Introduction
Transforming growth factor beta (TGFβ) is a pleiotropic cytokine that under normal conditions is involved in a range of cellular process including but not limited to regulating cell growth, proliferation and differentiation. The role of TGF-β in cancer progression is still a matter of debate, as it has been described as having both tumour suppression and tumour promoting potential, depending on stage of cancer progression [
1]. Moreover, TGF-β signalling and/or hypoxia have also been reported to drive epithelial to mesenchymal transition (EMT) in many cancers contributing to cancer progression (reviewed in refs. [
2,
3]), including thyroid cancers were EMT makers have been found to be overexpressed in more aggressive and metastatic thyroid tumours in vivo and in vitro [
4‐
8]. In the thyroid, TGF-β1 has also been reported to increase the expression of HMGA1 at both the gene and protein level in the thyroid cancer cell line SW579. While HMGA1 has been reported to be involved in the progression of several types of cancers including thyroid, this study supports a role for TGF-β1 in its induction [
9].
SP cells have been shown to be present in a number of different cancers and cancer cell lines and have been demonstrated to be more tumorigenic and drug resistant than non-SP cells (NSPs) [
10]. SP cell behaviour has been reported to be regulated at some levels when exposed to endogenous TGF-β1 in several cancers including breast cancer, ovarian cancer and pancreatic cancer [
11‐
13]. For example in the breast cancer cell line MCF7 SP it was shown that MCF7 SP abundance could be depleted by TGF- β1 directed EMT and that this was associated with a decrease in
ABCG2 expression. Moreover, removal of TGF-β1 resulted in restoration of
ABCG2 expression and SP cell numbers [
11]. While SP cells of the pancreatic cancer cell lines PANC-1 and Capan-2 were more responsive than NSPs (bulk cells without the SP fraction) to TGF-β1 switching their phenotypic traits for epithelial to mesenchymal and back upon reversal of TGF-β1 treatment [
13].
We have previously reported on the presence of SP cells in the normal thyroid cell line N-thy ori-3-1, papillary thyroid cancer (PTC) cell line BCPAP and the ATC cell line SW1736 and reported that these cancer SP cells are responsive to hypoxia, with an increase in cell number upon hypoxia exposure [
14]. In addition, we reported on functional differences in terms of migration and invasion potential in the SW1736 SP above that of the N-thy ori-3-1 SP [
15].
The objectives of this study were to determine the impact on thyroid SP cells exposed to endogenous TGF-β1 and then to determine the impact of removal of endogenous TGF-β1 on these cells. We hypothesized that fluctuations in TGF-β1 levels would show a transient effect on SP cell behaviour and might go some way in explaining why targeting of the TGF-β pathway alone has had little impact in the clinical setting for successfully treating a number of different cancers [
16].
Materials and methods
Cell lines
Cell lines used in this study included the PTC cell line BCPAP and the ATC cell line SW1736, gifts from G. Brabant (Universitats Klinikum, Germany) and C. McCabe (University of Birmingham, UK), while the N-thy ori-3-1 was purchased (ECACC, UK). All cell lines were authenticated prior to use using STR fingerprinting.
Culturing of cells lines
Cell culture was carried out as described previously [
15]. But in brief cells were cultured in appropriate base media, RPMI 1640 for the N-thy ori-3-1 and BCPAP and DMEM GlutaMAX for the SW1736 (both from Gibco, UK). All media was supplemented with 10% FBS L-glutamine and penicillin/streptomycin. Once 80% confluent cells were harvested and used for analysis.
Ethics and tissue collection
This study was performed according to the amended Declaration of Helsinki and informed consent from patients. As part of patient consent, all patients consented for results of this study to be used for scientific publication. Tissue used in this study was surplus to diagnostic requirements and collected under ethical approval REC number 13/NE/0026 2013. Tissue was made available for research purposes on the day of surgery.
Isolation of RNA from tissue
In brief, 1 ml of Tri reagent (Sigma, UK) was added to 50–100 mg of tissue, this was then dissociated using a MACS dissociator (MIlentyi Biotec, UK) programme RNA-01, the aqueous layer was then removed and centrifuged 11,000 g for 10 min. 200 μl of chloroform was then added to this and left at room temp for 3 min. This was then centrifuged as above for 15 min and clear aqueous layer containing RNA collected for further processing using the RNeasy MinElute Kit (Qiagen, UK) as per manufactures instructions.
Semi-quantitative PCR analysis
RNA from tissue and from cells was used for cDNA synthesised using the Tetro-cDNA synthesis kit (Bioline, UK), as per manufacturer’s instructions. PCR was performed as describe previously [
15]. Primers used in this study included
ABCB1 F:5′-CTGACGTCATCGCTGGTTTC-3′, R:5′- ATTTC CTGCTGTCTGCATTGTGA-3′;
ABCG2 F:5′-GAGCGCACGCATCCTGAGAT-3′, R:5′ TCATTGGAAGCTGTCGCGGG-3′; and
GAPDH F: 5'-GCCTTCTCCATGGTGGTGGTGAA-3', R: 5'-GCACCGTCAAGGCTGAGAAC-3', which was used as a loading control.
Isolation and culture of primary tissue-derived cells
Tissue was minced into @2 mm pieces manually, this was then digested in collagenase IV 20 mg/ml (Gibco, UK) and DNase 0.01% (Worthington, UK) in 20 ml of complete Roswell Park Memorial Institute 1640 (RPMI-1640) media in a shaking water bath at 37 °C for 90 min. This was the filtered using a 70 µM cells strainer, pelleted and the resulting pellet treated with lysis buffer (23 ml of 0.15 m NH
4Cl pH 7.5) to remove red blood cells. The cells were then re-pelleted, lysis buffer discarded and the cells, isolated from all tissue types, were cultured in Humanised seven homoeostatic additives thyroid media (h7H media—see Table
1 for additives), under standard tissue culture conditions until 80% confluent, then used for further analysis. All dissociation steps in primary tissue-derived cells were performed using the Tryple/Glucose (1 g/L) combination.
Table 1
Humanised seven media additives
Somatostatin | 50 ng/l |
Thyroid-stimulating hormone (TSH) | 40 mIU/l |
Insulin | 25 mIU/l |
Cortisol | 23 nM/l |
Recombinant human growth hormone protein | 0.2 μg/l |
Apotransferrin | 5 mg/l |
Sodium iodide | 10 μg/l |
Sodium selenite | 75 μg/l |
Zinc | 863 μg/l |
Iron | 834 μg/l |
Sodium L-ascorbate | 15 mg/l |
L-glutathione reduced | 0.2 mg/l |
DL ± -α-tocopherol | 0.5 mg/l |
DL-α-tocopherol acetate | 0.5 mg/l |
Sodium pyruvate | 220 mg/l |
Glucose | 1800 mg/L |
Ethanol (final concentration contributed from diluents of the above reagents) | 0.0177% |
Real-time PCR
Real time was performed as described previously [
17]. Taqman primers/probes (Applied Biosystems, UK) were used to examine gene expression, with primer/probes being specific for:
TGFB1 (Hs_00998133_m1),
TGFBR1 (Hs_00610320_m1),
TGFBR2 (Hs_00234253_m1),
CDH1 (Hs_0.023894_m1),
CDH2 (Hs_00983056_m1),
ABCG2 (Hs_01053790_m1) and
GAPDH (Hs_02758994_g1). The comparative ΔΔCT method was used to assess relative mRNA expression by normalising to
GAPDH and expressing values as fold change relative to N-thy ori-3-1 or to untreated cells. Real-time PCR was performed using the Quant Studio™7 Flex Real-Time PCR Machine (Life Technologies).
SP assay
For tissue-derived SP cells, h7H was removed from cultures and cells were washed three times with phosphate buffered saline (PBS) before being detached from the flask using Tryple Express (Gibco, UK). Resulting cell suspension were stained, 1 × 10
6 cells per ml of RPMI-1640 media, containing 1 µg/ml Hoechst 33342 dye. Cells were incubated for 60 min at 37 °C. Assay was then completed, data analysed, and SP phenotype confirmed by the addition of Verapamil as described previously [
18]. Assay and data analysis for cell line SP was performed as described previously [
15]. Conditions to obtain the highest SP percentage have been optimised for each cell line used in this study. The N-Thy ori-3-1, BCPAP and SW1736 needed 7, 3 and 5 μg/mL Hoechst 33342 respectively. The incubation time for N-thy ori-3-1 and BCPAP was similar at 60 min, while the SW1736 was incubated for 90 min. Verapamil was used to inhibit dye efflux, with all cells being incubated with 100 μM/mL of verapamil for 15 min before the final wash and analysis.
Gating strategy was performed using the forward (related to cell size) and side scatter (related to cell granularity) dot plots to exclude debris as described [
18]. Next, dead cells were exclude based on propidium iodine (PI) dye uptake. This was done by adding PI at 2 μg/ml to all samples prior to FACs analysis as described previously [
15]. PI is unable to pass through intact cell membranes and therefore only stains cells that have compromised membranes allowing for gating out of dead cells based on their PI positivity, leaving only inclusion of viable PI negative cells for analysis/sorting. This was followed by gating for single cells using the width to height ratio on Hoechst labelling plotted on Hoechst blue (3-355/405/50-A) versus Hoechst red (3-355/405/50-H) axis. This gating strategy was designed to analyse only live, single cells.
TGF-β1 treatment and reversal of treatment of cell lines cells and tissue-derived cells
Cells for all cell lines were plated into 100 mm dishes (Corning) and 24 h after plating, treated with pre-optimised concentrations of TGF-β1 [R&D Systems, Inc., Abingdon, UK] 10 ng/mL TGF-β1 for the N-thy ori-3-1 and 1 ng/ml for BCPAP and SW1736 and incubated for 72 h. Following this, SP analysis was performed as described previously [
15].
To study the impact of TGF-β1 reversal on the SP percentage, media containing exogenous TGF-β1 was removed. Cells were then washed with PBS and new pre-warmed media added before being incubated for another 72 h, the cells were then assayed for the presence of SP. In every step, following treatment and reversal of treatment of TGF-β1, untreated cells were used as control samples.
For tissue-derived cells, at 80% confluency, plated cells were treated with 1 ng/ml TGF-β1 for 14 days and then assayed for SP percentage as described previously [
15]. Controls were cells cultured for 14 days but not exposed to exogenous TGF-β1.
To confirm that any changes in SP cell percentage following treatment with TGF-β1, were specifically a result of involvement of TGF-β1, cells of the SW1736 cell line were treated with 3 mM SB-505124 (Sigma, UK).
Statistical analysis
One-way ANOVA and Tukey post-hoc test was used to determine the significance of the difference seen in SP % obtained from the normal, benign and malignant thyroid tissue-derived cells.
P-value of <0.05 was considered significant. In this study
R2 is a measure of explanatory power in this logistic regression model were
R2 is used to estimate whether the regression model can be reliably used or not to explain the relationship between the studied variables. Therefore, this was used to explain the relationship between the percentage of SP in relation to gender, age and nodule size. The regression model was run in a stratified manner looking at the influence of each factor on SP percentage (see Table
2).
Table 2
Details of patients age, gender, source of tissue, size of tissue and percentage SP isolated from tissue
Source of normal tissue |
From completion thyroidectomy | 1 | 0.1 | Female | 50 | n/a |
With follicular adenoma | 2 | 0.2 | Female | 42 | n/a |
From completion thyroidectomy | 3 | 0 | Female | 35 | n/a |
With MNG | 4 | 0.2 | Female | 78 | n/a |
With follicular adenoma | 5 | 0.1 | Female | 56 | n/a |
With follicular adenoma | 6 | 0 | Female | 37 | n/a |
With follicular adenoma | 7 | 0.1 | Female | 45 | n/a |
With MNG | 8 | 0.1 | Female | 34 | n/a |
From completion thyroidectomy | 9 | 0.4 | Female | 32 | n/a |
With follicular adenoma | 10 | 0.1 | Male | 47 | n/a |
From completion thyroidectomy | 11 | 0.1 | Male | 48 | n/a |
Source of benign tissue |
Colloid nodule | 1 | 0.2 | Female | 30 | 26 |
Follicular adenoma | 2 | 0.2 | Female | 42 | 20 |
Graves’ disease | 3 | 0.7 | Female | 30 | n/a |
MNG | 4 | 0.2 | Female | 78 | 30 |
Follicular adenoma | 5 | 0.1 | Male | 47 | 20 |
Follicular adenoma | 6 | 0.1 | Female | 45 | 20 |
Follicular adenoma | 7 | 0.1 | Female | 37 | 18 |
Follicular adenoma | 8 | 0.1 | Female | 35 | 13 |
Source of malignant tissue |
Papillary cancer | 1 | 1.6 | Male | 48 | 75 |
Papillary cancer | 2 | 0.8 | Male | 64 | 13 |
Papillary cancer | 3 | 0.6 | Female | 56 | 30 |
Source of tissue for semi-quantitative PCR | Patient | n/a | Gender | Age | n/a |
Normal | 1 | n/a | Female | 27 | n/a |
Papillary cancer | 2 | n/a | Female | 17 | n/a |
Follicular adenoma | 3 | n/a | Female | 37 | n/a |
Follicular cancer | 4 | n/a | Female | 23 | n/a |
Discussion
In the thyroid, SP cells have been shown to be present in normal and nodular goitres and in normal and thyroid cancer cell lines [
19,
20]. In the present study, we have shown that we can isolate SP cells from primary cell cultures derived from NT, BT and PTC. Our SP analysis revealed the SP percentage to be significantly higher in PTC compared to NT and BT. Our statistical analysis revealed no correlation between SP cell number, patient age, gender or size of nodule/tumour, but this data should be treated cautiously as patient numbers were small. The presence of SP cells from thyroid tissue has previously been identified in primary human thyroid cell cultures established from human goitres at passage 2 where SP was 0.1% or from growth factor stimulated thyrospheres which are enriched for SP (increased to 5%) [
19]. In this study we may also have expanded SP cell numbers by culturing primary cells from all 3 tissue types in h7H media until 80% confluent.
ABC transporters, particularly ABCG2 and ABCB1, have been linked to the SP phenotype for SP isolated from several other tissue types [
21‐
24] and cancer cell lines [
17,
21,
25].
ABCG2 expression has been reported to be more highly expressed in a number of thyroid cancer cell lines above that of their NSP [
20] and
ABCB1 has been reported to be expressed in the N-thy ori-3-1 and BCPAP cell lines [
15]. In addition, higher ABCG2 expression has also been reported in the solid component of PTC (and while still controversial PTC containing solid components have been suggested to be linked to poorer prognostic outcomes) [
26]. Therefore, we also examined several thyroid tissues for expression of these genes and showed that regardless of tissue origin (normal, benign or cancerous) we could detect mRNA for both transporters.
Based on our previous published study showing we could isolate SP cells from the N-thy ori-3-1, BCPAP and SW1736 cell lines, that have characteristics of stem cells [
15], we employed these cell lines in this study. However, as with use of all cell lines, it is important to note that overtime their characteristics may change. For example, BCPAP was originally classified as a PTC cell line [
27]. However, during the in vitro evolution of this cell line, its DNA synthesis/replication mechanisms have been partially lost and it now more closely resembles a poorly differentiated thyroid cancer.
Therefore, this needs to be taken into consideration when interpreting results generated using this cell line [
28,
29]. We examined bulk cells for the expression of markers associated with TGF-β and EMT and observed expression of both
TGFβ ligand and receptors in all 3 cell lines. Our analysis showed that only in the case of
TGFBR2 was there significantly higher expression of this receptor in the BCPAP cell line above that of expression in the N thy ori-3-1. It has been reported in a study comparing
TGFB1 mRNA levels in both benign nodules and PTC that the levels of
TGFB1 expression were significantly higher in the PTC [
24]. While in an earlier study examining expression of
TGFB1 in both PTC and normal thyroid it was also shown that
TGFB1 was overexpressed in the PTC tissue [
8].
In terms of EMT marker expression,
CDH1 was expressed at higher levels in the N-thy ori-3-1 cell line whereas
CDH2 was more highly expressed in the cancer cell lines. These results are perhaps not unexpected as down regulation of
CDH1 and increased expression of
CDH2 is pivotal for the onset of EMT (reviewed in ref. [
30]).
Having determined that the thyroid cell line models we were employing expressed markers that predicted TGF-β responsiveness we then wanted to determine if TGF-β1 treatment impacted on
ABCG2 expression. We observed that following treatment mRNA levels of
ABCG2 were reduced significantly when comparing both cancer cell lines to control. We then went onto determine if this treatment impacted on SP cell percentage, as would be suggested by the reduction in
ABCG2 expression and were able to determine that for both the cancer cell lines there was a reduction in SP percentage, but this was only significant when comparing control and treated SW1736. To determine if this was a reversible loss of the SP phenotype, we then removed the exogenous TGFβ and observed restoration of the SP phenotype and an increase in SP abundance in both cancer cell lines compared to controls. To confirm that these changes were due specifically to the impact of TGF-β1, we treated cells of the SW1736 cell line with a TGF-β1 receptor inhibitor and demonstrated that in the presence of this inhibitor the SW1736 SP was restored. This is in line with a study of MCF7 breast cancer SP cells which also showed a reduction in
ABCG2 expression and a reduction in SP numbers following TGF-β treatment, both of which were reversable on removal of exogenous TGF-β1 [
11].
We then examined the impact of TGFβ treatment on SP cells of primary PTC and NT and observed that while treatment had little effect on SP of NT (see supplementary Fig. 2), the percentage of SP cells in primary PTC cultures were reduced to below detectable levels using our SP protocol and gating strategy.
While therapeutic targeting of TGF-β as a means of treating cancers has as yet not lived up to the results observed in some pre-clinical studies, we suggest limitations might be due in part to cell type being targeted and EMT being partial. For example, in ovarian cancer, higher numbers of SP cells have been reported to be present in ovarian cancer cell lines. When treated with TGF-β1 these SP cell numbers are reduced and driven towards a more mesenchymal cell phenotype. They display an increase in expression of some EMT markers. For example, snail1 an important regulator of EMT.
Silencing of snail1 then resulted in the ovarian cancer SP cells showing a change in mRNA expression increasing expression of epithelial markers and decreasing expression of mesenchymal markers, again suggesting that regulation of the EMT process can impact on SP cells’ ability to undergo mesenchymal transformation [
12].
Our data supports a transient role for TGF-β in regulating thyroid cancer SP cell behaviour and further points to the need for a better understanding of the complexed role of TGF-β in cancer, including in regulation of cancer stem cells.
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