Figure 1.
Spontaneous formation of heterotypic hybrids between lung cancer cells and MSCs.
(A) Fluorescent micrographs of a co-culture of A549-EGFP (green) and MSC-RFP (red), showing formation of double-labeled hybrid cell with two nuclei and a fibroblastic shape (arrows). Scale bar, 50 µm. (B) Fluorescent micrographs, taken at 2-hourly intervals, of co-cultures of SK-MES-1-EGFP (green) with MSC-RFP (red), showing apparent involvement of cell fusion (arrows) and change in color in the interaction of lung cancer cells with MSCs. Scale bar, 10 µm. (C) Quantification of spontaneous hybridization between lung cancer cells (A549-EGFP, H460-EGFP or SK-MES-1-EGFP) and MSC-RFP in high-density co-culture. The number of hybrids was assessed by flow cytometric analysis and expressed as a percentage of the total cells. Data represent the mean ± SEM of three experiments. (D) FISH analysis of HCC827/MSC co-cultures. Male MSCs were cultured with female HCC827 lung cancer cells for 8 days and fixed. FISH (Spectrum red-Y chromosome and Spectrum green-X chromosome) was performed and nuclei were stained with DAPI (blue). Green arrows show X chromosomes and red arrows, Y chromosomes. Male MSCs expressed one Y chromosome and one X chromosome whereas female HCC827 cells expressed only X chromosomes. In HCC827/MSC hybrids: example of two cells, one harboring two nuclei and the other with one nucleus. These two cells each possess a Y chromosome indicating an MSC-derived cell. Scale bar, 25 µm.
Figure 2.
Fusion between lung cancer cells and MSCs induces morphological changes.
(A) Phase-contrast images of heterotypic hybrids and respective parental lung cancer cells. After fusion with MSCs, lung cancer cells dispersed, lost their epithelial morphology and assumed a fibroblast-like appearance with elongated shape and front-to-back polarity. Scale bar, 500 µm. (B) Confocal laser microscopy imaging. A549/MSC hybrids assumed a fibroblast-like appearance and expressed both EGFP and RFP. Scale bar, 25 µm.
Figure 3.
Expression of epithelial and mesenchymal markers in heterotypic hybrids and respective parental lung cancer cells.
The MSCs and lung cancer cells were fluorescently tagged by retroviral transduction with RFP- and EGFP-expressing vectors, respectively. Spontaneously-formed heterotypic hybrids expressed both EGFP and RFP. Immunostaining was performed with primary antibodies against (A) E-cadherin (E-Cad), (B) pancytokeratin (pCK), (C) vimentin (Vim), (D) α-SMA, and (E) fibronectin (Fibro), and revealed using AlexaFluor 647-labelled secondary antibodies (purple); nuclei were stained with DAPI (blue). Scale bar, 25 µm. (F) The expression levels of the mRNAs encoding E-cadherin, vimentin, α-SMA, fibronectin, Foxc2, Slug, Snail, Twist, Zeb1 and Zeb2 in spontaneously formed A549/MSC, H460/MSC, SK-MES-1/MSC heterotypic hybrids relative to respective parental lung cancer cells as determined by QRT-PCR. GAPDH mRNA was used to normalize the variability in template loading. The data are reported as mean ± SEM.
Figure 4.
Migration and invasion assays of heterotypic hybrids and respective parental lung cancer cells.
The migratory and invasive potential of the cells was determined by counting the number of cells that had migrated to the lower surface of the filter in 12 randomly selected microscopic fields per insert. (A) Migration assays: microscopic images and quantification. (B) Invasion assays: microscopy images and quantification. The data are reported as mean ± SEM of three independent experiments. Asterisks depict statistically-significant differences between the heterotypic hybrids and respective parental lung cancer cells (*P<0.001). Scale bar, 50 µm.
Figure 5.
Expression of stemness markers in heterotypic hybrids and respective parental lung cancer cells.
(A) Flow cytometry analysis of CD133 expression in heterotypic hybrids and respective parental lung cancer cells. Percentages of CD133+ cells are indicated in control antibody and specific antibody-stained cells. Grey and black line indicated negative control and sample, respectively. (B) The expression levels of the mRNAs encoding CD133, Nanog, BMI1, Notch1, ALDH1, Sox2 and OCT4 in spontaneously-formed A549/MSC, H460/MSC and SK-MES-1/MSC heterotypic hybrids relative to respective parental lung cancer cells as determined by QRT-PCR. GAPDH mRNA was used to normalize the variability in template loading. The data are reported as mean ± SEM.
Figure 6.
Tumor sphere formation ability of heterotypic hybrids and respective parental lung cancer cells.
(A) Soft agar assays: Single cells (1×103 per well) were plated into soft agar in 6-well plates in triplicate. Microscopic images and quantification of heterotypic hybrids and respective parental A549, H460 or SK-MES-1 cells. The data are reported as mean ± SEM. (B) Pneumosphere assay: Microscopic images and quantification of heterotypic hybrids and respective parental A549, H460 or SK-MES-1 cells; n = 12. The data are reported as mean ± SEM. Asterisks depict statistically-significant differences between the heterotypic hybrids and respective parental lung cancer cells (*P<0.001). Scale bar, 100 µm.
Figure 7.
Tumorigenic potential of MSC-lung cancer hybrids.
(A) MSC-lung cancer hybrids are tumorigenic and regenerate similar tumors to their respective parental lung cancer cells in NOD/SCID mice. Hematoxylin and eosin (H&E) staining performed on tumor specimens derived from parental tumor or from tumors generated by subcutaneous injection of MSC-lung cancer hybrids in NOD/SCID mice (xenograft). Data are representative of three independent experiments. (B) Upper panel: Tumorigenic potential of 1×103 A549/MSC compared with 1×104 parental A549 cells. Cells were simultaneously injected into the right (A549/MSC) and left flank (A549) of the same mouse, and the mouse photograph was taken 12 weeks after injection. Lower panel: Gross pathology of tumor generated by subcutaneous injection of A549/MSC hybrids in the upper panel. Data are representative of three independent experiments. Scale bar, 100 µm.
Table 1.
Tumorigenic Potential of MSC-lung Carcinoma Hybrids.
Figure 8.
Reversion of A549/MSC hybrids from fibroblast-like to epithelial morphological characteristics in vitro and in vivo.
(A) Images, at successive time points, showing reversion of the fibroblastic morphological characteristics of A549/MSC hybrids to an epithelial, A549-like appearance in culture. Scale bars: 500 µm. (B) Immunohistochemical staining for the indicated antigens performed on tumor specimens derived from parental A549 tumor or from tumors generated by subcutaneous injection of A549/MSC hybrids in NOD/SCID mice. Explants of tumor xenografts of A549/MSC hybrids and parental A549 cells displayed similar lung cancer-like morphological characteristics. Scale bar, 100 µm.