Murine melanoma cells incomplete reprogramming using non-viral vector

Abstract

Objectives: The reprogramming of cancer cells into induced pluripotent stem cells or less aggressive cancer cells can provide a modern platform to study cancer-related genes and their interactions with cell environment before and after reprogramming. Herein, we aimed to investigate the reprogramming capacity of murine melanoma B16F10 cells.

Materials and methods: The B16F10 was transfected using non-viral circular DNA plasmid containing the genes Sox-2, Oct4, Nanog, Lin28 and green fluorescent protein (GFP). These cells were characterized by immunofluorescence, analysis RT-PCR and cell cycle.

Results: Our results demonstrated for the first time that reprogramming of B16F10 may be induced using non-viral minicircle DNA containing the four reprogramming factors Oct4, Sox2, Lin 28, Nanog (OSLN) and the GFP reporter gene. The resulting clones are composed by epithelioid cells. These cells display characteristics of cancer stem cells, thus expressing pluripotent stem cell markers and dividing asymmetrically and symmetrically. Reprogrammed B16F10 cells did not form teratomas; however, they showed the suppression of tumourigenic abilities characterized by a reduced tumour size, when compared with parental B16F10 cell line. In contrast to parental cell line that showed accumulation of the cells in S phase of cell cycle, the cells of reprogrammed clones are accumulated in G1 phase. Long-term cultivation of reprogrammed B16F10 cells induces regression of their reprogramming.

Conclusions: Our data imply that in result of reprogramming of B16F10 cells less aggressive Murine Melanoma Reprogrammed Cancer Cells may be obtained. These cells represent an interesting model to study mechanism of cells malignancy as well as provide a novel tool for anti-cancer drugs screening.

Figure 1

Transfection assay. Light microscopy and fluorescent imaging of the B16F10 cell line transfection procedures. A‐A2, GFP ⁺ cells highlighted in green fluorescence 18 h after the transfection. B‐B2, It is possible to see GFP ⁺ colonies formation 72 h after de transfection. C‐C2, iPSC‐like colony morphology, in which not all cells are GFP ⁺. D‐D2, GFP ⁺ in the cell nucleus. E‐E1, Negative control of B16F10 for GFP ⁻. Comparison of transfection efficiency between the B16F10 cells and fibroblasts (F and G) 18 h after the transfection, showing the GFP ⁺ cells. In (H), a comparative bar graph of the number of GFP ⁺ transfected cells from the B16F10, 3T3 and MEF, calculated by Wimasis Software

Figure 2

Incompletely reprogrammed cells asymmetric division. Light microscopy image of the different colonies morphology during growing on matrigel plate for 1 wk after transfection in (A) and (B). The zoom in (A′) and (B′) showed the asymmetric division between the cells, demonstrating the heterogeneity generated after reprogramming these cells

Figure 3

Isolated clones morphology. Light microscopy and fluorescent imaging of the clones, with the parental B16F10 cells in (A‐D). Phalloidin stain highlights differences in the cytoskeleton morphology (A1‐D1). The colonies of Clo1 (B‐B1) are composed by separate cells, resembling parental B16F10 (A‐A1). Meanwhile, the Clo2 (C‐C1) and Clo3 (D‐D1) colonies are juxtaposed, being similar to pluripotent cells colonies

Figure 4

Expression of ESC‐markers in MMRC clones. ESC markers via immunofluorescence in B16F10 cells and MMRC clones. Reprogrammed cells are positive for ESC markers: Oct4 (A), Nanog (B) and Sox2 (C). (D) Secondary antibody negative control. (E) RT‐PCR analysis of ESC‐marker genes in B16F10 cell line, MMRC clones and mES positive control. Primers used for Oct3/4, Sox2, Klf4 and c‐Myc specifically detect the transcripts of the interest genes

Figure 5

Histological tumour‐derived analysis. The B16F10 cells and the MMRC were subcutaneously transplanted into dorsal flanks of mice (C57Bl6). After twenty days, the animals were euthanized and the tumours were collected at the same day. In (A) comparison of the size tumours derived from MMRC and B16F10 parental cell line. (B) Haematoxylin and eosin staining of tumour derived from MMRC clones and B16F10 cell line. Compared to B16F10, the MMRC tumour‐derived histological analysis suggests lower tumourigenicity, reduced tissue necrosis and decreased cell heterogeneity

Figure 6

Cell cycle analysis. (A‐E) Histograms of cell cycle of control cells and MMRC, F representative graph for comparison of the cell cycle phases of MMRC and respective controls. B16F10 parental cell line showed accumulation of cells in S phase (A, F), while MMRC demonstrated a high number of cells in G1 phase, besides the reduction of cell number in S phase (B‐D, F). mESC were used as control and shows high number of cells in S phase, due to rapidly cell division (E, F)