Follicular thyroid carcinoma but not adenoma recruits tumor-associated macrophages by releasing CCL15

Patients

Paraffin embedded tissue specimens were obtained from 59 patients histologically confirmed
as FTC and 47 patients with FA, and eight fresh-frozen tissue samples (four FTCs and
four FAs) collected from surgical specimens, from 2010 to 2012 at Ruijin Hospital,
Shanghai Jiaotong University, School of Medicine. Written informed consents were obtained
from all patients. This study was approved by the board of medical ethics of Ruijin
Hospital.

Cell culture and reagents

FTC133 human cancer cell line was originally from Dr. Robert Gagel (University of
Texas, M. D Anderson Cancer Center, Houston, Texas), and WRO82-1 cell line was get
from Dr. Zhimin Liu (Chongqing Medical University). Both of them have been genetically
fingerprinted by either single-nucleotide polymorphism comparative genome hybridization
(SNP-CGH) and verified to be unique 23]. THP-1 cell line was obtained from the American Type Culture Collection (ATCC, Manassas,
VA). Both WRO82-1 and THP-1 were cultured in RPMI 1640 supplemented with 10 % heated
fetal bovine serum and 1 % LG and 1 % PS. FTC133 cell line was cultured in DMEM/modified
HAM-F12 medium. All medium were bought from Gibico (Rockville, MD). The passage numbers
of all cell lines were within 30 passages.

Recombinant human CCL15 was purchased from Peprotech (rocky hill, USA). Affinity-purified
anti-human CCL15 antibody and normal goat IgG were obtained from RD Systems (Minneapolis,
USA).

Tissue microarrays and immunohistochemistry

Tissue microarrays (TMAs) were constructed using 2 mm cores from formalin-fixed, paraffin-embedded
tissue blocks. Immunostaining of CD68 (1:150; Dako, Glostrup, Denmark), CD163 (1:75;
Vector Laboratories, Burlingame, CA), CD206 (1:5000; Abcam, Cambrige, UK) and CCL15
(1:250; Novus Biologicals, USA) were performed on section of 4 ?m thickness as previously
reported 13]. Briefly, the first antibody was incubated at 4 °C overnight before the secondary
antibody conjugated with horseradish peroxidase was added. DAB (3,3-diami-nopdbenzidine)
substrate was introduced and hematoxylin was used for counterstaining. The stained
sections were microscaned by nanozoomer2.0-RS (Hamamatsu) to get the photo of entire
sample. A single pathologist, who was blinded to the histological assessments of each
case, counted the number of CD68/CD163/CD206 positive cells. For tissue microarrays
sections, CD68, CD163 or CD206 positive cells were counted in five independent fields
under 400 magnifications (represent 0.06 mm
²
). For whole tissue sections, CD68
⁺
cells in ten independent fields under 400 magnifications (represent 0.06 mm
²
) were counted. To avoid any overestimation of the number of TAMs which could have
been due to extended cytoplasmic ramifications, we counted only cells with a visible
nucleus. The CCL15 staining level was evaluated by an expert pathologist and was classified
as negative (CCL15-) and positive (CCL15+, ++).

Cytokine antibody array

Surgically resected tissue samples were obtained from four FTC and two FA patients.
RayBio Human Cytokine Antibody Array G Series was purchased from RayBiotech (Norcross,
GA). According to the manufacturer’s instruction, array membranes were incubated for
30 min in blocking buffer and afterwards in tissue lysates with 120 ?g total protein
overnight at 4 °C. The membranes were washed and a diluted cocktail of biotin-conjugated
antibodies was added for 2 h. Membranes were washed again and the sandwiched antigens
were detected by incubation with a fluorescent dye-conjugated streptavidin solution
for 2 h. Signals were captured by laser scanner Axon GenenPix using Cy3 and analyzed
by RayBio Antibody Array Analysis Tool.

RNA extraction and qRT-PCR

Total RNA was isolated from FTC or FA tissue samples, and THP-1 cells under different
treatments using TRIZOL reagent (Invitrogen) and 1 mg total RNA was converted into
first-strand cDNA with the First Strand cDNA Synthesis Kit (Promega, USA) according
to the manufacturer’s instructions. qRT-PCR was performed in Light Cycler480 instrument
(Roche Diagnostics, Switzerland), using the SYBR Premix Ex TaqTM (TaKaRa, Japan).
The expression data were analyzed using ??Ct and ACTB was used as internal control.
Primers used were list in Additional file 1: Table S5.

Chemotaxis assays

Chemotaxis assays were performed using 8 ?m pore size cell culture inserts within
24-well plates (Corning, USA) as previously described 22]. FTC133 or WRO82-1 in exponential phase were cultured with FBS free medium (plus
0.2 % BSA, Genebase Gene-Tech, China) for 24 h and then the medium was harvested as
condition medium (CM). THP-1 cells (5?×?10
⁵
/ml) were seeded into the inserts pre-coated with collagen from rat tail (Sigma, USA),
and CM/ mock medium /CCL15 neutralizing antibody was added in the lower well. After
12 h of incubation, cells in the upper membrane surface were removed with cotton swabs.
Cells on the lower membrane surface were fixed with methyl alcohol and stained with
0.1 % crystal violet (Sigma, USA). The cells of five randomly selected fields per
well were counted using the Axiovert 25 microscope (Carl Zeiss, Germany) under 400
magnifications. Three replicate measurements were included in a single experiment.

Statistical analysis

Statistical analyses were performed by SPSS Version 17.0 and Prism Version 5.0. Data
was expressed as mean ± standard deviation(SD). Mann–Whitney test was used for continuous
variables between groups, while Fisher’s exact test for counts between categorical
variables. All p values were 2-tailed, and p??0.05 was accepted as statistical significance.