introduced prior to the amplification step to maximise primer specificity. Melting analysis (95 °C for 30s, 65 °C for 30s, 65–99 °C at a ramp rate of 0.11 °C/s with a continuous 1 acquisition/°C) was performed at the end of qPCR amplification to validate single product amplification in each well. Relative quantification of mRNA transcripts was calculated based on an objective method using the second derivative maximum algorithm (Roche). All target genes were normalised to two stable reference genes (YAP1 and POLR2A) validated previously to be uninfluenced by disease process [
20]. For further verification and comparison, some experiments were performed using Taqman gene expression assays for FOXM1 (Hs1073586_m1), CEP55 (Hs01070181_m1) and ACTB (Hs01060665_g1) using LightCycler® 480 Probes Master (#04707494001, Roche Diagnostics) with a pre-incubation of 50 °C, 2 min and 95 °C, 10 min hot-start followed by 50 cycles of 95 °C, 10s and 60 °C, 60s.
Western blotting
Primary Antibody used were: Alix(3A9) (1:1000 dilution, mouse monoclonal, mAb#2171, Cell Signalling), CD9 (1:200, rabbit monoclonal, ab92726, Abcam), CD63 (H-193) (1:1000, rabbit polyclonal, sc-15,363, Santa Cruz), CEP55 (1:10000, rabbit monoclonal, ab170414, Abcam), Calnexin (1:1000, rabbit polyclonal, ab22595, Abcam), Glypican 1 (1:500, rabbit polyclonal, ab55971, Abcam), FOXM1 (1:500, rabbit polyclonal, sc-502, Santa Cruz), GAPDH (1:10000, mouse monoclonal, ab8245, Abcam), HSC70(B-6) (1:10000, mouse monoclonal, SC-7298, Santa Cruz). Secondary antibody used were: Goat anti Rabbit IgG (1:1000; AP#132P, Millipore), Goat Anti-Mouse IgG (1:10000, A0168, Sigma). Additional information on immunoblotting methodology can be found in (Additional file
1).
Microarray gene expression
Normal primary oral keratinocytes (OK113) cells were seeded (1 × 105 per well) in 6-well plates 1 day prior to transfection with exogenous exosomes derived from 3 normal oral keratinocytes (OK113, NK4, NOK368) and 5 malignant (Ca1, CaLH2, SQCC/Y1, SVpgC2a and SVFN8) cell lines. Exosome concentrations were adjusted to 2 × 1010 particles/well. Untreated OK113 cells were used as a control. After 48 h of incubation with exosomes in SFM, the cells were washed with 1 × PBS and lysed in lysis buffer (RLT buffer) for total RNA extraction using RNeasy Micro Kit (#74004; Qiagen). Quality and quantity of total RNA was analysed on Nanodrop 1000 spectrophotometer and Agilent BioAnalyzer prior to transcriptome profiling using Illumina genome-wide gene expression Human HT-12 v4.0 Expression BeadChip surveying 47,231 transcripts per sample (performed at Barts and The London Genome Centre, core facility). The data from microarray was analysed on Genome studio version 3 Gene Expression Module. Raw transcriptome data have been deposited at NCBI’s GEO database (GSE89217).
Discussion
To our knowledge, this is the first study characterising exosomes secreted from primary human oral keratinocytes and compared with exosomes derived from HNSCC cell lines. Exosomes purified through differential ultracentrifugation from 3 different strains of primary normal human oral keratinocytes and 5 malignant cell lines were subjected to various nanoparticle characterisation methods including scanning electron microscopy, transmission electron microscopy, dynamic light scattering (Zetasizer), 3D Brownian motion (NTA) and biochemical membrane protein and mRNA cargo characterisations. All physical characteristics of our purified exosomes were consistent with published data [
18,
22].
During characterisation of exosomal proteins, we accidentally identified a potential unique cancer exosomal membrane protein, CEP55, which were present in all 5 cancer exosomes and absent in all 3 normal exosomes (Fig.
2a). Further analysis using immune-gold TEM confirmed that CEP55 protein was located on the membrane of cancer-derived but not normal exosomes (Fig.
2d). This appears to be consistent with the biosynthesis of endosome involving the ESCRT (endosomal sorting complex required for transport) membrane budding machinery [
28]. CEP55 (55 kDa) is a centrosomal protein involved in cytokinesis [
29] and a known downstream target of FOXM1 oncogene in HNSCC [
30] and breast cancer [
31]. Crystal structural study revealed that CEP55 is a partner of ALIX in ESCRT complex [
10] involved membrane abscission during viral budding [
26] and exosomal budding into multivesicular endosomes [
32]. Midbody (Flemming body) typically has a diameter of ~ 1 μm and length of 3–5 μm [
33] would have been removed in our exosome purification protocol, hence we could rule out midbody contamination in our exosome preparation. There was also reports demonstrating ESCRT independent mechanism for budding exosomes [
32]. The presence of CEP55 on cancer exosomes but not normal exosomes led us to suggest the involvement of different endosome biosynthetic pathways. The mechanism of exosomal budding is beyond the scope of this study, further investigations are required to delineate the role of CEP55 in cancer exosomes.
Consistent with published studies that mRNA cargos are protected within exosomes and resistant to RNase degradation [
2], we further showed that digestion of membrane proteins by proteinaseK may render the RNA cargo partially susceptible to RNase degradation presumably because digestion of transmembrane proteins could perforate the membrane of exosomes (Fig, 3a). When screening for candidate mRNA transcripts packaged within cancer exosomes, we found selective cargo loading of certain mRNA transcripts within exosomes, thereby providing evidence of selective sorting of mRNA into exosomes, consistent with existing data that protein and RNA molecules are not randomly loaded into exosomes [
34]. Nevertheless, we found evidence that full-length exogenous transcripts were packaged within exosomes derived from a cell line (SVFN8) constitutively overexpressing EGFP-FOXM1B (Additional file
1: Figure S2A) and that the mRNA cargo could be delivered into recipient cells albeit transiently (Additional file
1: Figure S2B), consistent with the finding that mRNA cargos are rapidly degraded upon entry into recipient cells [
35]. This has implications in tailored engineering of specific exosomal cargos for cancer therapeutics including self-homing targeted anticancer drug delivery and cancer immunotherapy [
36].
The morphological changes observed in recipient oral keratinocytes transfected with cancer exosomes resembled senescence but we could not find evidence of exosome-induced senescence as reported in other cell systems [
37]. Instead, we found some evidence of perturbed differentiation markers cornifin (CORN) and loricrin (LORI). As little is known about the functional differences and molecular consequences of normal cells responding to exosomes secreted by normal cells compared to those secreted by cancer cells, we therefore performed transcriptome profiling to investigate the global gene expression profile (47,231 genes) in an unbiased way. Transcriptome data showed that regardless of normal or cancer derived, exosomes altered molecular programmes involved in matrix modulation (MMP9), cytoskeletal remodelling (TUBB6, FEZ1, CCT6A), viral/dsRNA-induced interferon (OAS1, IFI6), anti-inflammatory (TSC22D3), deubiquitin (OTUD1), lipid metabolism and membrane trafficking (BBOX1, LRP11, RAB6A). Interestingly, cancer exosomes, but not normal exosomes, modulated expression of matrix remodelling (EFEMP1, DDK3, SPARC), cell cycle (EEF2K), membrane remodelling (LAMP2, SRPX), differentiation (SPRR2E), apoptosis (CTSC), transcription/translation (KLF6, PUS7). These results indicated that cancer exosomes elicited additional transcriptome programmes compared to normal exosomes. However, both normal and cancer exosomes induced a subset of common pathways in recipient cells, indicating a fundamental mechanism involved when responding to any exosomes.
It has previously been shown that activated T cell exosomes could upregulate MMP9 expression in murine melanoma cells [
38] and hepatocellular carcinoma-derived exosomes could increase MMP9 secretion in hepatocytes [
39]. Upon further dose- and time-dependent exosome transfection experiments validation by qRT-PCR, we found that cancer exosomes induced stronger upregulation of MMP9 and PGAM1 whilst suppressed BBOX1 and EFEMP1, compared to normal exosomes. Interestingly, TSC22D3 and EFF2K showed biphasic time-dependent effects in respond to cancer exosomes. Our results suggest that although some genes were commonly modulated (eg., MMP9 and PGAM1 were upregulated by both normal and cancer exosomes), the effects were significantly amplified by cancer exosomes in recipient cells. Activation of MMP9 may promote cell migration and invasion, whilst PGAM1 may reroute [
40,
41] and/or uncouple glycolytic pathways to promote cell migration [
42].
Apart from MMP9, none of the other genes (Fig.
6c) had been previously associated with exosomes. BBOX1 has been shown to be essential for transport of fatty acids across the mitochondrial membrane [
43]. Through meta-analysis of microarray data across 13 different types of cancers, BBOX1 has been proposed to have an important role in cancer development [
44]. EFEMP1, also known as fibulin 3, is a member of the fibulin family of secreted glycoprotein [
45] known to regulate cell morphology, adhesion, growth and motility [
46]. Recently, upregulation of EFEMP1 has been found in bladder cancer, correlating with increased tumour invasiveness, while knockdown of EFEMP1 restored the invasive and migratory potential [
47]. Another study showed that suppression of EFEMP1 reduced migration, invasion and promote apoptosis in brain cells (glioma) [
48]. Studies have also reported that EFEMP1 regulates matrix metalloproteinase (MMPs) and tissue inhibitors of MMPs [
49]. Our finding that BBOX1 and EFEMP1 were suppressed by cancer exosomes may indicate activation of a protective mechanism in the recipient cells against potentially harmful cargos in cancer exosomes [
50].
SPRR2E is part of the human epidermal differentiation complex on chromosome 1q21 and code for precursor proteins of the cornified cell envelop, a structural characteristic of terminally differentiated keratinocytes [
51]. In a study on epidermal squamous cell carcinoma, low expression of SPRR2 was noted in malignant keratinocyte cell lines compared to normal suggesting defective terminal differentiation, a characteristic of carcinogenic transformation [
52]. Similar expression of SPRR2 has been observed in neoplastic keratinocytes of the anal track [
53]. This is consistent with our finding that cancer exosomes had significantly lower transactivation onSPRR2E expression when compared to normal exosomes, suggesting a role of cancer exosomes in antagonising differentiation in recipient cells.
TSC22D3, also known as glucocorticoid-induced leucine zipper (GILZ), is a potent anti-inflammatory protein and plays a role in cell survival [
54]. Previous studies have reported increased expression of EEF2K in breast cancer [
55] and glioma [
56], where it plays a critical role in cell cycle, autophagy and apoptosis [
57] making it a potential target for cancer therapy. We found that both TSC22D3 and EEF2K exhibited biphasic dose- and time-dependent expression following cancer exosomes transfection. Given their roles in regulating inflammation and apoptosis, their expression patterns in recipient cells may indicate a complex dynamic network of interacting signals responding to cancer exosomes.