Supplemental Materials and Methods
Cell Lines
NTHY cells were cultured in RPMI (Invitrogen) supplemented with 10% FCS. All
other thyroid cell lines were cultured in DMEM (Invitrogen) with 10% FCS.
N-terminal 3XHA-tagged LPA receptors 1-4 in pcDNA3.1 were purchased from
Missouri S&T cDNA Resource Center (www.cDNA.org). The construct for
hemagglutinin tagged LPAR1 (Grp78>Hs.3xHA-LPA1) was generated by the Protein
Expression Laboratory, SAIC-Frederick, Inc. The pGIPZ LPAR1 silencing plasmids
#1 (Item# RHS4430-101160584) and #2 (Item# RHS4430-101161464) were from
Open Biosytems. Introduction of LPAR1 and CD97 shRNA into the cell lines was
done as described (14).
Real-Time RT-PCR
Total RNA was isolated using RNeasy Mini RNA isolation system (Qiagen). Reverse
transcription of cDNA used 3μg of total RNA with the SuperScript III kit (Invitrogen).
The amplification step used the Taqman Gene Expression Master Mix (Applied
Biosystems). Taqman probes for LPAR 1-6 were from Applied Biosystems.
Standard Taqman PCR protocol was used. All reactions were normalized to human
Gapdh and run in triplicate.
Mice
The bTg-CD97 expression construct was generated by subcloning full length CD975EGF into pSG5 (Stratagene) using EcoRI and BglII restriction sites. A fragment
containing a beta-globin intron, CD97-5EGF, and SV40-polyA was cut from the pSG5
construct and cloned into the pBSK vector (Stratagene) downstream of a bovine
thyroglobulin promoter using Cla1 and SalI restriction sites. The 6 kb transgene
DNA fragment was released by SpeI and KpnI digestion, isolated by gel purification,
and microinjected into fertilized FVB/N mouse eggs (SAIC, Frederick). Mice were
genotyped using the 5′-primer (HCD97-S):
5′-GAAGTCGACATGGGAGGCCGCGTCTTTC, and the 3′-primer (HCD97-AS): 5′GAACTGTCATCATATGCCGGACTCTGATGC. PCR reactions were carried out for 30
cycles (95°C, 30 sec; 62°C, 30 sec; 72°C, 2 min 35 sec) using TaKaRa Ex Taq
polymerase (Clontech). ThrbPV mice were genotyped as described (20). BTg-CD97
mice were crossed with ThrbPV mice to generate ThrbPV/PV CD97(+) and ThrbPV/+
CD97(+) lines. Animals were bred, housed, and used in accordance with the Policy
on Humane Care and Use of Laboratory Animals (Office of Laboratory Animal
Welfare, National Institutes of Health, Bethesda, MD).
Organoid Culture
To establish organoid cultures of thyroid epithelial cells, thyroid tumors were
minced and digested for 2hours in 1mg/ml DNAse, 1mg/ml collagenase D, and
1unit/ml dispase in F12/DMEM medium supplemented with 10% FCS (F12/D10).
Organoids were washed twice with F12/D10, reuspended in 2.4ml F12/D10) and
pulled through a 19 gauge needle prior to being seeded (200l per well) into 6-well
PrimariaTM (Becton Dickinson) culture dishes containing F12/D10. After one week,
thyroid epithelial cells were treated as required and prepared for western blot
analysis following standard protocol.
Supplemental Figure Legends
Supplemental Figure 1. CD97 expression and function in human thyroid cell lines
with known genetic alterations. Western blots showing CD97 expression and RHOGTP following starvation and stimulation with 25ng/ml EGF in four human thyroid
cell lines. Data is shown for parental cells (C) and cells with depleted CD97 (Sh).
pERK demonstrates strong activation by EGF.
Supplemental Figure 2. LPAR expression in human thyroid cell lines. (A) Relative
expression of LPAR1-6 in NTHY (N), Cal62 (C), Sw1736 (S), and BCPAP (B) as
determined by qRTPCR. (B) Relative silencing of LPAR1 in human thyroid cell lines.
(C) In vitro invasion of parental and two LPAR1 depleted (#1 and #2) thyroid cell
lines. The chemoattractant was LPA (1M). Average of three independent
experiments is shown. Error bars represent ±SEM. Proliferative capability of
parental and LPAR1 depleted thyroid cancer cell lines starved or stimulated with
LPA (10M). Proliferation was determined for SW1736 (D), BCPAP (E), and Cal62
(F) cells using Click-iT® EdU Imaging (Invitrogen). Percent positive cells were
quantified by counting 5 fields for each cell line at each condition. Error bars
represent ±SEM. ***P<0.001, **P<0.01, *P≤0.02 at a 95% confidence interval.
Supplemental Figure 3. Human thyroid cancer cell lines permanently transfected
with empty vector (-) or LPAR1-HA (+) were subjected to in situ proximity ligation
assays. Cells were either starved or stimulated for 2hours with 10M LPA. Results
were quantified using the Duolink Image Tool software. Average of three
independent experiments is shown. Error bars represent ±SEM. ***P<0.0001,
**P<0.001 at a 95% confidence interval.
Supplemental Figure 4. CD97 expression is decreased in metastatic lesions. (A)
Lung metastasis from ThrbPV/PV mouse stained with anti-mouse CD97 polyclonal
antibody. Inset is at higher magnification. Note low level of membrane staining
indicated by arrow. Endothelial cells and lung parenchyma express CD97. (B) Lung
metastases from CD97(-) and CD97(+) ThrbPV/PV mice. Note the presence of
scattered metastatic cells positive for CD97 in ThrbPV/PV CD97(+) mice.
Immunostaining was performed using a polyclonal antibody directed against human
CD97. Scale bars: 20M.
Supplemental Figure 5. Comparative survival of ThrbPV/PV mice expressing or
lacking the CD97 transgene. Kaplan-Meier survival curves for mice up to 68 weeks
of age. The lines are not significantly different P = 0.27.
Supplemental Figure 6. CD97 expression does not affect relative tumor size in
ThrbPV/PV mice. (A) Average tumor area was determined for two thyroid sections cut
through the center of the thyroid for each mouse. Slides were scanned on a
MicroTek ArtixScan 4000tf to generate images and area was quantified using
Axiovision software. Error bars represent SEM (B) Whole thyroids were weighed on
a Sartorius analytical balance. Error bars represent SEM.
Supplemental Figure 7. CD97 expression patterns in (A) papillary carcinomas
(PTC and FV-PTC) and (B) follicular carcinomas parallel expression of pERK, pAKT,
and Ki67 in TMA #1. The expression pattern was considered the same if both
components were negative or both were expressed (≥5% positive cells).