Reduced Expression of Interleukin 6 in Undifferentiated Thyroid ...€¦ · and mouse IgGI...

Vol. 4, 381-387, February 1998 Clinical Cancer Research 381

Reduced Expression of Interleukin 6 in Undifferentiated Thyroid

Carcinoma: In Vitro and in Vivo Studies’

Fulvio Basolo, Lisa Fiore, Luca Pollina,

Gabriella Fontanini, Pier Giullo Conaldi, and

Antonio Toniolo2

Institute of Pathological Anatomy. University of Pisa, 561 26 Pisa

[F. B., L. F., L. P., G. F.]: and Department of Clinical and Biological

Sciences. University of Pavia, 21 100 Varese [P. G. C.. A. TI. Italy

ABSTRACT

Cytokines appear to play an important role in the

development and progression of epithelial tumors. Cultured

normal human thyroid follicular cells constitutively releasehigh levels of interleukin-6 (IL-6) and IL-8, together with

low to moderate levels of transforming growth factor-a

(TGF-a) and TGF-�3. IL-6 appears to play multiple func-tions in thyroid physiology and disease. Because certain data

indicate an inverse relationship between IL-6 production

and epithelial tumor aggressiveness, we used both tissueculture methods and histochemical techniques to search forpossible alterations of cytokine expression in thyroid carci-

nomas. As compared to cultures from normal tissue andwell-differentiated carcinoma, production of IL-6 wasstrongly down-regulated in cultures derived from undiffer-

entiated carcinoma. In contrast, levels of IL-8, TGF-a, and

TGF-� produced by neoplastic TFC were similar to those

produced by normal cells. Actually, production of TGF-awas slightly enhanced in cultures from well-differentiated

carcinoma. Immunoassay results were confirmed by reversetranscriptase-PCR analysis. Immunohistochemistry of hu-man thyroid carcinomas (n 99) and normal thyroid tissue(n 85) showed that immunoreactive IL-6 was strongly

diminished in undifferentiated forms (n = 34) and slightly

reduced in well-differentiated carcinoma (n = 65). In agree-

ment with the in vitro results, TGF-a expression was signif-icantly increased in neoplastic thyrocytes, as compared totheir normal counterpart. The results indicate that, as in the

mammary and salivary glands, down-regulation of IL-6 ex-pression may represent a marker of undifferentiated thyroid

carcinoma.

Received 5/30/97; accepted 10/30/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with I 8 U.S.C. Section 1734 solely to

indicate this fact.

) This work was supported by the Italian Association for Cancer Re-

search (Milan, Italy) and by Istituto Superiore di Sanit#{224}(Rome, Italy)

Grant 9405-12.2 To whom requests for reprints should be addressed, at Department ofClinical and Biological Sciences. University of Pavia. 57. Viale Born.21 100 Varese, Italy. Phone: 39-332-278-309: Fax: 39-332-260-017.

INTRODUCTION

Normal human mammary epithelial cells constitutively

produce IL-63 and IL-8 (1 . 2). Transformation of these cells by

several different genes (e.g. , c-Ha-ras, c-erb-B2, and SV4O T

large antigen) is associated with loss of IL-6 production and

responsiveness (3, 4). Immunohistochemical studies showed

that, as compared to normal tissue and to in situ lesions, the in

vivo expression of immunoreactive IL-6 is significantly reduced

in invasive mammary carcinoma (5). We also observed that, in

neoplastic mammary cells, the in vitro response to IL-6 was

greatly diminished. Similar but less pronounced alterations were

found in the apparently normal breast tissue adjacent to card-

noma (5). Taken together, these data suggested that alterations

of IL-6 pathways are frequently found in mammary neoplasia.

IL-6 is mainly a multifunctional regulator of the immune

response, hcmatopoicsis. megakaryocyte maturation. and acute

phase reactions (6). A few data indicate that IL-6 can also

promote differentiation of cells that arc not derived from the

hemolymphopoictic system, e.g. , regulation of gene expression

in hepatocytes (7), conversion of phcochromocytoma cells and

PCI2 cells into neuron-like cells (8, 9), induction of alkaline

phosphatase expression in a lung carcinoma cell line (10), and

enhancement of corneal cell adhesion and migration ( I I ). Al-

though it is well established that IL-6 deregulation is involved in

a variety of diseases. including lymphatic malignancies (12),

there is little information on its role in solid tumors (5. 13-15).

Growth and functions of epithelial TFCs are positively

regulated by TSH, EGF, TGF-ce, and insulin-like growth factors.

TGF43 has an inhibitory role (16). Some of the above regulators

are directly produced by thyrocytes and may have an autocrine

function. In fact, cultured TFCs constitutively express several

different cytokines. including IL-liz, IL-6, and IL-8 (17).

Although some information is available on the role of

cytokines in both thyroid physiology and autoimmunc disease

(18), data on the possible relationship between cytokincs and

thyroid neoplasia are extremely scarce (19, 20).

Different types of epithelial cells have been shown to

produce IL-6. Among them, cells from the mammary and sali-

vary glands (I, 2, 15), retinal pigment cells (21). and renal

tubular cells.4 Studies of salivary and mammary cancers suggest

an association between increased tumorigenicity and loss of

IL-6 expression (2. 5. 15). Pilot experiments with cultured TFCs

prompted us to investigate if alterations of cytokinc pathways

could be detected also in a series of thyroid carcinomas. Immu-

3 The abbreviations used are: IL. interleukin: TFC. thyroid follicularcell: TSH, thyroid-stimulating hormone: EGF, epidermal growth factor:

TGF, transforming growth factor: WDC. well-differentiated carcinoma:

UC, undifferentiated carcinoma: IUDR, iodo-deoxyuridine: RT-PCR,reverse transcriptase-PCR: IL-6-R, IL-6 receptor.

4 P. G. Conaldi. unpublished observations.

Research. on April 24, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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382 IL-6 in Normal and Neoplastic Thyroid

nohistochemical studies have been carried out in parallel with in

vitro investigations of TFC cultures prepared from both non-

neoplastic and neoplastic thyroid tissue. This approach allowed

us to compare the in i’ivo findings with the results obtained by

studying cytokine release in pure TFC cultures.

MATERIALS AND METHODS

Thyroid Tissue Collection. Thyroid tissue was obtained

for diagnosis at the Institute of Pathology, University of Pisa,

(Pisa, Italy). Primary cultures were obtained from cancer pa-

tients undergoing total thyroidectomy. Unaffected (normal) thy-

roid tissue was obtained from the lobe opposite to the neoplastic

one (five cases). Thirty-nine tissue samples of thyroid carci-

noma were derived from previously untreated patients. This

group included 32 WDCs (papillary histotype, 29 cases; follic-

ular histotype, 3 cases) and 7 UCs. Immunohistochemical stud-

ics were performed on archival material that included 85 sam-

ples of normal tissue, 65 cases of WDC, and 34 cases of UC.

Primary Cultures of Normal and Neoplastic Thyroid

Tissue. Unless otherwise specified, chemicals, media, and se-

rum were from Sigma Chemical Co. (St. Louis, MO). whereas

tissue culture plasticware was from Costar (Cambridge, MA).

Sterile samples of thyroid tissue were obtained at surgery and

immediately subjected to mechanic and collagenase (CLSII, 150

units/ml; Worthington Biochemical Co., Freehold, NJ) dissoci-

ation. Cell aggregates and isolated cells were cultured in DMEM

supplemented with 10% fetal bovine serum (low-endotoxin

type) but no exogenous hormones/growth factors (22). Primary

cultures were used for experiments between 30 and 50 days after

plating, i.e., at a time when contaminant nonepithelial cells were

<5%, as judged by morphology and immunocytochemistry.

Cultures from “normal” thyroid tissue and the undifferentiated

ARO human thyroid carcinoma cell line were used as controls.

For characterization, samples of each culture were plated in

chamber slides, acetone-/methanol-fixed and stained by indirect

immunofluorescence with the following antibodies: thyroglob-

ulin (murine clone TGO3: Cis-Bio International, Paris, France);

calcitonin (rabbit policlonal; Dako Ltd., Bucks, United King-

dom); pancytokeratin (murine clone MNF 1 16: Sigma); chro-

mogranin (murine clone A-3; Dako); human leukocytes and

human granulocyte/monocyte (murine clones MB 1 and

CLB3O2: ICN Pharmaceuticals, Costa Mesa. CA).

Matrigel Colonization Assay. As previously described

(23), 500 pi of liquid Matrigel ( 10 mg/ml) was placed in 16-mm

wells of 24-well plates and polymerized at 37#{176}Cfor 30 mm.

Cells (5 X l0�) in complete DMEM were seeded in each well on

the top of the gel and incubated at 37#{176}Cwith 5% CO,. Cultures

were examined daily and photographed after 7-10 days.

Immunohistochemistry. After formalin fixation, prod-

essing, and paraffin embedding, 3-5-tim-thick sections were

prepared. The following antibodies, capable of staining paraffin-

embedded sections, were used: IL-6 (rabbit polyclonal antibody

and mouse IgGI monoclonal antibody, LP-7l6 and 1618-01:

Genzyme, Cambridge, MA); TGF-a mouse IgG1 monoclonal

antibody (Oncogene Science, Manhasset, NY). As reported (5),

sections were incubated with primary antibody and stained with

the avidin-biotin immunoperoxidase complex technique (Vector

Laboratories, Burlingame, CA). Sections were scored independ-

ently by two pathologists (F. B. and L. P.) using the following

method: the intensity of staining for each antigen was scored

from 0 to 3 (0, absent; 1, weak; 2, moderate and 3 strong); the

proportion of malignant cells positively stained was scored from

0 to 4 (0, no positive cells; 1, <10% positive cells; 2, 11-50%;

3, 5 1-75%; and 4, 76-100%). The two scores were then added

to yield the total score (0-7). Immunorcactivity was defined as

negative when the score was 0, weak when it was 1-3, and

strong when it was �4.

Measurement of Cytokine Levels and IL-6 Responsive-

ness. Conditioned medium of primary cultures in T25 flasks

from either normal or neoplastic thyroid tissue was used to

measure the release of IL-4, IL-6, IL-8, TGF-a, and TGF-�3l.

Immunocnzyme dosage kits were from: R&D Systems, Minne-

apolis, MN (IL-4, IL-6, and IL-8), Oncogene Science (TGF-a),

and Genzyme (TGF-�3l). Medium samples were taken at 3, 6,

and 9 days from plating; determinations were made in quadru-

plicate. Reported data refer to day 6.

The responsiveness of cultured TFCs to IL-6 was studied

under serum-free conditions, as reported previously (4). Recom-

binant human IL-6 (Gcnzyme) and IL-6-ncutralizing mono-

clonal antibody (anti-IL-6, clone 8; Collaborative Research,

Becton-Dickinson, Milan, Italy) were used. DNA synthesis was

measured as incorporation of [ ‘ 251]-IUDR (Amersham, Milan,

Italy) 4 days after stimulation with IL-6 (10 and 100 ng/ml) or

treatment with IL-6-neutralizing antibody (50 �i.g/ml).

Analysis of Cytokine Transcripts. Cytokinc-specific

mRNAs were detected as described (5, 24) in total RNA cx-

tracted from 106 cells by the guanidinium thiocyanate method.

After treatment with RNAsc-frce DNAsc for 1 h at 37#{176}C,cDNA

was produced with Moloney murine leukemia virus reverse

transcriptase in conjunction with random hcxamcr primers

(Clontech, Palo Alto, CA). eDNA was then amplified by PCR

using Taq polymerase and cytokine-specific primer pairs (Cy-

tokine MAPPing Amplimcrs; Clontech). Amplification was car-

ned out for the following human transcripts: IL-4 (amplified

product, 344 bp), IL-6 (amplified product, 628 bp), IL-6-reccp-

tor-a (amplified product, 251 bp), IL-8 (amplified product, 289

bp), TGF-a (amplified product, 297 bp), TGF-� (amplified

product, 161 bp). As a control reaction, RT-PCR of glyceralde-

hydc-3-phosphate dehydrogenase mRNA (amplified product,

983 bp) was carried out with all samples. For detecting IL-6

transcripts, the following primers were used (25): downstream

3’ primer (5’-GAAGAGCCCTCAGGCTGGACTG-3’); up-

stream 5’ primer, (5 ‘-ATGAACTCCTTCTCCACAAGCGC-

3 ‘). Amplification products were separated by clectrophoresis

on 2.5% agarose gels and visualized under UV light by staining

with 0.5 mg/ml ethidium bromide. �X174/HaeIII digest (72-

1353 bp) was used as a size marker.

RESULTS

Characterization of Cultured Cells. Primary cultures

from 5 samples of normal thyroid tissue and 39 thyroid carci-

nomas of different histotypes (32 WDCs and 7 UCs) were

studied. By day 10-15 postplating. TFC monolayers reached

confluence. Primary cultures were analyzed between 30 and 50

days after plating, i.e., at a time when contaminant nonepithelial

cells were not detected by immunostaining. As shown in Fig. 1,



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Clinical Cancer Research 383

Fig. I Phase-contrast photomicrograph of primary cultures of normal(A) and neoplastic (B) thyroid follicular cells 8 days after plating.

Thyroid follicular cells were oval or spindle-shaped with no significantdifferences between cultures derived from normal or neoplastic tissue.Original magnification, x 100 (A): X200 (B).

primary cultures from normal and neoplastic tissue had similar

morphology: TFCs were oval or spindle-shaped, without any

significant difference between normal and neoplastic tissue. By

immunocytochemistry, over 80% of cells from normal tissue

and WDCs were thyroglobulin positive, and over 95% were

positive for cytokeratin. All cells were calcitonin and chro-

mogranin negative. In cultures derived from UC. only a few

cells were stained by thyroglobulin antibody, but all cells had an

epithelium-like morphology. The ability of TFCs to form cob-

nics on reconstituted basement membrane was also evaluated.

The morphological appearance of cells cultured in Matrigel for

S days is shown in Fig. 2. Normal TFCs remained as single cells

or small clusters in Matrigel (Fig. 14), whereas cells obtained

from WDCs and UCs formed either large colonies or branching

structures (Fig. 2, B-D). These results are in agreement with

observations of Bond et a!. (26).

Cytokine Production by Cultured Epithelial Thyroid

Cells and Response to IL-6. Table I shows the levels of IL-4,

IL-6, IL-8, TGF-a, and TGF-�3l in the supernatant of cultures

derived from both normal and ncopla.stic thyroid tissue. IL-4

was not produced by TFCs. Primary cultures from both normal

tissue and WDC released high amounts of IL-6 and IL-8, to-

gether with very low levels of TGF-ca (i.e., < 100 pg/mb) and

medium amounts of TGF-�3 1 . As compared to normal cells, IL-6

production was significantly reduced in cells from UC (P <

Fig. 2 Growth in Matrigel of primary cultures of normal or neoplasticthyroid follicular cells 7 days after plating. Single cells or small clustersfrom normal thyroid tissue (A), large colonies and branching structures

from WDC (B and C). and large branching colony from UC (D).

Dark-field microscopy, original magnification. X32 (A-C): bright-field

microscopy. original magnification. X80 (D).

0.001). The undifferentiated ARO cell line did not release IL-6.

Production of TGF-a and TGF-�3l was not significantly differ-

ent in cells from either normal or neoplastic tissue. The above

results were confirmed by analysis of cytokine transcripts in

cultured cells. Fig. 3 shows representative results of RT-PCR

analysis of cytokine transcripts. IL-6 mRNA was expressed in S

of S normal cultures, 7 of I 1 cultures from WDC, and I of 6

cultures from UC. ARO cells failed to express IL-6 mRNA.

Comparable expression of IL-8, TGF-a, and TGF-�3 mRNA was

found in cultures from both normal and neoplastic tissues. With

the exception of TGF43, the ARO cell line showed a reduced

expression of cytokinc transcripts. Transcripts for IL-4 and

IL-6-Ra were not detected in any of the thyroid cultures exam-

med (data not shown).

DNA synthesis in TFCs was analyzed. under serum-free

conditions, as [‘251]IUDR incorporation after treatment with

either exogenous IL-6 (10 and 100 ng/ml) or high amounts of

IL-6-neutralizing antibody (10 and 50 p.g/ml). As compared to

serum-free medium, no significant modulation of DNA synthe-



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Table 1 Release of IL-4, lL-6, IL-8, TGF-a, and TGF-�3 in cultures of epithelial thyroid cells from normal and neoplastic tissue (pg/mI)

Evaluation by immunocnzyme assay 6 days after plating. Each determination was made in duplicate using two different ELISA kits. The lack

of sign indicates cytokine levels below test sensitivity (<10 pg/ml). Statistical analysis was performed by Student’s t test.

Primary cultures

Normal WDC UCCytokine (�1 = 5) (n 32) (n = 7) ARO cells

lL-4IL-6” >2100 >2100 433 ± 84

IL-8 3600 ± 1800 4600 ± 2100 6100 ± 1050 480

TGF-�3 1 650 ± 200 500 ± 200 455 ± 7 1730

TGF-a 45 ± 18 96 ± 64 20 ± 3 430

(2 lL-6 group: normal tissue vs. WDC. not significant: normal tissue s’s. UC, P < 0.001. (No significant differences were found among levels ofIL-8, TGF-a, or TGF-�3l secreted by cultures of normal tissue, WDC, or UC.)

1 2 3 4 5 6 7 8 9 10 11 12

1L4

1L6

1L8

TGFa

TGF�

G3PDH

Fig. 3 RT-PCR amplification of total cell RNA extracted from pure

cultures of thyroid tollicular cells. Lane 1. molecular size markers.Cultures were obtained from: normal tissue (Lanes 2-4), WDC (Lanes

5-7), UC (Lanes 8-10), and undifferentiated thyroid ARO cell line used

as control (Lane 11). Lane /2, positive control. PCR was carried outwith primers specific for IL-4. IL-6. IL-8. TGF-a, TGF-[3, and glycer-aldehyde-3-phosphate dehydrogenase. Ethidium bromide-stained agar-

ose gel.

sis was observed in cultures derived from both normal (three

cultures) and neoplastic tissues (three cultures; data not shown).

Thus, IL-6 appeared to have neither an autocrine nor a paracrine

effect on pure TFC cultures.

Immunohistochemical Studies. Sections of normal and

neoplastic thyroid tissue were studied for immunoreactivity with

antibodies to IL-6 and TGF-a. Eighty-five samples of normal

thyroid tissue, 65 cases of WDC, and 34 cases of UC were

evaluated (Table 2). As compared to normal tissue, IL-6 reac-

tivity was slightly diminished in WDC (P < 0.001) and severely

reduced in undifferentiated histotypes (P < 0.001). IL-6 anti-

bodies produced a defined cytoplasmic staining in WDC but

failed to stain UC (Fig. 4). In our hands, normal thyroid tissue

was not stained by antibody to TGF-a. Expression of immuno-

reactive TGF-a was, however, evident in most cases of WDC

and UC (P < 0.001; Table 2). No significant differences of

TGF-a expression were noticed between WDC and UC forms.

DISCUSSION

The role of cytokines in cancer is the subject of intense

investigation. As seen in other systems, proteins of the TGF-�3

family have a potent antiproliferative effect on TFC growth

(27). TFCs from multinodular goiter were shown to be fre-

quently unresponsive to the growth-inhibiting activity of TGF43

(28), and it has been proposed that escape from TGF-�3 control

may favor growth of thyroid tumors (29). Besides TGF-�3, other

cytokincs/growth factors appear to act on thyrocytes as regula-

tors of cell growth, differentiation, and hormone production.

IL- 1 has been shown to stimulate proliferation of the human

thyroid cell line NIM 1 (30) and to enhance the release of both

IL-6 and IL-8 by the human thyroid cell line Htori3 (17). IFN-�y

reduced significantly both insulin- and TSH-stimulated growth

of human fetal TFC, as well as thyroglobulin accumulation (31).

IL-8 is constitutively produced by TFCs (32), but so far, no

evidence has been produced for a direct effect of this cytokinc

on thyrocytes (17). Because IL-8 release is up-regulated by

IL- 1 , an abundant cytokinc in the inflammatory infiltrate, IL-8

might play a role in the induction and maintenance of autoim-

munc thyroid disease.

The differentiated thyroid cell lines WRO and NPA were

shown to produce mesenchymal growth factors (basic fibroblast

growth factor or platelet-derived growth factor-b) that did not

appear to act in an autocrinc manner but rather by stimulating

stromal cells (33). Recent immunocytochemical studies mdi-

cated that TGF-a is expressed in the cytoplasm of normal and

neoplastic human thyrocytes and that EGF-R is expressed on the

surface of TFCs (34). Experiments in vitro also showed that

exogenous TGF-a stimulates growth and invasivity of human

thyroid cancer cells (35). Growth stimulation has been con-

firmed in cultured porcine TFCs, and in this model, TGF-a

appeared also to inhibit the TSH-induccd iodide uptake (36).

As is IL-8, even IL-6 is constitutively produced by TFCs

( I 7, 37) and appears to influence differentiated thyrocyte func-

tions (18). In cultures of human TFCs, IL-6 inhibits TSH-




Table 2 IL-6 and TGF-a immunoreactivity in normal and neoplastic thyroid tissues

Statistical analysis was performed by X2-test with Yates’ correction.

Normal tissue,no. of cases (%)

WDC,no. of cases (%)

UC,no. of cases (%)

Total no. of cases 85 65 34IL-6 immunoreactivity4,

Negative I ( 1) 40 (62) 32 (94)Weak 78(92) 14(21) 2(6)

Strong 6(7) 11(17)TGF-a immunoreactivityb

Negative 83(98) 17(26) 12(35)Weak 1(1) 10(16) 3(9)

Strong 1 (1) 38 (58) 19 (56)

a IL-6 group: normal tissue vs. WDC, P < 0.001; normal tissue vs. UC, P < 0.001.b TGF-a group: normal tissue vs. WDC, P < 0.001 ; normal tissue vs. UC, P < 0.001).

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Fig. 4 Immunohistochemical staining for IL-6 of formalin-fixed, par-affin-embedded thyroid carcinoma; Harris hematoxylin counterstaining.WDC shows clear cytoplasmic immunoreactivity (A; X250); there is anabsence of immunoreactivity in UC (B; X 100).

induced peroxidase gene expression and reduces T3 secretion

(38), a finding confirmed in rats (39). In contrast, production of

thyroglobulin and DNA synthesis were not influenced by exog-

enous IL-6 (40). Here, transcripts relative to the a-chain of

IL-6-R could not be detected by RT-PCR in either normal or

neoplastic thyrocytes. This finding would imply that IL-6 cannot

act in an autocrine way on thyrocytes. However, several tissues

that do not produce 1L6-Ra are known to express an incomplete

1L6-R consisting only of the �3 chain (IL6-R�3; Ref. 41). In

principle, the latter type of cells could respond to IL-6 when this

cytokine is given not by itself but together with soluble forms of

1L6-Ra (6). The above concept could help us to understand the

conflicting results obtained by different authors studying IL-6

responsiveness of cultured thyrocytes. In vivo, a more complex

situation undoubtedly exists because mutual interactions with

stromal/vascular cells may strongly influence TFC differentia-

tion and receptor functions (17, 33). In our hands, primary

cultures of both normal and neoplastic TFC were not responsive

to exogenous IL-6. In addition, DNA synthesis was not modu-

lated by neutralizing antibody to this cytokine, indicating that

IL-6 does not influence TFC proliferation in vitro.

Although the influence of IL-6 on the thyroid has been the

subject of intense investigation both in culture and in vivo

(normal tissue, multinodular goiter, autoimmune pathology, and

systemic disease; Ref. 18), there are no data on the role of this

cytokine in thyroid neoplasia.

Our experiments confirmed that pure cultures of normal

TFCs release high amounts of IL-6 and IL-8, together with

medium levels of TGF-�3l. In addition, we report that human

TFCs accumulate in the supernatant measurable levels of

TGF-a that are close to detection limits. Production of IL-4, a

regulator of the immune response that also has a potent effect

against solid tumors (42), was not detected. The above findings

were confirmed by RT-PCR analysis of cytokine-specific iran-

scripts in primary TFC cultures.

As compared to cultures derived from normal tissue, IL-6

release was significantly down-regulated in cultures derived

from undifferentiated forms of thyroid carcinoma and essen-

tially nonexistent in the ARO cell line that derives from an

anaplastic thyroid cancer. In contrast, levels of other test cyto-

kines in TFC supernatants were not significantly reduced in

cultures of neoplastic cells.

The concept that a selective reduction of IL-6 expression

occurs in neoplastic TFCs has been confirmed by in vivo ob-

servations. Immunohistochemical studies of a large series of

thyroid carcinomas (n = 99) in comparison to normal thyroid

tissue (n = 85) revealed that IL-6 expression was frequently

normal in well-differentiated forms of thyroid carcinoma but

strongly suppressed in undifferentiated forms (only 2 of 34

cases were IL-6 positive). This is consistent with previous

findings on mammary tumors (5). Thus, even in thyroid pathol-




ogy. down-regulation of IL-6 production appears to be associ-

ated with advanced stages of tumorigenesis. The results suggest

that IL-6 can have a differentiative role in thyroid epithelial

cells; definition of this role awaits further investigation.

In vitro, TGF-a production occurred at low and not signif-

icantly different levels in TFC cultures derived from both nor-

mal and neoplastic tissue. In our hands, immunostaining re-

vcaled that follicular cells of normal thyroid tissue were TGF-a

negative, whereas approximately 65% of samples from both

WDC and UC were TGF-a-positive. Thus, TGF-a expression is

apparently induced or enhanced in cancerous thyroid cells. In

partial contrast with our findings, van der Laan et a!. (34)

reported that immunoreactive TGF-a and EGF-rcccptor are

widely expressed both in normal tissue and in a wide spectrum

ofthyroid diseases (i.e., multinodular goiter, follicular adenoma,

and carcinomas originating from follicular or parafollicular

cells).

The observation that many cases of thyroid cancer are

TGF-a immunoreactive suggests that this growth factor may

play a role in TFC transformation. Supporting this suggestion is

a report by HOlting et a!. (35): in a TFC line, blockage of

EGF-rcceptor by either monoclonal antibody or geninstein pre-

vents TGF-a-induced growth and invasion. TGF-a expression

has been also documented in the medullary forms of thyroid

carcinoma (43).

To our knowledge, this is the first study of IL-6 expression

in thyroid cancer. As in other epithelial tumors (2, 5, 15), loss of

IL-6 production in undifferentiated thyroid carcinoma might

play different roles: removal of an immunopotentiating factor

(6); removal of a growth-inhibiting signal (12); and removal of

a putative differentiation signal. Investigation of the above

mechanisms will require innovative experimental systems for

studying thyroid cell differentiation and will benefit from an

in-depth analysis of IL-6 receptor expression in thyroid tissue.

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1998;4:381-387. Clin Cancer Res F Basolo, L Fiore, L Pollina, et al. carcinoma: in vitro and in vivo studies.Reduced expression of interleukin 6 in undifferentiated thyroid

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