Generation and characterization of virus-free reprogrammed melanoma cells by the piggyBac transposon

Abstract

Purpose: Reprogramming of cancer cells to stem cell-like state provides a promising tool for the study of cancer pathogenesis and drug screening. However, most instances of direct reprogramming have been achieved by forced co-expression of defined transcription factors using viral vectors. Retroviral transduction as well as the ectopic expression of reprogramming factors may alter the differentiation potential of reprogrammed cancer cells or induce malignant transformation. Therefore, generation of reprogrammed cancer cells via virus-free reprogramming strategy needs to be studied.

Methods: Melanoma cells were reprogrammed by co-expression of doxycycline-inducible Oct4, Sox2, Klf4, and c-Myc using the piggyBac (PB) transposon system. The expression level of genes was analyzed through RT-PCR, Western blot, and immunofluorescence. Epigenetic modification of genes was detected by bisulfite genomic sequencing. Post reprogrammed melanoma cells were generated through differentiation of reprogrammed melanoma cells. Sensitivity to chemotherapeutic agents and metastasis potential were investigated in post reprogrammed melanoma cells.

Results: The virus-free reprogrammed melanoma cells were positive for stem cell markers including Oct4, Nanog, and SSEA-1, and the promoters of Nanog and Oct4 were demethylated. Moreover, reprogrammed melanoma cells gained differentiation potential and higher sensitivity to differentiation-inducing drugs. Post reprogrammed melanoma cells showed lower proliferation rate and metastatic potential compared with the parental cells.

Conclusions: Our results indicate that PB transposon-based method is applicable to generate virus-free reprogrammed melanoma cells. These cells can differentiate into other lineages with loss of malignant phenotypes, which may provide a more suitable source for molding of cancer pathogenesis.

Fig. 1

Reprogramming of mouse melanoma cells by co-expression of defined factors. a Flow chart illustrating the steps of reprogramming mouse melanoma cells was summarized. b Morphology of B16-2B2 cells in monolayer culture. Scale bar represents 200 μm. c Reprogrammed melanoma cells had round shapes that were different from B16-2B2 cells. Scale bar represents 200 μm. d Reprogrammed melanoma cells lacked contact inhibition and formed mounds in culture by single-cell passage. Scale bar represents 200 μm

Fig. 2

The expression levels of pluripotency-related genes in reprogrammed melanoma cells. a RT-PCR analysis of ES marker genes in B16-2B2 cells, ES cells, and reprogrammed melanoma cells (REP). b Immunofluorescence revealed that reprogrammed melanoma cells were positive for ES cell markers, including Nanog, Oct4, and SSEA-1. Scale bars represent 50 μm. c The mRNA level of melanocyte markers (TYR and DCT) was measured by Q-PCR. The expression of mRNA was normalized against GAPDH. Error bars denote the standard error (n = 3). d Western blot showed expression levels of tyrosinase and β-actin in B16-2B2 cells and reprogrammed melanoma cells (REP)

Fig. 3

Epigenetic modification of pluripotency-related genes evaluated with methylation analysis. The promoters of Nanog and Oct4 were analyzed by bisulfite genomic sequencing for DNA methylation status in B16-2B2 cells and reprogrammed melanoma cells (REP). White balls indicate unmethylated CpGs and black balls indicate methylated CpGs

Fig. 4

Differentiation potential of reprogrammed melanoma cells. a Flow chart illustrating the steps of generation of post reprogrammed melanoma cells (P-REP) from reprogrammed melanoma cells (REP) was summarized. b Q-PCR analysis verified the expression of differentiation markers, such as Alb, Fabp4, and Gfap in post reprogrammed melanoma cells (P-REP). The expression of mRNA was normalized against GAPDH. Error bars denote the standard error (n = 3). c Immunofluorescence confirmed the expression of Gfap, AFP, and α-SMA in the differentiated reprogrammed melanoma cells. Scale bars represent 10 μm. d Q-PCR confirmed the expression of the bone markers (alkaline phosphatase (AP), osteopontin, and osteocalcin) in REP after osteogenic differentiation. The expression of mRNA was normalized against GAPDH. Error bars denote the standard error (n = 3). e Q-PCR confirmed the expression of the specific cartilage markers (typeII collagen), highly expression of cartilage regulator gene Runx2 in reprogrammed melanoma cells (REP) after osteogenic differentiation. The expression of mRNA was normalized against GAPDH. Error bars denote the standard error (n = 3)

Fig. 5

Reprogrammed melanoma cells were sensitive to differentiation-inducing chemicals and anticancer drug a proliferation assay showed that REP were sensitive to the differentiation-inducing chemicals (NaB, DMSO, and RA). Error bars denote the standard error (n = 3, *p < 0.05, **p < 0.01). b Post reprogrammed melanoma cells (P-REP) showed sensitive to dacarbazine compare to B16-2B2 cells (n = 3, *p < 0.05)

Fig. 6

Post reprogrammed melanoma cells displayed reduced aggressive behavior. a The growth curves of B16-2B2 cells and post reprogrammed melanoma cells (P-REP). Error bars denote the standard error (n = 3, *p < 0.05). b B16-2B2 cells and post reprogrammed melanoma cells (P-REP) were cultured for 25 days in 0.5 % soft agar. Scale bar represents 200 μm. c Anchorage-independent growth of B16-2B2 cells and post reprogrammed melanoma cells (P-REP). 1 × 10³ cells were seeded in one well of six-well plate, and the number of soft agar colonies presented the mean of colonies counts in 3 wells of six-well plate. Error bars denote the standard error (n = 3, *p < 0.05). d In vivo tumor formation in C57BL/6 mice injected with B16-2B2 cells and post reprogrammed melanoma cells (P-REP). Error bars denote the standard error (n = 3, *p < 0.05). e Representative photos of the lungs from mice carrying tumors of B16-2B2 cells and post reprogrammed melanoma cells (P-REP) 30 days after tail vein injection. f Total numbers of lung metastatic nodules in individual mouse were counted. B16-2B2 cells and post reprogrammed melanoma cells (P-REP) were injected into the tail vein of C57BL/6 mice (n = 10, **p < 0.01)