Interferon-Alpha Decreases Cancer Stem Cell Properties and Modulates Exosomes in Malignant Melanoma

Abstract

Malignant melanoma (MM) can spread to other organs and is resistant in part due to the presence of cancer stem cell subpopulations (CSCs). While a controversial high dose of interferon-alpha (IFN-α) has been used to treat non-metastatic high-risk melanoma, it comes with undesirable side effects. In this study, we evaluated the effect of low and high doses of IFN-α on CSCs by analyzing ALDH activity, side population and specific surface markers in established and patient-derived primary cell lines. We also assessed the clonogenicity, migration and tumor initiation capacities of IFN-α treated CSCs. Additionally, we investigated genomic modulations related to stemness properties using microRNA sequencing and microarrays. The effect of IFN-α on CSCs-derived exosomes was also analyzed using NanoSight and liquid chromatography (LC-HRMS)-based metabolomic analysis, among others. Our results showed that even low doses of IFN-α reduced CSC formation and stemness properties, and led to a significant decrease in the ability to form tumors in mice xenotransplants. IFN-α also modulated the expression of genes and microRNAs involved in several cancer processes and metabolomics of released exosomes. Our work suggests the utility of low doses of interferon, combined with the analysis of metabolic biomarkers, as a potential clinical approach against the aggressiveness of CSCs in melanoma.

Keywords: biomarkers; cancer stem cells; exosomes; interferon-α; malignant melanoma; metabolomics.

Figure 1

Proliferation assay, tumorsphere and colony-forming ability of MM CSCs enriched subpopulations. (A) Proliferation curves of A375 and MEL-1 CSCs subpopulations after treatment with low and high doses of IFN-α compared with controls non-treated cells (Mock). The initial cell number plated was 25,000 cells per well in all conditions; (B,C) Number of secondary spheres formed by A375 and MEL-1 cells after treatment with low and high doses of IFN-α compared with Mock cells. Melanospheres were counted after 3 days under light microscopy; the diameter of spheres from A375 cell line and MEL-1 cell line were measured by ImageJ software. Representative light microscopy (4×) images of spheres formed have been included. (D) Representative optical image of the colonies formed by A375 and MEL-1 cells from secondary spheroids (previously IFN or Mock-treated), after 37 days of soft agar culture in P6 well plates, stained with 0.1% Iodonitrotetrazolium Chloride. Data are graphed as mean ± SD from experiments carried-out by triplicates (*** p < 0.001; ** p < 0.01; * p < 0.05).

Figure 2

Effect of IFN-α treatment on the stemness properties of MM CSCs enriched subpopulations. (A) Comparative analysis for ALDH1 activity, CD20+, CD44+ and CD133+ expression in A375 and MEL-1 CSCs subpopulations analyzed by flow cytometry after treatment with low and high dose of IFN-α. The data were analyzed by t-test, *** p < 0.001; ** p < 0.01; * p < 0.05. (B) Effect of IFN-α on SP percentage in A375 and MEL-1 CSCs subpopulations; (C) Wound healing assay on A375 and MEL-1 CSCs subpopulations. Cells migration was quantified by measuring the wound closure area in pixels at 24, 48 and 72 h with ImageJ software and graphed. Representative optical images (10×) show the cells migration. The data were analyzed by t-test, ## p < 0.0001; # p < 0.0005; *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 3

Array and RNAseq analysis of MM CSCs. (A) Gene expression scatter plots of control versus 24 h IFN-α treated CSCs in A375 and MEL-1 cell lines. Up- and downregulated genes are shown in red and green, respectively. Number of common up- and downregulated genes (Fold Change > 2 and FDR < 0.05) between A375 and MEL-1 secondary spheres after 24 h of IFN-α treatment. (B) Functional relationships among selected genes as displayed by String database. (C) Heatmap of expression values (log2 Read Per Million) of selected miRNAs in A-375 and MEL-1 secondary spheres after 24 h of IFN-α treatment.

Figure 4

In vivo tumor formation by IFN-α pre-treated MM CSCs. (A) Tumor volume and weight of MM tumors formed in NSG mice after inoculation of A375 CSCs subpopulations and MEL-1 CSCs subpopulations. Representative images of tumors are shown. Data are shown as mean ± SEM statistical analysis Student’s test comparison IFN vs. Mock. (B) Histopathology of tumors formed by IFN-α pre-treated spheres. Representative immunofluoresence images for CD133, CD44, p75 and SAMD9L of tumors. Original magnification: 20×. Scale bar = 100 µm; Graph of the quantification of the fluorescence intensities. The average fluorescence intensities were calculated from three parallel immunofluorescence images. Statistical significance indicated * (p < 0.05), ** (p < 0.01), *** (p < 0.001).

Figure 5

Characterization of exosomes derived from MM CSCs subpopulations under low dose IFN-α treatment. (A) Transmission electron microscopic images of isolated exosomes with a saucer-like shape limited by a lipid bilayer. Vesicles isolated from culture supernatant (diameter ranging from ~40 to 130 nm). (B) The size distribution of exosomes obtained from CSCs A375 subpopulations and CSCs MEL-1 subpopulations was analyzed by NTA. (C) Topography of exosomes derived from CSCs A375 subpopulations and CSCs MEL-1 subpopulations observed under atomic force microscopy (AFM). Exosomes on a mica surface revealed heterogeneity in size and shape as well as forming aggregates in both 2D (above) images and 3D profiles (below). Acquisition areas were 5 × 5 µm² and 5 µm long profile lines are shown in red. (D) Number of exosomes counts and quantification of area from exosomes analyzed by AFM Grain Mode. (E) Western blot analysis of representative CD9, CD63, Alix and Hsp70 exosomes markers and the CD44 MM stem cell marker in melanospheres-derived exosomes treated under low dose IFN-α. GAPDH was used as a positive control.Statistical significance indicated * (p < 0.05), ** (p < 0.01). The full western blot figures could be found in the Supplementary Materials.

Figure 6

PCA score plots (A) and heatmaps (B) for all the LC-HRMS analyzed sample groups of exosomes from both MM CSCs A375 and MEL-1 subpopulations treated with IFN versus Mock control.