Human Cell DOI 10.1007/s13577-014-0088-9

RESEARCH ARTICLE

Functional analysis of three novel cell lines derived from human papillary thyroid carcinomas with three different clinical courses Mayumi Ishikawa • Toshiaki Tachibana • Hisashi Hashimoto • Junko Toyomura Toshihide Ito • Kumiko Tsuboi • Kazutoshi Shibuya • Takahisa Hirose • Shiro Minami • Gen Yoshino

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Received: 16 December 2013 / Accepted: 27 December 2013 Ó Japan Human Cell Society and Springer Japan 2014

Abstract Papillary thyroid carcinoma (PTC) is the most frequent thyroid carcinoma. PTC cell lines have been of considerable value in studying aspects of thyroid cancer, such as gene expression, cell proliferation, and differentiation. Here we report three novel PTC lines established from three patients with different backgrounds. Case 1 was a 38-year-old woman with PTC in the right thyroid lobe, with no metastasis. The cell line was established from the resection sample and named D-PTC. The cell line consisted of epithelial cells with few lysosomes and showed a pavement structure and follicular formation at confluency. There was a little pilling up. The secretion of free thyroxin (fT4) and thyroglobulin (Tg) was increased by TSH, or GH and IGF-I treatment. Case 2 was a 22-year-old woman with PTC initially in the right thyroid lobe, but 4 years after the right lobe resection, PTC metastasis was observed in left lobe. The cell

line was established from a sample of the second resection and named UD-PTC. This cell line consisted of small epithelial cells with evident lysosomes and exhibited floating cell clusters. The secretion of fT4 and Tg was slightly increased by TSH, or GH and IGF-I treatment. Case 3 was an 85-year-old man with PTC and with acromegaly. Metastasis was observed at cervical lymph nodes. The cell line was derived from the metastasis region and named A-PTC. This cell line consisted of small epithelial cells and many lysosomes. The cells frequently showed pilling up. The secretion of fT4 and Tg was significantly increased by GH and IGF-I treatment. We have established three PTC cell lines with substantial variation in their phenotype. The cell lines may be useful for thyroid cancer research. Keywords

Papillary thyroid carcinoma
Cell line

M. Ishikawa is formerly affiliated to Division of Diabetes, Metabolism and Endocrinology, Toho University School of Medicine, Tokyo, Japan M. Ishikawa (&)
S. Minami Center of Endocrinology, Diabetes and Arteriosclerosis, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki 211-8533, Japan e-mail: [email protected] T. Tachibana Core Research Facilities, Jikei University School of Medicine, Tokyo, Japan H. Hashimoto Department of Anatomy, Jikei University School of Medicine, Tokyo, Japan

T. Ito Division of Breast and Endocrine Surgery, Toho University School of Medicine, Tokyo, Japan K. Tsuboi
T. Hirose
G. Yoshino Division of Diabetes, Metabolism and Endocrinology, Toho University School of Medicine, Tokyo, Japan K. Shibuya Department of Pathology, Toho University School of Medicine, Tokyo, Japan

J. Toyomura Department of Life Sciences, Faculty of Life Dentistry, Nippon Dental University, Tokyo, Japan

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Introduction Papillary thyroid carcinoma (PTC) is the commonest thyroid carcinoma, representing 80–90 % of thyroid cancer [1, 2]. Usually the clinical course is benign, but there is variation, and 1–8 % of patients with PTC present with distant metastases [3]. Cell lines are useful for understanding tumor biology and are needed to exploit therapeutic methods. Several cell lines derived from PTC have been reported [4–9]. However, it is important to note that there is variation in these PTC lines. For example, cell lines derived from well-differentiated PTC express variable amounts of thyroid differentiation markers, such as thyroglobulin (Tg), thyroid peroxidase (TPO), and TSH receptors (TSHR) [3], and cytokines, such as IL6, IL8, TGF-beta, and LIF [1, 10–12]. We established and examined the characteristics of three novel PTC cell lines derived from three patients with different clinical courses, such as with or without metastasis, or with acromegaly and hence GH and IGF-I excess [13]. One line (D-PTC) displayed follicular formation and TSHresponsive Tg, free triiodothronine (fT3), and free thyroxin (fT4) secretion. Materials and methods These studies were authorized by the Ethical Committee of Toho University Omori Medical Center (No 21-117).

pathologies are shown in Table 1. Completion of thyroidectomy and lymph node dissection (D2a) revealed a contralateral recurrence in the left thyroid lobe and local recurrence in the neck lymph node. Subsequently, further metastases have not been detected (pT1 N0 M0 Stage1). A cell line was established from a biopsy at the second operation and named UD-PTC. Case 3 An 82-year-old man who had been suffering from acromegaly since the age of 60 years was given total thyroidectomy for PTC. Octreotide therapy was discontinued because of diarrhea so that his serum concentrations of GH and IGF-I were persistently high. Subsequently, PTC metastases in the cervical lymph nodes were revealed at the age of 85 years (pT4 pEx1 pN1b M0, stageIV), and central neck resection was performed. At 85 years of age, his height was 166 cm and weight 66 kg. His blood pressure was 130/84 mmHg and pulse 60/min. He had acromegalic facies with enlarged hands and feet. Two hard nodules of 2 cm diameter were palpable on the neck. The blood pathologies are shown in Table 1. The cell line was derived from those lymph nodes and designated A-PTC. PTC metastases developed bilaterally in the lungs and the patient died of lung metastasis and respiratory failure at the age of 88.years Primary culture of PTCs and establishment of novel cell lines

Case reports Case 1 A 37-year-old woman presented with right thyroid nodule, which was solid and 4 cm in diameter. Her height was 164 cm and weight 55 kg. Her blood pressure was 118/74 mmHg and pulse 66/min. Fine needle aspiration biopsy of the nodule revealed PTC, and subtotal thyroidectomy and neck lymph node dissection (D2a) were performed. Blood pathologies are shown in Table 1. Histological diagnosis was well-differentiated PTC. No metastasis has been detected (pT2 N1 M0 Stage1). A cell line was established from the resection sample and named D-PTC. Case 2 A 18-year-old woman received right hemi-thyroidectomy for PTC. At 22 years of age a nodule was found in her left thyroid lobe. Her height was 165 cm and weight 68 kg. Her blood pressure was 110/74 mmHg and pulse 64/min. Blood

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The tumors derived from these three PTC patients were cut into small pieces with razor blades and dispersed in 0.01 % trypsin-0.02 % EDTA solution for 30 min at 37 °C. Dissociated cells were then obtained by vigorous pipetting. In order to remove the digestive enzymes, single cells and small cell aggregates were washed several times with growth medium [GM: DMEM/F12 medium supplemented with 10 % FBS (Cellular Mate, Gunma, Japan, Gibco, Grand Island, NY, USA, and Sigma, St. Louis, USA), 50 U/mL of penicillin, 50 lg/mL of streptomycin (Pen Strep; Gibco, Grand Island, NY, USA), 0.25 lg/mL Amphotericin B (Gibco, Grand Island, NY, USA), 1 % non-essential amino acids (MEM NEAA (1009); Gibco, Grand Island, NY, USA)] and then centrifuged (350 9 g for 5 min). Pellets from the dissociated tumor cells were resuspended with GM and cells were cultured in 60 mm dishes (Falcon) using GM as the primary culture. The GM was changed twice weekly. Most of the cells died; however, some colonies enlarged during the 3-month culture period. The colonies were picked up and then novel cell lines were established from these. The novel cell lines were named D-PTC, UD-PTC, and A-PTC-3 derived from case 1, 2, and 3, respectively.

Novel cell lines from papillary thyroid carcinomas Table 1 Diagnostic laboratory tests and studies of patients

Case 1 Blood chemistry

Urinalysis

Endocrinological examination

RBC

4.49 9 106/lL

CRP

0.1 mg/dL

pH 7

TSH

1.50 lIU/mL

Hb

13.2 g/dL

Na

137 mM

Glucose (-)

FT3

2.89 pg/mL

Ht

39.9 %

K

3.4 mM

Protein (-)

FT4

1.40 ng/dL

PLt

541000/lL

Ca

9.0 mg/dL

Blood (-)

Tg

69 ng/mL

WBC

9100/lL

P

3.6 mg/dL

Ketone (-)

TgAb

B0.3 U/mL

Baso

1.2 %

TP

7.4 g/dL

TPOAb

B0.3 U/mL

Eos

9.9 %

Alb

4.4 g/dL

NE

53.8 %

BUN

6 mg/dL

Ly

28.9 %

Cr

0.46 mg/dL

Mono

6.3 %

Case 2 Blood chemistry

Urinalysis

Endocrinological examination Under LT4 treatment

RBC

4.52 9 106/lL

CRP

0.1 mg/dL

pH 7

TSH

1.90 lIU/mL

Hb

12.5 g/dL

Na

134 mM

Glucose (-)

FT3

2.81 pg/mL

Ht

38.3 %

K

4.2 mM

Protein (-)

FT4

1.27 ng/dL

PLt

230000/lL

Ca

8.7 mg/dL

Blood (-)

Tg

23 ng/mL

WBC

5600/lL

P

2.3 mg/dL

Ketone (-)

TgAb

B0.3 U/mL

Baso Eos

0.0 % 6.6 %

TP Alb

7.4 g/dL 4.8 g/dL

TPOAb

B0.3 U/mL

NE

54.1 %

BUN

7 mg/dL

Ly

30.7 %

Cr

0.56 mg/dL

Mono

8.5 %

Case 3 Blood chemistry

Urinalysis

Endocrinological examination Under LT4 treatment

3.44 9 106/lL 11.9 g/dL

CRP Na

0.2 mg/dL 141 mM

pH 7 Glucose (-)

TSH FT3

4.40 lIU/mL 0.97 pg/mL

Ht

34.9 %

K

4.1 mM

Protein (-)

FT4

1.40 ng/dL

PLt

109000/lL

Ca

8.5 mg/dL

Blood (-)

Tg

B5.0 ng/mL

WBC

10500/lL

P

4.7 mg/dL

Ketone (-)

TgAb

B0.3 U/mL

Baso

0.0 %

TP

7.4 g/dL

GH

32.1 ng/mL

Eos

1.0 %

Alb

4.4 g/dL

IGF-I

330 ng/mL

NE

64.3 %

BUN

17 mg/dL

Ly

27.9 %

Cr

0.88 mg/dL

Mono

6.8 %

RBC Hb

Follicular construction with D-PTC

Cytological studies

When cultures cell reached confluency, the monolayer displayed balloon structures (100–700 lM diameter), similar to follicular formation, and some detached into the culture medium. These balloons were collected by pipet aspiration under a phase contrast microscope and then cultured in a fresh dish.

The cultures were observed under phase contrast microscopy. For electron microscopic observation, the cells cultured on 6 cm tissue culture plates were fixed with 2.5 % glutaraldehyde in 0.1 M phosphate buffer for 30 min at room temperature and then post fixed with 1 % osmium tetroxide

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in the same buffer at 4 °C for 30 min. Dehydration was carried out using a graded series of ethanol, then the cultures were placed in propylene oxide, and subsequently embedded in Epon araldite. Ultrathin sections were prepared, stained with uranium acetate and lead citrate solution, and observed by JEOL 1200 EX-II electron microscope. Secretion of Thyroglobulin and thyroxine by PTC cell lines The cell lines were treated with 1 lg/mL of TSH, GH, or 100 ng/mL of IGF-I. After 3 h, samples of medium were collected, and the concentration of both free thyroxine (fT4) and thyroglobulin (Tg) in was measured by ELISA (Beckman Coulter; Brea, CA, USA). Secretion of fT4, free tri-iodothyronine (fT3), and Tg by the follicular D-PTC cell line were also investigated. This was both for the medium and for the contents of epithelial balloon structures, captured on the end of small glass tubes under a stereomicroscope using micromanipulator. Samples were stored at -80°Cuntil assay. The concentrations of fT4 and Tg were measured as above, and fT3 was measured by ELISA (Beckmancoulter; Brea, CA, USA). Table 2 Primers used in the present study

Primer b-actin Tg TSHR

The three cell lines were homogenizes in Isogen (Nippon Gene, Toyama-ken, Japan), and RNA was prepared as per the manufacturer’s instructions. The RNA was reverse transcribed using GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA, USA), and used for PCR with the primers indicated in Table 2. Statistics One-way Anova was performed with Tukey’s comparison test using Prism (version 5).

Results Morphological study Under phase contrast microscope, D-PTC cells were median size and showed pavement in shape. UD-PTC cells were small overlapping cells and showed many Sequence

References [14]

Forward

AGCACAATGAAGATCAAGAT

Reverse

TGTAACGCAACTAAGTCATA

Forward

GGCTAATGCTACATGTCCTG

Reverse

GCTTCTGTTGGAGATGCTGG

[15]

Forward

TGAAGCTGTACAACAACGGC

Reverse

TCAGTTCCTTCAGGTGCTCC

TPO

Forward

GAGAACCTGCGTAGACTCCG

[16]

onfFN

Reverse Forward

TCCCAGCACTCTCCTGCTC TCTTCATGGACCAGAGATCT

[17]

Reverse

TATGGTCTTGGCCTATGCCT

PAX8

Forward

TTGAGTCATGTGGTAATGGCA

Reverse

CCCCAAGGCCTGACTGTATA

TTF-1

Forward

GCCGTACCAGGACACCATGAG

Reverse

CAGGTACTTCTGTTGCTTGAAG

NIS TERT Oct3/4 Nanog p63 Sox2

123

mRNA expression of thyroid cell markers and oncogenes

Forward

TGCGGGACTTTGCAGTACATT

Reverse

TGCAGATAATTCCGGTGGACA

Forward

TGAACTTGCGGAAGACAGTG

Reverse

GAGGCTGTTCACCTGCAAAT

Forward

GACAGGGGGAGGGGAGGAGCTAGG

Reverse

CTTCCCTCCAACCAGTTGCCCCAAAC

[15]

[18] [19] [16] [17] [20]

Forward

CAGCCCCGATTCTTCCACCAGTCCC

Reverse

CGGAAGATCCCAGTCGGGTTCACC

[21]

Forward Reverse

CCACCTGGACGTATTCCACTG TGGGGTCATCACCTTGATCTG

[16]

Forward

CCCCCGGCGGCAATAGCA

[22]

Reverse

TCGGCGCCGGGGAGATACAT

Novel cell lines from papillary thyroid carcinomas Fig. 1 Morphological study with phase contrast micrographs (a–c) and electron micrographs (d–f). D-PTC cells derived from case 1 are shown in a and d. The cells of D-PTC were polygonal dark cells. It was the feature of this cell line to form blank spaces in the places of cell sheet (a). A number of phagosomes and lysosomes were observed (d). UD-PTC derived from case 2 are shown in b and e. Small angular cell and large spherical cells were observed. The feature of this cell line is forming calcificated deposits (b). A-PTC cells derived from case 3 are shown in c and f. Small dark angular cells and light large cells were observed (c). Microvilli (arrow) and cell projections were evident (f). (The bar in phase contrast microscopic photos was 100 lm. And the bars in microscopic photos were 2 lm in d and f, and 5 lm in e.)

calcifications (Fig. 1b). A-PTC cells were small-median size overlapping cells (Fig. 1c). Electron microscopy revealed D-PTC cells had few lysosomes and many lipid droplets (Fig.1d), whereas UD-PTC cells had lysosomes, but their number was fewer than that of D-PTC cells (Fig. 1e). A-PTC cells possessed many lysosomes (Fig. 1f). Secretion of fT4 The concentration of fT4 in conditioned media was measured after treatment with TSH, GH, IGF-I, and GH and IGF-I co-treatment (Fig. 2). TSH increased fT4 secretion in every cell line (Fig. 2a, c, e). In A-PTC cells, fT4 secretion was increased by GH, or GH and IGF-I cotreatment, remarkably (Fig. 2e). In the other cell lines, fT4 secretion was only modestly increased by GH, or GH and IGF-I cotreatment (Fig. 2a, c).

Secretion of Tg The concentration of the Tg in conditioned media was measured after TSH, GH, IGF-I, and GH and IGF-I cotreatment (Fig. 2). In A-PTC, Tg was increased by GH, or by GH and IGF-I cotreatment, remarkably (Fig. 2f). In other cell lines, Tg was increased only marginally by GH and IGF-I cotreatment (Fig. 2b, d). These results were similar to the results for fT4. mRNA expression for markers of thyroid cell differentiation The mRNA level of Tg and oncofetal fibronectin (onfFN) was lower in UD-PTC compared to the other cell lines, and there was tendency for TSH receptor (TSHR) mRNA level to be lower in UD-PTC compared to the

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M. Ishikawa et al.

b

fT4 of D-PTC

20

Tg ng/µg DNA

a fT4 ng/µg DNA

Fig. 2 Secretion of fT4 and Tg from the cell lines. The results of D-PTC, derived from case 1, are shown in a and b, the results of UD-PTC, derived from case 2, are shown in c and d, and the results of A-PTC, derived from case 3, are shown in e and f (Mean ± SD. Sample number is 6 in each group.)

15 10 5

*

*

#

*

TSH

IGF-I

GH

GH/IGF-I

control

10 5

control

fT4 of UD-PTC

d

*

*

Tg ng/µg DNA

20

TSH

IGF-I

15

10

* 5

*

*

*

IGF-I

GH

control

TSH

15 10

* 5

*

*

#

GH

control

GH/IGF-I

TSH

IGF-I

GH

f

fT4 of A-PTC

20

Tg ng/µg DNA

15 10

* 5

GH/IGF-I

*: p