Conversion to stem-cell state in response to microenvironmental cues is regulated by balance between epithelial and mesenchymal features in lung cancer cells

Abstract

Cancer cells within a tumor are functionally heterogeneous and specific subpopulations, defined as cancer initiating cells (CICs), are endowed with higher tumor forming potential. The CIC state, however, is not hierarchically stable and conversion of non-CICs to CICs under microenvironment signals might represent a determinant of tumor aggressiveness. How plasticity is regulated at the cellular level is however poorly understood. To identify determinants of plasticity in lung cancer we exposed eight different cell lines to TGFβ1 to induce EMT and stimulate modulation of CD133(+) CICs. We show that response to TGFβ1 treatment is heterogeneous with some cells readily switching to stem cell state (1.5-2 fold CICs increase) and others being unresponsive to stimulation. This response is unrelated to original CICs content or extent of EMT engagement but is tightly dependent on balance between epithelial and mesenchymal features as measured by the ratio of expression of CDH1 (E-cadherin) to SNAI2. Epigenetic modulation of this balance can restore sensitivity of unresponsive models to microenvironmental stimuli, including those elicited by cancer-associated fibroblasts both in vitro and in vivo. In particular, tumors with increased prevalence of cells with features of partial EMT (hybrid epithelial/mesenchymal phenotype) are endowed with the highest plasticity and specific patterns of expression of SNAI2 and CDH1 markers identify a subset of tumors with worse prognosis. In conclusion, here we describe a connection between a hybrid epithelial/mesenchymal phenotype and conversion to stem-cell state in response to external stimuli. These findings have implications for current endeavors to identify tumors with increased plasticity.

Keywords: CDH1; Cancer initiating cells; Lung cancer; Microenvironment; Plasticity; SNAI2.

Figure 1

TGFβ1 induces heterogeneous increase of CD133 + CICs in lung cancer cells. (A) Representative analysis by Flow cytometry of CD133 antigen expression in eight different lung cancer cell lines. (B) In vivo tumor growth after s.c injection of 5 × 105 A549, LT73, H460 or LT259 cells into the flanks of nude mice (n = 10 for each cell line). (C) Fold change of CD133+ cells after TGFβ1 treatment relative to untreated control. The data represent three independent experiments and replicates are expressed as mean ± SD. *p < 0.05. (D) Effect of TGFβ1 treatment on cancer initiating cells frequency in LT73 cells. Scalar doses of LT73 cells were subcutaneously injected in SCID mice and tumor growth observed for up to two months. (E) Relative expression levels of EMT‐related genes after TGFβ1 treatment: S=SNAI2, F=Fibronectin (FN1), V=Vimentin (VIM), Z = ZEB1 and E‐cadherin (CDH1). Expression levels were compared to untreated cells and normalized using HPRT as housekeeping gene. The Real‐Time PCR data represent the results of three independent experiments each with three replicates and are represented as mean ± SD. (F) Bright‐field microscopy (magnification 40×) of representative cell lines with different phenotypes, before and after 5 ng/ml TGFβ1 for 3 days.

Figure 2

NSCLC cells display different phenotypic states and can be classified according to the ratio of expression of CDH1 and SNAI2 genes (RES index). (A) Basal expression levels of EMT‐related genes analyzed by Real‐Time PCR. Average of gene expression of each cell line was used as reference sample. The data are the results of three independent experiments with three replicates each represented as mean ± SD. (B) Alignment of cell lines on the basis of RES index classification. (C) Scheme of different phenotypic state classification of lung cancer cell lines.

Figure 3

Epigenetic changes regulate cellular plasticity. (A) miR‐200c expression levels evaluated by Real‐Time PCR (relative to scramble transfected cells). RNU48 was used as housekeeping. The data represent two independent transfections each with three replicates and are represented as mean ± SD. (B) Quantitative Real‐Time PCR analysis of CDH1 and SNAI2 expression levels analyzed relatively to scramble transfected cell lines used as reference. Data are representative of three independent experiments with three replicates each. (C) Alignment of miR‐200c transfected cells on the basis of RES index classification. (D) Fold change of CD133 in miR‐200c transfected cells after TGFβ1 treatment relative to scramble transfected untreated cells. Data are the results of three independent experiments expressed as mean ± SD. *p < 0.05. (E) Expression levels of CDH1 analyzed by Real‐Time PCR relative to control after 5‐aza‐2'deoxycytidine treatment, miR‐200c or miR‐200c+5‐aza‐2'deoxycytidine treatment. Real‐Time PCR data represent the results of three independent experiments each with three replicates, expressed as mean ± SD. (F) Fold change of CD133 in H1299 transfected and/or 5‐aza‐2'deoxycytidine treated cells after TGFβ1 treatment (relative to untreated cells). Plots represent fold changes ± SD of three independent experiments. *, p < 0.05.

Figure 4

The frequency of cells co‐expressing epithelial and mesenchymal markers defines cellular plasticity. (A,B) Representative immunofluorescence analysis of cells of the different cell lines stained with CDH1 (green) and SNAI2 (red). Images are at 63× (A) and 40× (B) magnification. Scale bar 10 μm. C) Relative percentage of different cell subpopulations in different cell lines calculated with ImageJ software. 100 cells in two independent experiments were counted.

Figure 5

De novo generation of CD133+ cells in CICs depleted cells. (A) Fold change of CD133+ cells in LT73CD133Neg cells (a) and LT73‐CD133Neg_miR‐200c cells (b) after TGFβ1 treatment (relative to untreated control). Bars represent three independent experiments mean ± SD. *p < 0.05. (B) Representative result of three independent experiments performed by flow cytometry of spontaneous formation of CD133+ cells in H460CD133Neg or H460CD133Neg_miR‐200c after 30 days of in vitro culture (a). Fold change of frequency of CD133+ cells in H460CD133Neg (b) cells and H460CD133Neg_miR200 cells (c) after TGFβ1 treatment (relative to untreated control). Bars represent three independent experiments. *p < 0.05.

Figure 6

Microenvironmental stimuli promote selective in vivo tumor growth and spread of lung cancer cells with plastic phenotype. (A) Fold change of CD133+ cells in lung cancer cell line treated with five different fibroblasts conditioned media (n = 5). The results are represented as the mean value of CD133+ cells increase after different CM treatments compared to untreated control (±SD) Significant increase was seen only in A549 and LT73 cells (*p < 0.05). (B) (a) Kaplan–Meier curves representing tumor free animals injected with LT73 cells alone (n = 16, dashed line) or co‐injected with fibroblasts (n = 32, solid line). p value was calculated using the log‐rank test. Statistical significance was set at p < 0.05. (b) Frequency of CD133+ cells is significantly different between tumors originated from LT73 alone or co‐injected with fibroblasts. The results are expressed as the mean ± SD of frequency of CD133+ cells in control xenografts and co‐injected xenografts. *p < 0.05 was calculated with Student's t. (c) Percentage of human cancer cells detected in the lungs of mice carrying co‐injected tumors (n = 10) compared to control mice (n = 4) (p = 0.05). The data represent the mean of percentage of human cells ± SD. p value was calculated with Student's t. (C) (a) Kaplan–Meier curves representing tumor free animals injected with H460 cells alone (n = 14, dashed line) or co‐injected with fibroblasts (n = 32, solid line). p value was calculated using the log‐rank test. Statistical significance was set at p < 0.05. (b) Frequency of CD133+ cells is not significantly different between xenografts obtained from H460 cells injected alone or co‐injected with fibroblasts. The results are expressed as mean ± SD. p value was calculated with Student's t. (c) Percentage of human cancer cells detected in the lungs of mice carrying co‐injected tumors (n = 4) is not different compared to control mice (n = 4) (p = 0.1). The data represent the mean of percentage of human cells ± SD. p value was calculated with Student's t. (D) (a) Kaplan–Meier curves representing tumor free animals injected with H460_miR‐200c cells alone (n = 16, dashed line) or co‐injected with fibroblasts (n = 16, solid line). Difference between curves is significant with an increased number of events in co‐injected cells p = 0.02. p value was calculated using the log‐rank test. (b) Frequency of CD133+ cells is significantly different between xenografts from H460_miR‐200c, alone or co‐injected with fibroblasts. *p < 0.05 was calculated with Student's t.The results were expressed as the mean ± DS. p value was calculated with Student's t (c) Percentage of human cancer cells detected in the lungs of mice carrying co‐injected tumors (n = 4) compared to control mice (n = 4) (p = 0.06). p value was calculated with Student's t.

Figure 7

Specific pattern of CDH1 and SNAI2 immunostaining correlates with clinical outcome in lung cancer patients. (A) (a) Representative images of lung cancer sections stained with anti‐SNAI2 (negative, intermediate = 1 or high = 2) or (b) anti‐E‐cadherin (Low, MI = 1 E = 1, intermediate MI = 1 E = 2, MI = 2, E = 1, High MI = 2, E = 2). Scale bar 50 μm (magnification 40×). (c) Stratification of patients by tumor phenotypic state. (B) (a) Kaplan–Meier analysis of overall survival (OS) for patients stratified by negative (0), intermediate (I) or high (H) intensity of SNAI2. (b) Kaplan–Meier analysis of OS for patients stratified by low or high membrane intensity of E‐cadherin. (c) Kaplan–Meier curves of patients stratified by phenotypic states (E+,M+ or E/M) of tumors classified by the combination of SNAI2 and CDH1 scores. The colored bars indicate the high expression levels (SNAI2 High = Red M+, CDH1 High = Green E+) or intermediate expression levels (SNAI2 or CDH1 = Yellow E/M) of each marker. (d) Kaplan–Meier curves of patients with plastic phenotype tumors (hybrid E/M) vs. others phenotypic states (E+ and M+). p values were calculated using the log‐rank test and are indicated and statistical significance was set at p < 0.05.