Mast cell exosomes promote lung adenocarcinoma cell proliferation – role of KIT-stem cell factor signaling

Background Human cells release nano-sized vesicles called exosomes, containing mRNA, miRNA and specific proteins. Exosomes from one cell can be taken up by another cell, which is a recently discovered cell-to-cell communication mechanism. Also, exosomes can be taken up by different types of cancer cells, but the potential functional effects of mast cell exosomes on tumor cells remain unknown. Methods and results Exosomes were isolated from the human mast cell line, HMC-1, and uptake of PKH67-labelled exosomes by the lung epithelial cell line, A549, was examined using flow cytometry and fluorescence microscopy. The RNA cargo of the exosomes was analyzed with a Bioanalyzer and absence or presence of the c-KIT mRNA was determined by RT-PCR. The cell proliferation was determined in a BrdU incorporation assay, and proteins in the KIT-SCF signaling pathway were detected by Western blot. Our result demonstrates that exosomes from mast cells can be taken up by lung cancer cells. Furthermore, HMC-1 exosomes contain and transfer KIT protein, but not the c-KIT mRNA to A549 cells and subsequently activate KIT-SCF signal transduction, which increase cyclin D1 expression and accelerate the proliferation in the human lung adenocarcinoma cells. Conclusions Our results indicate that exosomes can transfer KIT as a protein to tumor cells, which can affect recipient cell signaling events through receptor-ligand interactions.


Introduction
Exosomes are nano-sized membrane vesicles (30-100 nm) constitutively released by various cells, such as B lymphocytes [1], dendritic cells [2,3], mast cells [4], natural killer (NK) cells [5], intestinal epithelial cells [6], tumor cells [7,8], and neuronal cells [9]. These extracellular vesicles have a lipid bilayer structure, and carry multiple proteins and RNA molecules [10]. Exosomes are known to shuttle their cargo, including proteins and lipids between cells [11], and in addition, we showed in 2007, that exosomes also shuttle functional RNA from one cell to another [12]. Importantly, the transfer of these molecules can change a recipient cell phenotype in many ways, and the exosome-mediated signal from the original cell may be different under different circumstances [13]. Exosomes are present in all human body fluids so far investigated, including saliva [14], blood plasma [15], breast milk [16], cerebrospinal fluid [17] and urine [18], and are abundant in the tumor microenvironment [19,20], indicating their importance for tumor biology.
The human c-KIT oncogene codes for the protein mast/stem cell growth factor receptor Kit (KIT), a member of the tyrosine kinase family of growth receptors [21]. KIT is expressed on a variety of hematopoietic cells, such as mast cells and bone marrow progenitor cells. Stem cell factor (SCF) dependent activation of KIT is critical to maintain homeostasis and function of mast cells [22]. In clinical lung cancer research, it has been shown that non-small cell lung cancer more rapidly leads to death if the tumor is KIT positive [23]. For example, if tumors are positive for KIT at the time of surgery, the disease is associated with short term survival, compared to those that are KIT negative [24]. In addition, coexpression of KIT and other tumor-promoting molecules such as EGFR tend to increase mortality further [25]. During some circumstances it is less clear how tumor cells become KIT positive, but one possibility is that non-tumor cells in the tumor microenvironment could shuttle such molecules between cells [26].
Tumors also harbor many other cells beside tumor cells, including inflammatory cells such as dendritic cells and mast cells [27,28], as well as fibroblasts and endothelium [29,30]. Furthermore, co-cultures of mast cells and non-small cell lung cancer leads to increased proliferation of the cancer cells both in vitro and in vivo [31]. In this study we therefore hypothesized that KIT could possibly be transferred to tumor cells via exosomes from one or several of the surrounding cells. To test this, we used a mast cell line (HMC-1) constitutively expressing the active form of the KIT receptor, and a nonsmall cell cancer lung epithelial tumor cell line (A549), to determine whether KIT can be transferred from mast cells to the epithelial cancer cell via exosomes, and whether those exosomes can influence the function of the recipient cell.

Isolation of exosomes
Exosomes were isolated from the supernatant of HMC-1, HEK 293 and NIH 3T3 cells by differential centrifugation and a filtration step as described by Lässer et al. [32]. In brief, cell supernatant were harvested, centrifuged at 300 × g for 10 minutes to eliminate cells and at 16,500 × g for 20 minutes, followed by filtration through 0.2 μm filter (Sarstedt, Numbrecht, Germany) to eliminate cellular debris and larger vesicles. Exosomes were pelleted by ultracentrifugation at 120,000 × g for 70 minutes. Exosomes were measured for their protein content using the BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA).

Uptake of HMC-1 exosomes by A549 cells
To monitor uptake kinetics, exosomes derived from HMC-1, as well as the control cells HEK 293 and NIH 3T3 cells, were labelled with the green fluorescent dye PKH67 (Sigma-Aldrich) according to the adjusted protocol outlined in Lässer et al. [14]. Briefly, 20 μg of the PKH67-stained exosomes were washed five times using 300 kDa Vivaspin filters (Sartorius AG, Göttingen, Germany) to remove excess dye and then added to 2 × 10 5 A549 cells in culture. Cells were harvested at different time points (1,2,4,8,12,24 and 48 hours), washed three times and analyzed by flow cytometry using a BD FACSAria (BD Biosciences). Additionally, cells were visualized at 8 or 12 hours with a fluorescence microscopy (Zeiss Axioplan 2 microscope, Carl Zeiss) or at 4 hours with confocal laser scanning microscope (LSM 700, Carl Zeiss). As control for non-specific labelling of the cells, PBS was PKH67 stained, washed and added to the cells in a parallel experiment. For analysis with flow cytometry, the A549 cells were washed twice with PBS, treated with 0.25% trypsin-EDTA solution (Sigma-Aldrich) to detach the cells and washed twice with 1% FBS in PBS before acquired in a BD FACSAria flow cytometry running BD FACS Diva version 6.0 Software (BD Biosciences) and analyzed with the FlowJo Software (Tree Star Inc., Ashland, OR, USA). For microscopy, the cells were washed twice with PBS, fixed with 4% formaldehyde solution for 15 minutes and washed again twice with PBS. For the fluorescence microscopy the cells were mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA, USA) supplemented with 3% 7-Aminoactinomycin (7-ADD; BD Biosciences) to label cell nuclei and for confocal microscopy cells were mounted with ProLong® Gold Antifade Mountant containing 4',6-diamidino-2-phenylindole (DAPI; Life Technology).

Transfer of KIT protein and c-KIT mRNA to A549 cells
To determine if the KIT protein could be transferred via exosomes, HMC-1 exosomes (80 μg) were added to A549 cells (4 × 10 5 ) and the cells were incubated for 24 hours. The A549 cells were harvested, washed and the total proteins were isolated using RIPA buffer (Cell Signaling Technology, Danvers, Massachusetts, USA). As a control, cells were treated with only media. The presence of KIT was determined using Western blot.
To determine if the c-KIT mRNA could be transferred via exosomes, HMC-1 exosomes (80 μg) were added to A549 cells (4 × 10 5 ). The cells were harvested at different time points (30 minutes, 2, 4, 24 and 48 hours), washed and isolation of total RNA was performed. Presence of c-KIT mRNA was determined using reverse transcription polymerase reaction (RT-PCR).

RT-PCR
Total RNA was isolated using miRCURY™ RNA Isolation Kit (Exiqon¸Vedbaek, Denmark) according to the manufacturer's protocol and as previously described [33]. Detection and quantity of RNA was determined using a Bioanalyzer and RNA 6000 Nano chips according to the manufacturer's protocol (Agilent Technologies, Santa Clara, CA, USA).

Reverse migration assay
To evaluate the functional effect on lung epithelial cells (A549) induced by HMC-1 derived exosomes, reverse migration assay was performed using a 46 well Boyden chamber (Neuroprobe Inc.). Briefly, A549 cells (32,500 cells/well) were added in the lower chamber and allowed to adhere onto the gelatin (0.1%) coated polycarbonate membrane by inverting the Boyden chamber upside down (Neuroprobe Inc.). After 3 hours of incubation the assembly was placed in correct orientation and exposed to various HMC-1 exosomes dosage on the upper side of the membrane for 12 hours. Migrating cells towards the upper side of membrane were fixed in ethanol, and stained with Giemsa (Histolab, Gothenburg, Sweden) and images were acquired using a light microscope (Zeiss Axioplan, Germany). Adhered cells, present on lower side of membrane (non-migrated side), were wiped out carefully before imaging.

Detection of cell proliferation
Bromodeoxyuridine (BrdU) is incorporated into the newly synthesized DNA strands of actively proliferating cells. We therefore determined cell proliferation using a BrdU labeling ELISA kit (Calbiochem, San Diego, CA, USA) according to the manufacturer's protocol. Shortly A549 cells were seeded at the density of 1 × 10 5 in a 96-well plate and incubated overnight to allow cells to attach to the plate. Cells received BrdU (20 μl) and 20 μg of either the HMC-1 exosomes or the control exosomes (isolated from NIH 3T3 cells) at the same time. After 4 hours of incubation the absorbance was obtained at dual wavelength (450 nm and 595 nm) with a spectrophotometer (Spectra Max; Molecular Devices, Sunnyvale, CA, USA). The results were normalized as percent of control.

Statistical methods
The statistical analyses were performed using SPSS 16.0 statistical software (SPSS, Chicago, IL, USA). Student's t-test was used to analyze the difference between two groups, and one-way ANOVA followed by Dunnett's test or Kruskal-Wallis test followed by Dunn´s multiple comparisons test was employed for the comparisons among three or more groups. To estimate correlation, a Spearman ranked correlation test was performed. Data are presented as the mean ± SEM for all statistical tests. Results were considered to be statistically significant when p ≤0.05.

Identification of mast cell exosomes and their effect on lung adenocarcinoma cells
After isolation of exosomes from the supernatant of cultured HMC-1 cells, using ultracentrifugation, CD63 positive vesicular round structures could be visualized by electron microscopy ( Figure 1A). The protein fraction isolated from the pellet after ultracentrifugation was positive for the traditional exosome markers CD81 and TSG101, while negative for a endoplasmic reticulum protein, calnexin ( Figure 1B), confirming our previous data showing that HMC-1 cells can release exosomes [12,34,35]. Furthermore, exosomes isolated from HEK 293 and NIH 3T3 cells were also positive for the exosomal markers and negative for the endoplasmic reticulum protein calnexin ( Figure 1B). These exosomes were therefore considered to be appropriate to be used as control exosomes in this study.
To examine whether exosomes from human mast cells can be taken up by lung cancer cells, the HMC-1 exosomes were labeled with PKH67 dye and added to cultures of A549 cells. Flow cytometry analysis (Figure 2A) showed an increased fluorescence intensity of the A549 cells after the addition of mast cell-derived exosomes, indicating cellular uptake. This uptake was reduced at 4°C, arguing that the uptake was an active process ( Figure 2B). The fluorescence of A549 cells was obvious already two hours after addition of the stained exosomes, and increased over time ( Figure 2C and D), indicating initiation of uptake of the exosomes by A549 cells. The uptake of the fluorescent exosomes by the A549 cells was also visualized using fluorescence microscopy ( Figure 2E) and confocal microscopy ( Figure 2F). Additionally, fluorescence microscopy demonstrated that control exosomes isolated from HEK 293 and NIH 3T3 cells were taken up by A549 cells in a similar manner as HMC-1 exosomes ( Figure 2G). This verifies their validity to be used as control exosomes in the later functional experiments.
To determine whether mast cell exosomes can influence lung adenocarcinoma, both proliferation and migration was assessed in the A549 cells in the presence of HMC-1 exosomes. Firstly, HMC-1 exosomes were added to A549 cells in culture for 4 hours in the presence of BrdU to evaluate their capacity to influence proliferation. The incorporation of BrdU was significantly increased by 142% in the presence of HMC-1 exosomes compared to the medium control and by 38% compared to the control exosomes, as quantified by an ELISA kit ( Figure 3A). Secondly, the A549 cells were seeded on the membrane of the lower chamber with different doses of HMC-1 exosomes present in the upper chamber to evaluate the exosomal capacity to induce migration. Significantly more cells migrated into the upper chamber in a dose response dependent manner in the presence of HMC-1 exosomes, with the highest doses being statistically significant compere to the control and with a correlation coefficient of 0.91 (p <0.0001) ( Figure 3B).

The presence of KIT protein in mast cell exosomes and their shuttling to lung adenocarcinoma cells
The presence of the KIT protein in HMC-1 exosomes is illustrated in Figure 4A. By contrast, the A549 cells had no detectable KIT, as determined by Western blot. However, when HMC-1 exosomes were incubated with the A549 cells for 24 hours, KIT could be detected in the A549 cells by Western blot ( Figure 4A). Stem cell factor (SCF), the ligand for KIT, was however present in the A549 cells, but absent in HMC-1 exosomes. By contrast, we were unable to detect any c-KIT mRNA in the exosomes, but only in the HMC-1 cells ( Figure 3B). Furthermore, the c-KIT mRNA was not detected in the A549 cells at any of the time points analyzed. GAPDH were used as positive controls, and GAPDH mRNA was present in all samples, except in the HMC-1 exosomes ( Figure 3B). Our identification of KIT protein, but not c-KIT mRNA, in the HMC-1 exosomes, and the transfer of the protein to lung adenocarcinoma cells, suggests that the presence of KIT in the A549 cells after exosomes depend on transfer of KIT protein, rather than transfer of its mRNA ( Figure 4A and B).

Mast cell exosomes activate the KIT-SCF signaling pathway in recipient lung adenocarcinoma cells
Next, we sought to determine whether transfer of KIT by HMC-1 exosomes could influence A549 intracellular KIT-associated pathways, proposing a molecular explanation for the observed enhanced proliferation seen in Figure 3A. As shown in Figure 5A-E, we indeed found that PI3K and its downstream targets, AKT, and GSK3β showed increased phosphorylation in A549 cells when HMC-1 exosomes were added, compared to media alone or control exosomes. However, the total amounts of these three proteins were unchanged according to the Western blot analysis. When PI3K is phosphorylated and thus activated it phosphorylates AKT, which in turn inactivates GSK3β by phosphorylating the S 9 residue on GSK3β. Cyclin D1 is a cell-cycle regulator downstream of GSK3β and is inhibited by GSK3β. Therefore an increase in deactivation of GSK3β by phosphorylation will lead to an increase in cyclin D1, which will result in cells moving from the G1 phase into the S phase of the cell cycle. Importantly, cyclin D1, was indeed increased in A549 cells after the addition of HMC-1 exosomes, which then can be associated to increased proliferation of the lung adenocarcinoma cells.
When cyclin D1 is phosphorylated it is transported out of the nucleus and degraded, and as shown in Figure 5A, this inactivated stage is attenuated by HMC-1 exosomes, which thus further can enhance the activation of cyclin D1.
These experiments together indicate that KIT positive exosomes from HMC-1 cells have the capability to induce PI3K/AKT signaling and influence cell proliferation in A549 cells.

Discussion
Tumor tissues include many different cells, and little is known how they interact in a clinical situation to influence cancer development. It has been suggested that exosomes and other extracellular vesicles can shuttle many molecules between cells, which is a process likely to be very active in cell-to-cell signaling in tumors. Tumor tissue also contains many inflammatory cells, and it is known that mast cells can be part of the tumor microenvironment in lung tumors such as non-small cell lung cancer. However, little is known how mast cells might act to specifically promote cancer proliferation. Here, we found that exosomes derived from the human mast cell line, HMC-1, contain the receptor KIT, which can be transferred to lung adenocarcinoma cells by exosomes. In particular, we show that mast cells release exosomes with typical exosome markers such as TSG101 and CD81. The mast cell exosomes are rapidly taken up by lung adenocarcinoma cells (A549 cells), a process that peaks after approximately 12 hours. Importantly, the exosomes released by human mast cells contain the natural growth factor receptor KIT, but not c-KIT mRNA. Addition of mast cell exosomes containing KIT enhance proliferation and migration of the lung adenocarcinoma cells, and enhance the KIT-SCF signaling pathway activity. Overall, these data suggest that exosomes from mast cell can enhance proliferation of lung adenocarcinoma cells, putatively by enhancing KIT-SCF signaling in tumor cells.
In this study, we confirm the ability of the mast cell line HMC-1 to release exosomes into their microenvironment [34], and extend these findings by demonstrating their ability to be taken up by human lung adenocarcinoma cells (A549 cells). The observed uptake is rapid, as more than 70% of cells have taken up some exosomes Figure 2 Uptake of human mast cell exosomes by human lung adenocarcinoma cells. Twenty microgram of the PKH67-labelled HMC-1, HEK 293 or NIH 3T3 exosomes, or a PKH67-PBS control were added per 2 ×10 5 A549 cells and incubated at 37°C or 4°C for 1-48 hours. The uptake of the fluorescently labelled exosomes by A549 was detected with flow cytometry (at all the time points), fluorescence microscopy (at the 12 hour time point) and confocal microscopy (at the 4 hour time point). A) Representative graph of uptake at 4 hours at 37°detected with flow cytometry. Black curve, control cells; blue curve, PKH67-PBS control; green curve, PKH67-labelled HMC-1 exosomes. B) Representative graph of uptake at 8 hours detected with flow cytometry. Black curve, control cells; red curve, PKH67-labelled HMC-1 exosomes at 4°C; green curve, PKH67-labelled HMC-1 exosomes at 37°C. C and D) Percent positive cells and relative fluorescence intensity (rFI) data for all time points determined with flow cytometry are shown as mean ± SEM (n =3). E) Uptake of PKH67-PBS control and PKH67-labelled exosomes at 37°C imaging with fluorescence microscopy. Nuclei were stained with 7-AAD (red). F) Uptake of PKH67-labelled exosomes at 37°C imaging with confocal microscopy (bar is indicating 20 μm). Nuclei were stained with DAPI (blue). G) Uptake of PKH67-labelled NIH 3T3-and HEK 293-derived exosomes at 37°C imaging with fluorescence microscopy. Nuclei were stained with 7-AAD (red).  already after one hour, and after four hours almost all cells are positive for exosomes. However, the uptake continues over time, measured as relative fluorescent intensity, and peaks at 12-24 hours after addition of exosomes. This time course of uptake is slightly faster then what we have previously reported [34].
Less is known about the functionality of mast cell exosomes. In the current experiments, we have described the presence of the KIT tyrosine kinase receptor in mast cell exosomes, a molecule that sometimes, but not always, is expressed in lung tumor tissues [36]. Importantly, we were unable to detect the c-KIT mRNA in the exosomes, suggesting that any functional shuttling of this molecule between cells is at the protein level. KIT was present in the lung adenocarcinoma cell line after addition of mast cell exosomes, documented using Western blot analysis. KIT has been considered to be a marker of progenitor cells [37] and can be more expressed in preneoplastic tissue and tumors, such as gastrointestinal stromal tumor (GIST) [38], small cell lung cancer (SCLC) [39], colorectal cancer [40,41] and pancreatic neoplasms [42]. KIT has also been implied in non-small cell lung cancer mortality, indicating that it may influence tumor growth and metastasis [23].
In the present study we confirm that addition of KITcontaining mast cell exosomes can enhance the proliferation of lung adenocarcinoma cells, shown as uptake of BrdU. A similar finding was recently published in which patient tumor exosomes containing KIT protein could transfer that protein to gastrointestinal stromal tumor cells and smooth muscle cells in vitro, which enhanced signs of invasiveness [26]. In our study, we have also included an appropriate exosome control in all Figure 5 Mast cell-derived exosomes can activate the KIT-SCF signaling pathway in A549 cells. A549 cells treated with exosomes from control cells (HEK 293) or HMC-1 cells were analyzed using Western blot. Phosphorylated and total PI3K, AKT, GSK3β and cyclinD1 were measured by Western blot and to normalize protein loading, samples were also probed for GAPDH. (A). Relative intensity was calculated as follows for p-PI3K (B), p-AKT (C) and p-GSK3β (D); (phosphorylated protein/GAPDH)/(total protein/GAPDH), and for cyclin D1 (E); cyclin D1/GAPDH. All the above data are representative of three independent experiments (n =3). *P <0.05 and **P <0.01. experiments, and provided evidence that the KIT containing mast cell exosomes can enhance the KIT-SCF signaling pathways intracellularly.
We could here detect enhanced KIT-SCF signaling pathways, by up-regulation of phosphorylated PI3K as well as AKT and GSK3β. These data suggest, collectively, that the KIT containing mast cell exosomes indeed can activate cascades downstream from the tyrosine kinase receptor KIT. We therefore propose that the tumor cellproduced SCF can help tumor growth via a PI3K dependent pathway, if mast cell KIT-containing exosomes are delivered to the cells. Mast cell exosomes carrying KIT may bind SCF molecules produced by the tumor cells, and activate the cell in an autocrine fashion via PI3K and AKT. It is known that AKT can enhance tumor growth, by phosphorylating and inactivating GSK3β. This can in turn lead to a decrease in the nuclear exclusion of cyclin D1, which thereby allow the molecule to functioning as a cell cycling promoter by driving the cells from G1 phase to S phase [43,44]. Overall, our data imply that mast cell KIT-containing exosomes can influence the PI3K signaling pathway in recipient tumor cells.
There are of course shortcomings with in vitro studies such as our, as results may or may not be applicable for clinical disease. However, in a previous clinical study of non-small cell lung tumors, we found that KIT positivity was associated with higher mortality [23].
Our current series of experiments have demonstrated the release of KIT-containing exosomes from a human mast cell line, and shows that these exosomes can be taken up by lung adenocarcinoma cells. This leads to enhanced proliferation in recipient tumor cells, by the activation of the PI3K signaling pathway. Future work can highlight the molecular mechanisms leading to the release of exosomes from mast cells, and their uptake into tumor cells. Further, it is essential to understand the diverse possibilities of exosome-based intercellular communication among many different types of cells in the tumor microenvironment.
Competing interests JL is a co-owner of using exosomes as therapeutics in disease, otherwise no other conflict of interest are present.
Authors' contributions HX performed a majority of the in vitro cell culture experiments. CL performed the electron microscopy. HX, CM and LHL optimized and performed the flow cytometry experiments and analysis. JW, HX, LHL and JLi participated in Western blot analyses and supported cell culture experiments. CL, TRL and GS supported the study by developing optimized exosome isolation protocols, and participated actively in the exosome isolations. GS supported the fluorescence microscopy experiments and performed the migration assay. TRL supported the Western blot and the RT-PCR experiments. MR and CL participated in the design of experiments and interpretation of data. LL conceived the study, and LL and JLö participated in the overall design of appropriate experiments. JLö together with HX and CL wrote the first draft and final versions of the manuscript, but all authors participated in the process. All authors have read and approved the final version of the manuscript.