Reprogramming mediated radio-resistance of 3D-grown cancer cells

Abstract

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine.

Keywords: 3D growth microenvironment; matrigel; radio-resistance; reprogramming; β-catenin.

Fig. 1.

The morphology and clonogenic survival of 2D- and 3D-grown A549 cells. (A) Phase-contrast images of the 2D monolayer (above) and the 3D-cultured (below) A549 cells. (B) Survival fraction of 2D- and 3D-cultured A549 cells after exposure to 0, 1, 2 or 4 Gy of X-rays . Data are presented as mean ± SE. Experiments were independently repeated at least three times.

Fig. 2.

The percentage of stem cell–like cells in 2D- (left) and 3D- (right) cultured A549 cells assayed using Rho/FACS; the percentage of Rho^low cells is shown in the picture.

Fig. 3.

The expression of reprogramming transcription factors in 2D- and 3D-grown A549 cells. (A) qRT-PCR for the expression of reprogramming transcription factors OCT4, SOX2, NANOG, c-MYC and LIN28 in 2D- and 3D-grown A549 cells. (B) Western blotting for expression of OCT4, SOX2 and NANOG in 2D- and 3D-grown A549 cells. (C) Gray analysis the result of Figure 3B. (D) qRT-PCR for the expression of reprogramming factor miR-302a expression in 2D- and 3D-grown A549 cells. (E) Western blotting for the expression of β-catenin in 2D- and 3D-grown A549 cells. Data are presented as mean ± SE. Experiments were independently repeated at least three times.

Fig. 4.

The morphology and stem cell-like phenotype of MCF7 and PC3 cancer cells in a 3D microenvironment, compared with 2D monolayer cells. (A) Phase-contrast images of the 2D- (left) and 3D- (right) grown MCF7 (above) and PC3 (below) cells. (B) The percentage of stem cell–like cells in 2D- (left) and 3D- (right) grown MCF7 (above) and PC3 (below) cells were assayed using Rho/FACS; the percentage of Rho^low cells are shown in the picture.