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

Abstract

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.

Figure 1

Identification and characterization of HMC-1 exosomes. Exosomes were isolated using differential centrifugation. A) The electron micrographs of the exosomes revealed rounded structures with a size of approximately 30–100 nm. Arrows indicate 10-nm gold-labeled anti-CD63. The scale bar indicates 200 nm. B) Western blot analysis of exosomes derived from HMC-1 cells supernatant shows presence of the common exosome proteins, CD81 and TSG101, but absence of the endoplasmic reticulum protein, calnexin. Also control exosomes isolated from NIH 3T3 and HEK 293 cells were positive for TSG101 and CD81, but negative for calnexin. Cells were used as a control.

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 ×10⁵ 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).

Figure 3

Mast cell-derived exosomes induce proliferation and migration in A549 cells. (A) BrdU cell proliferation assays were used to detect proliferating A549 cells after co-culturing for 4 hours with HMC-1 exosomes or control exosomes (derived from NIH 3T3 cells). *P <0.05. (B) A549 cells were added to the lower chamber of a Boyden chamber (32 500 cells/well). To the upper chamber 30 μl of the different doses of HMC-1 exosomes were added. Media was used as a control. After 12 hours the number of cells migrated to the lower chamber of the 8 μm pore-sized membrane were analyzed by taking photos and counting the number of cells per visual field. Kruskal-Wallis test followed by Dunn’s multiple comparisons test were used to determine significant differences where all concentrations were only compared to the control. Spearman’s rank correlation coefficient was 0.91 (p <0.0001). p-values; * <0.05, *** <0.001.

Figure 4

HMC-1 exosomes transfer KIT protein to A549 cells. Eighty microgram of HMC-1 exosomes were added per 4 ×10⁵ A549 cells and incubated at 37°C for 24 hours for the Western blot experiments (A) and for 30 minutes - 48 hours for the RT-PCR experiments (B). A) Western blot confirmed the presence of the KIT protein in HMC-1 exosomes, but not in the A549 cells. SCF expression was detected in A549 cell but not in the HMC-1 exosomes. SCF and KIT were both detected in A549 cells 24 hours after the addition of HMC-1 exosomes. B) RT-PCR showed that c-KIT mRNA was present in HMC-1 cells, whereas it was absent in both HMC-1 exosomes and A549 cells. GAPDH mRNAs were detected as positive control and were detected in all samples except in HMC-1 exosomes.

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.