The Next Generation of Orthotopic Thyroid Cancer Models:
Immunocompetent Orthotopic Mouse Models
of BRAFV600E-Positive Papillary
and Anaplastic Thyroid Carcinoma
Pierre Vanden Borre,1David G. McFadden,2Viswanath Gunda,1Peter M. Sadow,1
Shohreh Varmeh,1Maria Bernasconi,1Tyler Jacks,3and Sareh Parangi1
Background: While the development of new treatments for aggressive thyroid cancer has advanced in the last
10 years, progress has trailed headways made with other malignancies. A lack of reliable authenticated human
cell lines and reproducible animal models is one major roadblock to preclinical testing of novel therapeutics.
Existing xenograft and orthotopic mouse models of aggressive thyroid cancer rely on the implantation of highly
passaged human thyroid carcinoma lines in immunodeficient mice. Genetically engineered models of papillary
and undifferentiated (anaplastic) thyroid carcinoma (PTC and ATC) are immunocompetent; however, slow and
stochastic tumor development hinders high-throughput testing. Novel models of PTC and ATC in which tumors
arise rapidly and synchronously in immunocompetent mice would facilitate the investigation of novel thera-
peutics and approaches.
Methods: We characterized and utilized mouse cell lines derived from PTC and ATC tumors arising in
genetically engineered mice with thyroid-specific expression of endogenous BrafV600E/WTand deletion of either
Trp53 (p53) or Pten. These murine thyroid cancer cells were transduced with luciferase- and GFP-expressing
lentivirus and implanted into the thyroid glands of immunocompetent syngeneic B6129SF1/J mice in which the
growth characteristics were assessed.
Results: Large locally aggressive thyroid tumors form within one week of implantation. Tumors recapitulate
their histologic subtype, including well-differentiated PTC and ATC, and exhibit CD3+, CD8+, B220+, and
CD163+ immune cell infiltration. Tumor progression can be followed in vivo using luciferase and ex vivo using
GFP. Metastatic spread is not detected at early time points.
Conclusions: We describe the development of the next generation of murine orthotopic thyroid cancer models.
The implantation of genetically defined murine BRAF-mutated PTC and ATC cell lines into syngeneic mice
results in rapid and synchronous tumor formation. This model allows for preclinical investigation of novel
therapeutics and/or therapeutic combinations in the context of a functional immune system.
matic rise, thyroid cancer is both common and escalating
(1,2). While highly effective for the treatment of papillary
thyroid cancer (PTC), traditional therapies, including surgery
and radioactive iodine, are ineffective against advanced ra-
dioactive iodine-resistant PTC and undifferentiated (ana-
plastic) thyroid carcinoma (ATC). Approximately 45% of
ith an estimated 60,000 new cases to be diagnosed
in the United States in 2013 and incidence on a dra-
PTCs and 20–40% of ATCs harbor a transversion point
to-glutamate substitution at amino acid 600 of the pro-
tein (BRAFV600E) and ultimately a constitutively active
kinase (3,4). While true that BRAFV600Eplays a critical role
in tumor behavior, it is also clear that not all patients with
BRAF-mutant tumors have clinically aggressive thyroid
cancer (5,6). Other known and putatively undiscovered gene
pathways and immune factors interact with mutant BRAF
signaling and contribute to the development of aggressive
1Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts.
2Thyroid Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts.
3Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge,
Volume 24, Number 4, 2014
ª Mary Ann Liebert, Inc.
characteristics in thyroid tumors. Among the additional ge-
netic events identified to drive dedifferentiation and tumor
progression are mutations affecting the tumor suppressor p53
and the PI3K-AKT pathway (5). The inactivation of p53 is
detected in the vast majority of ATC (7). Though less prev-
alent than mutations of p53, the inactivation of the tumor
suppressor PTEN leads to the activation of the PI3K-AKT
pathway and is observed in *15% of cases of ATC (8).
Further, additional relevant signaling pathways and driver
mutations will putatively be discovered by large-scale efforts
such as The Cancer Genome Atlas (http://tcga-data.nci.nih
.gov/tcga/). Mouse models have proven very useful for
studying thyroid cancer progression. Both PTC and the more
aggressive andlethal formofthyroidcancer, ATC,havebeen
faithfully modeled in mice using orthotopic and, more re-
cently, genetically engineered approaches. Each of these
approaches has both advantages and disadvantages.
Our laboratory has previously shown that BRAFV600E
plays an important role in the aggressive behavior of thyroid
cancer cells and that targeted pharmaceutical inhibition of
BRAFV600Eresults in impressive decreases in tumor volume
and metastasis in an orthotopic animal model of ATC (6,9–
12). Orthotopic placement of human thyroid cancer cell lines
in the native thyroid gland is simple and inexpensive and
allows metastatic spread; however, the animals are impera-
tively immunodeficient to prevent rejection of the human
cells. Orthotopic implantation can be performed on a large
number of mice allowing homogeneous cohorts of tumor-
bearing animals, which are useful for investigating potential
therapeutics. This model has proven useful in preclinical
testing of BRAF inhibitors in the treatment of thyroid cancer
and contributed to the initiation of a phase I clinical trial of
vemurafenib, a selective BRAFV600Einhibitor, in patients
with advanced thyroid cancer (3,9–11). While very practical
and valuable, the absence of a functional immune system in
these models unfortunately precludes the study of the native
immune response to tumorigenesis and tumor progression in
the presence of mutated BRAF.
Genetically engineered models are immunocompetent and
elegant in their basic approach with exquisite control of ge-
netic initiating events and timing of tumor development.
However, tumors often take months to develop and thera-
of similar sizes, making use of these elegant genetically en-
gineered models for preclinical testing time-consuming, ex-
pensive, and ultimately challenging. The first transgenic
model of BRAFV600E-positive PTC was developed by over-
expression of oncogenic BRAF using the thyroid-specific
bovine thyroglobulin promoter (13). These mice developed
well-differentiated PTC, and some transitioned to a more
poorly differentiated state over 5 months. Since this initial
transgenic overexpression model, PTC tumors have been
generated by Cre-mediated expression of BrafV600Efrom the
endogenous Braf locus in the thyroid gland. PTC develops in
young mice (approximately five weeks of age) when
BRAFV600Eexpression is induced in the embryo by crossing
Braftm1Rimamice to transgenic mice expressing constitutive
thyroid-specific Cre recombinase (14) and in adult mice (six
months after induction of BRAF expression) by crossing
specific CreER transgenic mice (15). PTC tumors have been
rapidly induced (within one week) in adult mice by doxy-
cycline treatment to drive doxycycline-inducible transgenic
expression of BRAFV600E, though continuous drug admin-
istration is required (14–16). A genetically engineered model
of ATC has been generated in immunocompetent mice by
double-knockout mice develop follicular thyroid carcinoma
that progresses to dedifferentiated ATC characterized by
pleomorphism, aneuploidy, and epithelial-to-mesenchymal
transition by 9 months of age (17). Given the timeframe and
stochastic nature at which these mice develop tumors, it was
suggested that acquired mutations spontaneously arose,
though alterations were not found in various mutation hot-
spots, including those within the Braf locus (17).
A model of BRAFV600E-positive ATC was recently de-
veloped by inducing thyroid-specific expression ofBrafV600E
from the endogenous locus along with biallelic thyroid-
specific deletion of either p53 or Pten (18). These novel mice
develop either PTC or ATC. In this study, we utilized cell
lines derived from tumors arising in this genetically en-
gineered model for orthotopic implantation into syngeneic
mice. In this ‘‘best of both worlds’’ approach, murine
cell lines with well-defined genetic changes known to be
important in thyroid cancer were implanted orthotopically
into the thyroid environment of immunocompetent mice.
This approach, which results in rapid tumor development,
allows for the generation of cohorts suitable for preclinical
investigation in the context of a functional immune system,
thereby meeting the needs for studying the interaction of
tumor cells, the tumor microenvironment, and the immune
Materials and Methods
The murine cell lines (TBP-3868, TBP-3743, TBPt-3403,
and TBPt-3610R) were cultured from autochthonous tumors
with either PTC-like or ATC-like pathologic features (18). In
brief, TPOCreER; Braftm1Mmcm/WTmice (B6129F1/J) with ei-
ther homozygous floxed p53 alleles (Trp53tm1Brn/tm1Brn) or
biallelic floxed Pten alleles (Ptentm1Hwu/tm1Hwu) were treated
with tamoxifen to induce thyroid-specific expression of Cre
recombinase resulting in the expression of BrafV600Efrom the
endogenous Braf promoter and the deletion of either p53 or
Pten (18). In addition, human 8505c ATC cells (Deutsche
Sammlung von Mikroorganismen und Zellkulturen) and
human BCPAP PTC cells (previously provided by Dr. G.
were maintained in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum and penicillin/
streptomycin and incubated at 37?C with 5% CO2.
The murine thyroid cancer cell lines were transduced with
lentiviral vectors encoding luciferase and GFP in order to
detect the primary tumor and metastases. HEK-293 cells
were cotransfected with a lentiviral plasmid encoding firefly
luciferase (pLenti CMV Puro LUC, w168-1) obtained from
Addgene, a lentiviral plasmid encodingGFP (HIV-U6-GL3B-
GFP) kindly provided by Carmelo Nucera, Beth Israel
706VANDEN BORRE ET AL.
was collected 24 and 48 hours after transfection and filtered
with 0.44lm Millex-HP PES filters. The murine lines (TBP-
3868, TBP-3743, TBPt-3403, and TBPt-3610R) were grown
to 40–60% confluence and infected with the filtered medium
for 16 hours in the presence of 8lg/mL polybrene (Sigma, St.
Louis, MO). The infected cells were treated with 1–3lg/mL
puromycin before sorting for GFP-positive cells by flow cy-
tometry using equivalent gating (MoFlo/FACSAria Sorting;
Beckman Coulter, Fullerton, CA).
To determine tumor take and metastatic potential in our
orthotopic models, we implanted the BRAFV600E-positive
murine tumor cell lines in the thyroid glands of syngeneic
mice, as previously described (12). In brief, B6129SF1/J
mice were anesthetized with 2.0–2.5% isoflurane, 100mg/kg
ketamine, and 10mg/kg xylazine with 0.1mg/kg buprenor-
phine preemptive analgesic. The thyroid gland was exposed
and 104, 105, or 106cells suspended in 10lL serum-free
media were unilaterally injected into the left thyroid gland of
each mouse using a Hamilton syringe attached to a 27-gauge
needle. The right side of the thyroid gland was not manipu-
lated and was used as an internal control. Mice were eutha-
nized and tumor volume was calculated as (p/6)·length·
width·height (19). Prism (GraphPad Software, San Diego,
CA) was utilized for statistical analysis of tumor volumes.
Statistical difference was determined with one-way analysis
of variance followed by the Tukey post hoc multiple com-
parison test, and p-values <0.05 were considered significant.
All animal work was done at Massachusetts General Hospital
(Harvard Medical School) in accordance with federal, local,
and institutional guidelines.
Bioluminescence and GFP imaging
Luciferase activity was detected in mice anesthetized with
using a multispectral fluorescence scanner (CRi Maestro 500,
Woburn, MA) able to detect and image fluorophores between
450 and 900nm. Primary tumor and lung specimens were
imaged immediately after necropsy using a fluorescence mi-
croscope (Leica Microsystems, Bannockburn, IL).
and immunohistochemical analysis
Cultured cells and stained and immunohistochemical tissue
sections were photographed using an Olympus BX41 micro-
scope and an Olympus Q COLOR 5 photo camera (Olympus
Corp., Lake Success, NY). Tissue specimens were fixed with
10% buffered formalin phosphate immediately after necropsy
and embedded in paraffin blocks. Sections of the dissected
orthotopic thyroid tumor were hematoxylin and eosin stained
before histopathologic evaluation by an endocrine pathologist
(P.M.S.). Sections of formalin-fixed tissues were also pro-
cessed for immunohistochemical analysis as previously de-
scribed (10). Immunohistochemical analysis was performed
CA), B220 (BD Pharmingen, San Jose, CA), CD3, CD8, and
CD163 (Leica Microsystems) antibodies.
Western blot analysis
plates with RIPA buffer, separated bysodium dodecyl sulfate
polyacrylamide gel electrophoresis, transferred to nitrocel-
lulose membranes, and ultimately probed with antibodies
against ERK, phosphorylated ERK, AKT, phosphorylated
AKT, PTEN, TTF-1, E-cadherin, N-cadherin, and tubulin
(Cell Signaling Technologies, Beverly, MA) diluted 1:1000
in Tris-buffered saline with TWEEN containing either 5%
milk or bovine serum albumin. Horseradish peroxidase-
conjugated secondary antibodies (Cell Signaling Technolo-
gies) were then used, and the membranes were treated with a
peroxidase substrate for enhanced chemiluminescence
(Thermo Scientific, Rockford, IL) before film exposure.
Cellular growth rate curve
For eachcellline, 2.5·104cells wereseededintriplicatein
day (day 0) and at days 2, 4, and 6, cells were washed with
phosphate-buffered saline, fixed with 10% formalin, and
stored at 4?C. The fixed cells were washed with phosphate-
methanol. After washing with water, the cells were dried and
1mL of 10% acetic acid was added to each well. The absor-
bance at 595nm was analyzed for each line and time point.
The MAPK and PI3K-AKT signaling pathways
are active in BRAFV600E-positive thyroid cancer
cell lines derived from genetically engineered
mouse models of PTC and ATC
We characterized four distinct murine thyroid cancer cell
lines derived from tumors that developed in genetically en-
gineered models of PTC and ATC. These cell lines were
derived from genetically engineered mice with alterations in
Braf, p53, and Pten, genes known to play an important role in
aggressive thyroid cancer (5). The cell lines are listed as
TBP-3868, TBP-3743, TBPt-3403, and TBPt-3610R (Table
1). In brief and to be published elsewhere, the genetically
engineered mice received tamoxifen to induce thyroid-
specific expression of Cre recombinase and consequent
recombination and expression of BrafV600Efrom the endog-
enous Braf promoter in the thyroid gland. Additionally, these
mice were homozygous for either p53 or Pten floxed alleles,
and the tamoxifen-induced expression of Cre resulted in
established TBP and TBPt cell lines were named to reflect
their derivation from tumors arising in either TPOCreER;
Braftm1Mmcm/WT; Trp53tm1Brn/tm1Brn(TBP) or TPOCreER;
Braftm1Mmcm/WT; Ptentm1Hwu/tm1Hwu(TBPt) mice (Fig. 1).
Phosphorylated ERK, indicative of activated MAPK signal-
ing, was detected by Western blot analysis of each of the
murine thyroid cancer cell lines at levels comparable to those
in the BRAFV600E-positive human ATC cell line, 8505c
(Fig. 2A). Loss of PTEN protein expression was evident in
the Pten-deleted lines, TBPt-3403 and TBPt-3610R, and
consequently elevated levels of phosphorylated AKT were
detected. Modest levels of AKT phosphorylation are also de-
(Fig. 2A). Bona fide human thyroidcancer cell lines 8505c and
IMMUNOCOMPETENT ORTHOTOPIC MOUSE MODELS OF THYROID CANCER 707
lines, confirming the thyroid origin of each of the murine lines
(Fig. 2B). The murine PTC line, TBP-3868, expresses the ep-
ithelial marker E-cadherin (CDH1) and low levels of the
of N-cadherin (Fig. 2B). Interestingly, neither E-cadherin nor
human ATC line, 8505c, shows low N-cadherin only at long
exposure (Fig. 2B). The mesenchymal marker vimentin is de-
tected in all of the lines examined (Fig. 2B).
The murine thyroid cancer cell lines exhibit varied
cell morphologies and exhibit higher growth rates
than human thyroid cancer lines
Each murine cell line examined grew as a monolayer with
morphology reflecting the tumor of origin. TBP-3868 was
initially derived from a well-differentiated papillary tumor
and, consistent with the expression of E-cadherin, continued
to exhibit an epithelial growth pattern in culture with cells
growing in close association with each other. The murine cell
lines derived from anaplastic tumors, TBP-3743, TBPt-3403,
and TBPt-3610R, exhibit more mesenchymal-like morphol-
ogies and less cohesive growth patterns (Fig. 2C).
Reaching confluence four days postplating, all four of the
murine lines grow faster than the human PTC and ATC lines,
plating (Fig. 2D). Though the p53-deficient ATC line, TBP-
3743, grows moderately faster than both the PTEN-deficient
ATC lines and the PTC line, TBP-3868, the relative growth
rates of the murine lines do not markedly vary (Fig. 2D).
Orthotopic tumor implantation results in rapid tumor
formation in syngeneic B6129SF1/J mice
One million luciferase- and GFP-labeled mouse tumor
cells from each of the lines were implanted into one of the
thyroid lobes of the immunocompetent common strain
B6129SF1/J mice. The implantation of each cell line resulted
in rapid tumor development. Tumor size could be followed
in vivo using luciferase activity (Fig. 3A) and ex vivo using
GFP(Fig.3D). Consistent withluciferaseactivity
Table 1. Murine Thyroid Cancer Cell Lines
Cell lineType MutationsGenotype TTF-1 staining
(mm3, 4 weeks)
Focally positive211No CD3, CD8,
TBP-3743 ATC BRAFV600E/WT
34 NoNot tested
TBPt-3610R ATC BRAFV600E/WT
aTumor volume measured at 2 weeks.
ATC, anaplastic thyroid cancer; PTC, papillary thyroid cancer.
rived from genetically engineered mouse models are or-
thotopically implanted into syngeneic mice. (A) Thyroid
tumors developing in the initial genetically engineered mice
after the induction of thyroid-specific Cre expression and
consequent thyroid-specific recombination and expression
of BrafV600Efrom the endogenous Braf promoter in
TPOCreER; Braftm1Mmcm/WTmice (B6129F1/J) with either
biallelic p53 deficiency (Trp53tm1Brn/tm1Brn) or Pten defi-
ciency (Ptentm1Hwu/tm1Hwu). (B) Cells were cultured from
either a p53-deficient tumor with PTC characteristics or
ATC tumors arising in the p53-deficient and Pten-deficient
backgrounds. (C) After transduction with luciferase and
GFP lentivirus, each cell line was surgically implanted into
the thyroid bed of syngeneic B6129SF1/J mice. ATC, ana-
plastic thyroid cancer; PTC, papillary thyroid cancer.
BRAFV600E-postive thyroid cancer cell lines de-
708VANDEN BORRE ET AL.
representing actual tumor growth, the greatest level of lu-
ciferase activity was observed in mice implanted with the
p53-deficient ATC line, TBP-3743, at both one and two
weeks postimplantation (Fig. 3A). Metastasis to the lungs
was not detected during the two- to four-week observation
period in any of the cell lines despite the presence of very
large and locally aggressive primary tumors (Fig. 3A, D).
While direct measurement of the tumor is the most faithful
way to determine tumor volume, the correlation between
luciferase activity and tumor growth allows live-tracking of
tumor growth during experiments while GFP allows visual-
ization of tumor tissue after necropsy (Fig. 3D).
Previously, we and others reported that the implantation of
human 8505c ATC cells into SCID mice leads to the for-
mation of thyroid tumors (60–250mm3) within four to five
weeks postimplantation (9,10,20,21). The growth curves of
the murine Pten-deficient lines (TBPt-3403 and TBPt-
3610R) are approximately equivalent to those of the human
ATC line, 8505c, with tumors reaching the sizes of 34–
134mm3one month after implantation. The implantation of
the most aggressive murine line examined, TBP-3743, which
has both a BRAF and p53 mutation, leads to the formation of
large tumors within one week after implantation with mice
becoming moribund and requiring euthanasia approximately
two weeks postimplantation. Tumors arising from the only
murine PTC cell line, TBP-3868, show well-differentiated
pathology with clear-cut papillary architecture but with an
aggressive in vivo growth pattern and achieve volumes sim-
ilar to those of the TBPt lines one month postimplantation. It
is notable that this line, which was derived from a papillary
thyroid tumor, recapitulates the architectural characteristics
of this tumor subtype after implantation.
The unique in vivo growth characteristics and histologic
profile of each implanted cell line is described below.
TBP-3868: PTC cell line (BrafV600E/WTand p53-/-). TBP-
3868 cells developed into well-differentiated PTCs with
distinct papillary architecture featuring serrated follicular
was 21mm3at one week, and by four weeks postimplantation
AKT signaling under basal conditions, and exhibit varied morphologies. (A) The phosphorylation status of ERK and AKT
was assessed under basal conditions in the immortalized human ATC line, 8505c, and the murine PTC and ATC lines by
probing whole cell lysates with antibodies detecting total and phosphorylated levels of ERK and AKT. The absence of
PTEN protein expression was verified in the Pten-deficient lines, and the levels of phosphorylated AKT were highest in
these lines. Whole cell lysates were probed for tubulin to ensure even protein loading. (B) The 42kDa thyroid marker TTF-1
was detected in the human ATC and PTC lines (8505c and BCPAP) as well as in each of the murine cell lines by Western
blot analysis. A higher molecular weight nonspecific band was also observed. The epithelial marker E-cadherin was
detected in the murine PTC line, TBP-3868. The mesenchymal marker N-cadherin was readily detected in the murine ATC
lines and in 8505c and TBP-3868 upon longer exposure. Vimentin was detected in each line examined. Tubulin was
individually probed for on the sets of lysates used for TTF-1 and for E-cadherin, N-cadherin, and vimentin as a loading
control. (C) Each cell line exhibits a unique morphology. 200· magnification. (D) Absorbance at 595nm over time for the
murine and human thyroid cancer cell lines. Data are expressed as mean–standard error of the mean (SEM).
The murine BRAFV600E-postive PTC and ATC cell lines are of thyroid origin, feature active MAPK and PI3K-
IMMUNOCOMPETENT ORTHOTOPIC MOUSE MODELS OF THYROID CANCER709
the tumor volume had dramatically increased to 211mm3
(Fig. 3B, C). Despite the well-differentiated architecture, the
nuclei of the cells predominantly exhibit high-grade nuclear
morphology with hyperchromasia, marked pleomorphism,
and atypia. TTF-1 expression was focally positive in tumors
arising from TBP-3868 (Fig. 4).
TBP-3743: ATC cell line (BrafV600E/WTand p53-/-). Mice
implanted with the p53-deficient ATC cells, TBP-3743, ex-
hibited large palpable neck masses and a significantly larger
mean tumor volume, 238mm3, one week postimplantation
when compared with mice implanted with the other three cell
lines (Fig. 3B, C). In fact, tumors grew so rapidly that mice
implanted with 106TBP-3743 cells were euthanized two
weeks postimplantation, and at necropsy the mean tumor
volume was 390mm3. To determine if this rapid tumor for-
mation can be reduced by injecting fewer cells, additional
mice were implanted with either 104or 105TBP-3743 cells.
Luciferase activity indicates the proliferation and take of each of the four injected mouse cell lines one week postimplantation.
Mice implanted with TBP-3743 exhibit the highest signal (photons/sec). (B) Mice were implanted with 106cells of each line
and euthanized at one (n=3 for each line), two (n=2), and four (n=2–4) weeks postimplantation. Mice implanted with either
104(n=2) or 105(n=9) TBP-3743 cells developed tumors and were euthanized two weeks postimplantation. Tumors were
measured directly. The TBP-3743 line showed the most aggressive growth in vivo. Data are expressed as mean–SEM and
were analyzed by one-way analysis of variance followed by the Tukey multiple comparison test. *p<0.05 of TBP-3743 (106
cells) relative to the other lines at one week postimplantation of 106cells.#p<0.01 of TBP-3743 (104cells) relative to TBP-
3743 (106cells) two weeks postimplantation. (C) Gross morphology after euthanization at two to four weeks postimplantation
of 106cells. Scale bar is 25mm. (D) Dissection and visualization of GFP at four weeks postimplantation indicates that the
tumor is comprised of the GFP-expressing thyroid cancer cells and that the lungs are clear of metastatic spread. The results
shown are of mice implanted with TBP-3868 cells and are representative of the mice implanted with the ATC cell lines.
Dashed lines delineate trachea. Color images available online at www.liebertpub.com/thy
Thyroid tumors form within one week of orthotopic implantation of PTC and ATC cell lines in syngeneic mice. (A)
710 VANDEN BORRE ET AL.
After two weeks, the orthotopic implantation of 104and 105
cells gave rise to tumors with mean volumes of 92mm3
(n=2) and 231mm3(n=9), respectively (Fig. 3B). Whereas
the tumor size in mice implanted with 105cells was not
significantly lower than that inmice implanted with 106cells,
the implantation of 104cells results in the formation of sig-
nificantly smaller tumors (p<0.01). Further, mice implanted
with 104cells did not present with poor body condition two
weeks postimplantation and would putatively exhibit in-
creased survival relative to mice implanted with greater
numbers of cells had they not been euthanized for tumor
measurement. Histology of this p53-deficient ATC line,
TBP-3743, showed aggressive spindled and epithelioid
neoplasms with marked nuclear atypia, pleomorphism, and
hyperchromasia. These tumors readily infiltrated skeletal
muscle and entrapped normal thyroid follicles (Fig. 4, data
not shown). TTF-1 immunostaining of tumors arising from
TBP-3743 was weakly and diffusely positive (Fig. 4).
TBPt-3403: ATC cell line (BrafV600E/WTand Pten-/-).
The mean volume of tumors arising in mice implanted with
TBPt-3403 cells was 72mm3one week after implantation,
and though the mean tumor volume was 34mm3four weeks
postimplantation, the decrease in size was not significant
(Fig. 3B, C). Tumors arising from this line featured mixed
spindled and epitheloid morphology characterized by
abundant nuclear pleomorphism, hyperchromasia, and nu-
clear atypia (Fig. 4). Tumors arising from this ATC-derived
cell line exhibited weak and diffuse TTF-1-positive im-
munostaining (Fig. 4).
TBPt-3610R: ATC cell line (BrafV600E/WTand Pten-/-).
Tumors arising from TBPt-3610R measured 14mm3one
week postimplantation and grew to a mean tumor volume of
134mm3at four weeks postimplantation (Fig. 3B, C). This
Pten-deficient line also gave rise to lesions featuring mixed
spindled and epitheloid morphology and abundant nuclear
pleomorphism, hyperchromasia, and nuclear atypia. There
was alsoinfiltration ofboththyroidgland and skeletalmuscle
with malignant cells along with a marked inflammatory re-
sponse with neutrophils and lymphocytes inside the tumors
(Fig. 4). TTF-1 immunostaining was weakly and diffusely
positive upon pathologic examination (Fig. 4).
Tumors arising from the PTC and ATC lines
exhibit immune cell infiltration
Todetermine if endogenous immune cells infiltrate tumors
arising in mice implanted with PTC (TBP-3868) or ATC
plastic. (A–D) TTF-1 protein expression was detected in sections of tumors established from each line in patterns consistent
with the type of tumor of origin. TTF-1 expression is positive in tumors arising from the PTC line TBP-3868 and weakly
and diffusely positive in the lines arising from ATC tumors, with strongly positive cells around normal follicles. A region of
skeletal muscle (sm), negative for TTF-1, is included in (A). 400· magnification. (E–H) H&E staining of tumors one week
postimplantation, 100· magnification. (E) Well-differentiated papillary thyroid carcinoma in mice implanted with TBP-
3868. (F) High-grade carcinoma infiltrating skeletal muscle in mice implanted with TBP-3743. (G) TBPt-3403 mass
entrapping normal thyroid follicles. (H) TBPt-3610R showing a single mass with malignant infiltration of skeletal muscle.
(I–L) H&E staining of tumors two to four weeks postimplantation, 400· magnification. H&E, hematoxylin and eosin;
TTF-1, thyroid transcription factor 1. Color images available online at www.liebertpub.com/thy
Histopathologic features indicate the formation of aggressive tumors ranging from well differentiated to ana-
IMMUNOCOMPETENT ORTHOTOPIC MOUSE MODELS OF THYROID CANCER711
(TBP-3743) cells, tumor tissue was probed for CD3, CD8,
B220, and CD163 to detect T cells, cytotoxic T cells, B cells,
and macrophages, respectively. Both the PTC and ATC tu-
mors exhibited abundant CD3+ and CD8+ T cell infiltration
(Fig. 5). Though less abundant than the T cells, infiltrating
B220+ B cells and CD163+ macrophages were also de-
tected in the TBP-3868 and TBP-3743 tumors (Fig. 5).
Here we show that immunocompetent mice implanted
with four distinct murine BRAFV600E-positive cell lines,
TBP-3868, TBP-3743, TBPt-3403, and TBPt-3610R, devel-
oped thyroid tumors efficiently, reliably, and within the
context of a functional immune system. The genetically
engineered mice from which these cell lines were initially
positive ATC (18). Because of the rapid and simultaneous
formation of tumors with known genetic backgrounds, the
orthotopic models of BRAFV600E-positive PTC and ATC
presented herein are highly suitable for investigating novel
While oncogenic BRAF has been implicated in the initi-
ation of PTC, the alteration of additional signaling pathways,
including p53 and PI3K-AKT, is believed to be required for
progression and dedifferentiation (5,7,22). Thus far, muta-
tions in the thyroid cancer cell lines used in research reflect
what grows out in primary culture of tumors and undergoes
furtheralterations whencellsaremaintained intissueculture.
Our novel methods allow selection of thyroid cancer cell
lines with specific genetic alterations in Braf, p53, and Pten.
The fact that these cell lines have defined alterations in
multiple signaling pathways is invaluable in studying the
complex relations between the pathways when it comes to
tumor progression, metastasis, or response to drugs. The
murine lines used in the novel models presented here are
heterozygous for the Braf mutation. (BrafV600E/WT); thus, in
addition to the BRAFV600E-positive PTC TBP-3868 tumors,
the tumors arising from TBP-3743, TBPt-3403, and TBPt-
3610R are the first orthotopic models of ATC to harbor both
wild-type and oncogenic Braf alleles. As such, the use of
these BrafV600E/WTmurine thyroid cancer lines will permit
investigation into the efficacy of BRAFV600Einhibitors in the
context of heterozygous BrafV600E. While the clinical sig-
nificance of BRAFV600Ezygosity in the thyroid gland is lar-
gely unknown, evidence suggests that BRAFV600Einhibition
enhances the tumor immune response of BRAFV600Ehomo-
zygous melanoma cells but not heterozygous cells (23).
Previously reported orthotopic models of PTC and ATC have
utilized either PTC cells heterozygous for BRAFV600E
(BCPAP) or ATC cells hemizygous for BRAFV600E(8505c)
(9,10,20,21). The human ATC line SW1736 is heterozygous
for BRAFV600E; however, to date, this line has not been or-
thotopically implanted in mice and the human derivation
would prevent implantation in an immunocompetent mouse.
In addition to the expression of oncogenic BRAF, the TBP
and TBPt cell lines harbor inactivations of the tumor sup-
pressors p53 and Pten, respectively. We observed a notable
difference in growth rate between the tumors with p53 and
Pten deletions. Mice implanted with the BRAFV600E-positive
ATC cell line harboring a biallelic deletion of p53 (TBP-
3743) grow tumors significantly more rapidly than mice
implanted with BRAFV600E-positive ATC cell lines biallelic
p53-deficient PTC cell line, TBP-3868, gave rise to differ-
entiated tumors that were as large as those arising from the
less-differentiated PTEN-deficient ATC cell lines. It is of
significant note that the orthotopic tumors recapitulate the
histology of the source tumors from which each line was
initially derived. Implantation of the TBP-3868 PTC line
results in the formation of large but well-differentiated tu-
mors, whereas undifferentiated tumors arise after the
TBP-3868 PTC tumor four weeks postimplantation, probed with T cell marker CD3 (A), cytotoxic T cell marker CD8 (B), B
cell marker B220 (C), and macrophage marker CD163 (D). (E–H) 400· magnification of TBP-3743 ATC tumor two weeks
postimplantation, probed with T cell marker CD3 (E), cytotoxic T cell marker CD8 (F), B cell marker B220 (G), and
macrophage marker CD163 (H). Color images available online at www.liebertpub.com/thy
Immune cell infiltration into TBP-3868 papillary and TBP-3743 anaplastic tumors. (A–D) 400· magnification of
712 VANDEN BORRE ET AL.
implantation of the ATC lines. While distant metastases were
not observed in any of our newly developed orthotopic
models within a month of implantation, in vitro analysis of
the cell lines suggests that PTEN loss may convey a greater
migratory capacity than p53 loss (data not shown). This ob-
servation may be explained by the commensurate increase of
phosphorylated AKT that is observed with a loss of PTEN
expression, and putative downstream activation of signaling
that increases cellular motility.
Beyond the genetic mutations that alter signaling, the
tumorigenesis (12,22,24,25). An advantage of the orthotopic
approach to modeling PTC and ATC is the faithful recapit-
ulation of the tumor microenvironment and further, with the
immune cells and their local cytokines. We detect abundant
CD3+ T cell infiltration in tumors arising from TBP-3868
PTC and TBP-3743 ATC cells and, importantly, a large
portion of these are CD8+ cytotoxic T cells. Increased
tumor-associated macrophage density correlates with poor
prognosis in patients with advanced thyroid cancer, and the
inhibition of tumor-associated macrophage recruitment at-
tenuates disease progression in an immunocompetent trans-
genic mouse model of PTC (26,27). Whereas the tumors
arising from the ATC line TBP-3743 do not present with
abundant CD163+ tumor-associated macrophages, tumors
arising from the aggressive but well-differentiated PTC line
TBP-3868 exhibit robust monocyte/macrophage infiltration.
Additionally, though evidence suggests that inflammatory
conditions, including those resulting from Hashimoto’s
thyroiditis, may promote tumorigenesis, several studies
demonstrate that PTC is less advanced in patients with con-
infiltration, suggesting that an immune response may atten-
uate tumor progression (28–30). This duality aside, the im-
mune response to tumorigenesis has emerged as a potential
avenue of therapy for solid tumors. Approaches for generat-
ing targeted patient-specific T cells for prostate cancer and
for modulating T cell activity in melanoma patients have
been approved by the Federal Drug Administration (31–33).
There is potential to refine these approaches, broaden
them to other types of cancer, and develop additional
Our model is not perfect and there are several caveats
that should be noted. For one, the implanted cells are
monoclonal and lack the cellular heterogeneity found in
human tumors. Further, while our cells harbor well-defined
cancer,additionalundescribedmutationsare most certainly
playing a role in both our cell lines and human tumors.
Other pertinent differences exist between the described
orthotopic mouse model and human tumors, including
those pertaining to angiogenesis, the stromal environment,
and the functioning of the immune system, though these
must be considered for any mouse model of human cancer.
With regard to invasion, tumor cell implantation, whether
orthotopically or under the skin, creates a wound and
therefore may confound the analysis of local invasion
through the thyroid capsule. Most of our tumors have very
rapid growth, which, while very practical for determining
initial efficacy of novel therapeutics and elucidating mo-
lecular mechanisms, does not allow study of later events
such as metastasis. The slowest growing cell line in vivo,
TBPt-3403, may not be an accurate representation of rap-
idly growing and progressing anaplastic thyroid tumor;
however, given the relatively slow growth of the primary
tumor and the putative longer lifespan of implanted mice,
there may be an opportunity for distant metastases to arise
at later time points, and therefore this model may warrant
Thus far, both the limited number of human thyroid
cancer cell lines and the need for implantation into immu-
nodeficient mice have hindered the ability to study the ef-
fects of targeted therapies aimed at specific mutated
pathways in the context of a normal immune system. We
have characterized four novel and distinct mouse PTC and
ATC cell lines and demonstrated that implanting these
immunocompetent B6129SF1/J mice results in rapid tumor
formation. We suggest that our new models of aggressive
murine thyroid cancer in immunocompetent mice allow for
the rapid investigation of potential therapeutics.
P.V.B. is a recipient of the Ruth L. Kirschstein National
Research Service Award for Individual Postdoctoral Fel-
lows (Parent F32) from the National Institutes of Health
and National Cancer Institute. S.P. is funded through the
National Institutes of Health, the American College of
Surgeons, and the American Thyroid Association. The
murine thyroid cancer cell lines were derived from mice
generated by D.G.M., recipient of a K08 award from the
National Institutes of Health and National Cancer Institute,
in the laboratory of T.J. We also thank Derrick Jeon at the
Center for Systems Biology at Massachusetts General
Hospital for assistance with live imaging and Patricia Della
Pelle in the Department of Pathology at Massachusetts
General Hospital for histological and immunohistochemi-
Author Disclosure Statement
All authors certify that they have no competing financial
interests pertaining to any of the data or statements given in
1. Chen AY, Jemal A, Ward EM 2009 Increasing incidence of
differentiated thyroid cancer in the United States, 1988–
2005. Cancer 115:3801–3807.
2. National Cancer Institute. Thyroid Cancer. www.cancer
.gov/cancertopics/types/thyroid (last accessed on June 3, 2013).
3. Xing J, Liu R, Xing M, Trink B 2011 The BRAFT1799A
mutation confers sensitivity of thyroid cancer cells to the
BRAFV600E inhibitor PLX4032 (RG7204). Biochem
Biophys Res Commun 404:958–962.
4. Bhaijee F, Nikiforov YE 2011 Molecular analysis of thy-
roid tumors. Endocr Pathol 22:126–133.
5. Nikiforov YE, Nikiforova MN 2011 Molecular genetics
and diagnosis of thyroid cancer. Nat Rev Endocrinol 7:
6. Nucera C, Goldfarb M, Hodin R, Parangi S 2009 Role of B-
Raf(V600E) in differentiated thyroid cancer and preclinical
IMMUNOCOMPETENT ORTHOTOPIC MOUSE MODELS OF THYROID CANCER713
validation of compounds against B-Raf(V600E). Biochim Download full-text
Biophys Acta 1795:152–161.
7. Fagin JA, Matsuo K, Karmakar A, Chen DL, Tang SH,
Koeffler HP 1993 High prevalence of mutations of the p53
gene in poorly differentiated human thyroid carcinomas.
J Clin Invest 91:179–184.
8. Hou P, Liu D, Shan Y, Hu S, Studeman K, Condouris S,
Wang Y, Trink A, El-Naggar AK, Tallini G, Vasko V, Xing
M 2007 Genetic alterations and their relationship in the
phosphatidylinositol 3-kinase/Akt pathway in thyroid can-
cer. Clini Cancer Res 13:1161–1170.
9. Nehs MA, Nagarkatti S, Nucera C, Hodin RA, Parangi S
2010 Thyroidectomy with neoadjuvant PLX4720 extends
survival and decreases tumor burden in an orthotopic
mouse model of anaplastic thyroid cancer. Surgery
148:1154–1162; discussion 1162.
10. Nehs MA, Nucera C, Nagarkatti SS, Sadow PM, Morales-
Garcia D, Hodin RA, Parangi S 2012 Late intervention with
anti-BRAF(V600E) therapy induces tumor regression in an
orthotopic mouse model of human anaplastic thyroid can-
cer. Endocrinology 153:985–994.
11. Nucera C, Nehs MA, Nagarkatti SS, Sadow PM, Mekel M,
Fischer AH, Lin PS, Bollag GE, Lawler J, Hodin RA,
Parangi S 2011 Targeting BRAFV600E with PLX4720
displays potent antimigratory and anti-invasive activity in
preclinical models of human thyroid cancer. Oncologist
12. Nucera C, Porrello A, Antonello ZA, Mekel M, Nehs MA,
Giordano TJ, Gerald D, Benjamin LE, Priolo C, Puxeddu E,
Finn S, Jarzab B, Hodin RA, Pontecorvi A, Nose V, Lawler
J, Parangi S 2010 B-Raf(V600E) and thrombospondin-1
promote thyroid cancer progression. Proc Natl Acad Sci
13. Knauf JA, Ma X, Smith EP, Zhang L, Mitsutake N, Liao
XH, Refetoff S, Nikiforov YE, Fagin JA 2005 Targeted
expression of BRAFV600E in thyroid cells of transgenic
mice results in papillary thyroid cancers that undergo de-
differentiation. Cancer Res 65:4238–4245.
14. Franco AT, Malaguarnera R, Refetoff S, Liao XH, Lund-
smith E, Kimura S, Pritchard C, Marais R, Davies TF,
Weinstein LS, Chen M, Rosen N, Ghossein R, Knauf JA,
Fagin JA 2011 Thyrotrophin receptor signaling dependence
of Braf-induced thyroid tumor initiation in mice. Proc Natl
Acad Sci USA 108:1615–1620.
15. Charles RP, Iezza G, Amendola E, Dankort D, McMahon
M 2011 Mutationally activated BRAF(V600E) elicits
papillary thyroid cancer in the adult mouse. Cancer Res
16. Chakravarty D, Santos E, Ryder M, Knauf JA, Liao XH,
West BL, Bollag G, Kolesnick R, Thin TH, Rosen N,
Zanzonico P, Larson SM, Refetoff S, Ghossein R, Fagin JA
2011 Small-molecule MAPK inhibitors restore radioiodine
incorporation in mouse thyroid cancers with conditional
BRAF activation. J Clin Invest 121:4700–4711.
17. Antico Arciuch VG, Russo MA, Dima M, Kang KS,
Dasrath F, Liao XH, Refetoff S, Montagna C, Di Cristofano
A 2011 Thyrocyte-specific inactivation of p53 and Pten
results in anaplastic thyroid carcinomas faithfully recapit-
ulating human tumors. Oncotarget 2:1109–1126.
18. McFadden DG, Vernon A, Santiago PM, Martinez-McFaline
R, Bhutkar A, Crowley DM, McMahon M, Sadow PM, Jacks
TE 2014 p53 constrains progression to anaplastic thyroid
carcinoma in a Braf-mutant mouse model of papillary
thyroid cancer. Proc Natl Acad Sci USA (in press).
19. Tomayko MM, Reynolds CP 1989 Determination of sub-
cutaneous tumor size in athymic (nude) mice. Cancer
Chemother Pharmacol 24:148–154.
20. Chan CM, Jing X, Pike LA, Zhou Q, Lim DJ, Sams SB,
Lund GS, Sharma V, Haugen BR, Schweppe RE 2012
Targeted inhibition of Src kinase with dasatinib blocks
thyroid cancer growth and metastasis. Clin Cancer Res 18:
21. Nucera C, Nehs MA, Mekel M, Zhang X, Hodin R, Lawler J,
Nose V, Parangi S 2009 A novel orthotopic mouse model of
human anaplastic thyroid carcinoma.Thyroid 19:1077–1084.
22. Xing M 2013 Molecular pathogenesis and mechanisms of
thyroid cancer. Nat Rev Cancer 13:184–199.
23. Sapkota B, Hill CE, Pollack BP 2013 Vemurafenib en-
hances MHC induction in BRAF homozygous melanoma
cells. Oncoimmunology 2:e22890.
24. Nucera C, Lawler J, Parangi S 2011 BRAF(V600E) and
microenvironment in thyroid cancer: a functional link to
drive cancer progression. Cancer Res 71:2417–2422.
25. Liotti F, Visciano C, Melillo RM 2012 Inflammation in
thyroid oncogenesis. Am J Cancer Res 2:286–297.
26. Ryder M, Ghossein RA, Ricarte-Filho JC, Knauf JA, Fagin
JA 2008 Increased density of tumor-associated macro-
phages is associated with decreased survival in advanced
thyroid cancer. Endocr Relat Cancer 15:1069–1074.
27. Ryder M, Gild M, Hohl TM, Pamer E, Knauf J, Ghossein
R, Joyce JA, Fagin JA 2013 Genetic and pharmacological
targeting of CSF-1/CSF-1R inhibits tumor-associated
macrophages and impairs BRAF-induced thyroid cancer
progression. PloS One 8:e54302.
28. Marotta V, Guerra A, Zatelli MC, Uberti ED, Di Stasi V,
Faggiano A, Colao A, Vitale M 2013 BRAF mutation
positive papillary thyroid carcinoma is less advanced when
Hashimoto’s thyroiditis lymphocytic infiltration is present.
Clin Endocrinol (Oxf) 79:733–738.
29. Schaffler A, Palitzsch KD, Seiffarth C, Hohne HM, Ried-
hammer FJ, Hofstadter F, Scholmerich J, Ruschoff J 1998
Coexistent thyroiditis is associated with lower tumour stage
in thyroid carcinoma. Eur J Clin Invest 28:838–844.
30. Konturek A, Barczynski M, Wierzchowski W, Stopa M,
Nowak W 2013 Coexistence of papillary thyroid cancer with
31. Graziani G, Tentori L, Navarra P 2012 Ipilimumab: a novel
immunostimulatory monoclonal antibody for the treatment
of cancer. Pharmacol Res 65:9–22.
32. Lipson EJ, Drake CG 2011 Ipilimumab: an anti-CTLA-4
antibody for metastatic melanoma. Clin Cancer Res 17:
33. Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ,
Penson DF, Redfern CH, Ferrari AC, Dreicer R, Sims RB,
Xu Y, Frohlich MW, Schellhammer PF 2010 Sipuleucel-T
immunotherapy for castration-resistant prostate cancer.
N Engl J Med 363:411–422.
Address correspondence to:
Sareh Parangi, MD
Department of Surgery
Massachusetts General Hospital
Harvard Medical School
Wang ACC 460, 15 Parkman Street
Boston, MA 02115
714 VANDEN BORRE ET AL.