Received: 9 June 2016
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Revised: 26 February 2017
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Accepted: 14 March 2017
DOI: 10.1002/hed.24807
ORIGINAL ARTICLE
Structural alterations in tumor-draining lymph nodes before papillary thyroid carcinoma metastasis Andrew M. Hinson, MD1
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Nicole A. Massoll, MD2 | Lee Ann Jolly, PhD3 |
Brendan C. Stack Jr, MD1 | Donald L. Bodenner, MD, PhD4 | Aime T. Franco, PhD3 1 Department of Otolaryngology - Head and Neck Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Abstract Background: The purpose of this study was to define and characterize the thyroid tumor-draining
2
Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 3 Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 4
Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas Correspondence Aime T. Franco, Department of Physiology and Biophysics, Biomedical Research Building II, Room 264-2, Little Rock, AR 72205. Email
[email protected] Funding information This work was supported by the University of Arkansas for Medical Sciences grant NIH UL1TR000039; The American Thyroid Association/Thyca research grant (A. Franco); UAMS Envoys Seeds of Science Award (A. Franco).
lymph nodes in genetically engineered mice harboring thyroid-specific expression of oncogenic BrafV600E with and without Pten insufficiency. Methods: After intratumoral injection of methylene blue, the lymphatic drainage of the thyroid gland was visualized in real time. The thyroid gland/tumor was resected en bloc with the respiratory system for histological analysis. Results: Although mice harboring BrafV600E mutations were smaller in body size compared with their wild-type (WT) littermates, the size of their thyroid glands and deep cervical lymph nodes were significantly larger. Additionally, the tumor-draining lymph nodes showed increased and enlarged lymphatic sinuses that were distributed throughout the cortex and medulla. Tumorreactive lymphadenopathy and histiocytosis, but no frank metastases, were observed in all mice harboring BrafV600E mutations. Conclusions: The tumor-draining lymph nodes undergo significant structural alterations in immunocompetent mice, and this may represent a primer for papillary thyroid carcinoma (PTC) metastasis. KEYWORDS
cancer, lymph node, metastasis, mouse, thyroid
1 | INTRODUCTION
is the most common genetic alteration in thyroid cancer and occurs in 44% of PTC and in 24% of PTC-derived poorly differentiated thyroid
By the year 2019, thyroid cancer is expected to become the third most
carcinoma or anaplastic thyroid carcinoma (ATC) cases.6–8 In addition to
common cancer in women and will cost the United States approxi-
the BRAFV600E mutation, ATC also frequently harbors one or more
1
mately 20 billion dollars. Papillary thyroid carcinoma (PTC), which
genetic alterations in the phosphatidylinositol-4,5-bisphosphate 3-kinase
accounts for 85%-90% of all thyroid cancer, is generally considered a
– protein kinase B pathway (eg, PIK3CA, AKT1, and PTEN).9,10 ATC is one
relatively indolent and highly curable disease.2 However, initial treat-
of the most lethal types of cancer, with a median survival of just
ment is not always curative, and persistent or recurrent disease occurs
5 months.11 Although metastasis is observed in 10%-20% of cases,
3
in up to 30% of patients. The most common site for PTC metastasis
most patients with ATC die because of locally invasive disease, which is
and recurrent disease is the locoregional lymph nodes.3 Although PTC
generally refractory to conventional treatment modalities (ie, radiation
lymph node metastasis is associated clinically with both a higher risk
and chemotherapy).11
for disease recurrence and disease-specific mortality, the mechanisms underlying lymphatic dissemination of tumor cells remain elusive.3–5
The lack of successful treatment options for advanced thyroid cancer underscores the need for novel therapeutic strategies, as well
BRAFV600E mutation, which constitutively activates the mitogen-
as in vivo models that can help predict their clinical utility. In the past,
activated protein kinases – extracellular signal-regulated kinases pathway,
in vivo models were developed by injecting human thyroid cancer cell
Head & Neck. 2017;1–8.
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C 2017 Wiley Periodicals, Inc. V
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lines into immunodeficient mouse strains.2,7,12 Immunodeficient mod-
Euthanasia was achieved by 100% carbon dioxide insufflation, followed
els, however, fail to mimic the natural tumor microenvironment,
by cervical dislocation.
because they do not evoke protective immune responses or tumorpromoting inflammation.13 For this reason, genetically engineered
2.2 | Lymph node mapping
strains have been developed to study tumors that arise in situ in immunocompetent hosts.2,7,12 In order to rapidly induce tumor development, orthotopic injection of tumor cell lines derived from genetically engineered mice have also been used.2,7,12 Although such models are practical for defining the underlying molecular mechanisms of tumor development, the rapid progression of disease does not allow for a 12
step-wise analysis of later events, such as lymph node metastasis.
Moreover, subcutaneous or orthotopic injection of tumor cells creates a wound, which may confound interpretation of the mechanisms involved in disease progression, such as, thyroid capsule invasion, extrathyroidal extension, regional lymph node metastasis.
Mouse models have demonstrated that physiological levels of oncogenic Braf
expression alone is sufficient to initiate de novo 14
thyrocyte tumorigenesis in immunocompetent mice.
the lymphatic system in dissemination of PTC. To trace the regional lymphatic drainage, a 25-microliter Hamilton syringe (Hamilton Company, Reno, NV) and a 26-gauge standard double-wing needle (pointed in a rostral and medial direction) was used to inject (depth 2-3 mm; velocity approximately 40-50 microliters/minute) a mixture of 1% methylene blue (Akorn, Lake Forest, IL) and 10 microliters phosphatebuffered saline into the left thyroid lobe/tumor approximately 15 minutes before euthanasia. Alternatively, the footpad was injected subcutaneously with the needle pointed in a caudal direction. The total
12
V600E
We used these models as a first step to examine the involvement of
In general, the
volume of dye injected in each case was 2 microliters. Distilled water was used to flush any extravasation of dye away from the surrounding soft tissue. Lymph nodes were classified based on their anatomic location
latency and extent of these tumors depends on the presence of an intact thyroid-stimulating hormone signaling axis at the time of tumor initiation.15 Despite the fact that physiological levels of oncogenic BrafV600E expression result in de novo thyrocyte tumorigenesis and 14,15
invasive PTC with relatively short latency times,
the tumor-
draining lymph nodes have not yet been comprehensively examined in genetically engineered mouse models.10,14–17 Unfortunately, it is not
using the nomenclature summarized by Van den Broeck et al19 and Shao et al20: mandibular lymph nodes, accessory mandibular lymph nodes, superficial parotid lymph nodes, cranial deep cervical lymph nodes, caudal deep cervical lymph nodes, proper axillary lymph nodes, accessory axillary lymph nodes, cranial mediastinal lymph nodes, tracheobronchial lymph nodes, and popliteal lymph nodes.
always clear from the literature whether this is because lymphatic disease was not present in prior studies or whether the tumor-draining
2.3 | Tissue preparation and histology
lymph nodes were not routinely examined.
At necropsy, the primary tumor was resected en bloc with the respira-
In this study, we developed a technique to identify the lymphatic
tory system for processing and analysis. The neck and lung tissue
drainage of the murine thyroid gland in real time in order to facilitate
specimens were fixed in 10% formalin-buffered acetate overnight,
our studies of lymph node responses to thyroid cancer. Herein, we
dehydrated, and embedded in paraffin. The embedded specimens were
demonstrate that the tumor-draining lymph nodes undergo significant
cut into 5-lm serial sections, stained with hematoxylin-eosin, and
structural alterations in mice harboring PTC, and this may represent a
reviewed by a high-volume thyroid pathologist (N.M.).
priming of the lymph nodes for metastasis.
2.4 | Subcapsular sinus width 2 | MATERIALS AND METHODS
The width (lm) of the marginal or subcapsular sinus (SCS) was meas-
2.1 | Experimental animals
using ImageJ (National Institutes of Health, Bethesda, MD). Statistical
ured in hematoxylin-eosin section images at 340-3200 magnification
All animal experiments were performed at the University of Arkansas for Medical Sciences and approved by the Institutional Animal Care V600E
and Use Committee. The LSL-Braf
fl/fl
, Pten
, and thyroid peroxidase
promoter (TPO)-Cre strains have previously been described.15,18 Mice
analysis was performed using GraphPad Prism 7 software (La Jolla, CA). Differences between groups were assessed by paired Student’s t test. All data were presented as the mean 6 SD, and P values of .05 were considered statistically significant.
were on mixed C57BL6/129SVJ (B6) genetic backgrounds. Genotypes were determined by reverse transcription-polymerase chain reaction, as previously described.18 Three male and 6 female mice: (a) wild-type (WT; 1 male and 2 female), 8-10 weeks old; (b) LSL-BrafV600ECre1
3 | RESULTS 3.1 | Primary tumor
(1 male and 2 female), 3-5 weeks old; and (c) LSL-BrafV600E/
BrafV600E mice were significantly runted compared with their WT litter-
Pten1/-/Cre1 (1 male and 2 female), 10-12 weeks old; were maintained
mates, consistent with previous reports because of hypothyroidism in
in a barrier facility on high efficiency particulate air-filtered racks.
BrafV600 animals.15 Histologic examination of thyroid tumors obtained
All surgical procedures were performed with the mice under general anesthesia with 2.5% isoflurane followed by immediate euthanasia.
from
3-week-old
V600E
Braf
1/-
/Pten
LSL-BrafV600E/TPO-Cre
and
12-week-old
LSL-
/TPO-Cre mice demonstrated multifocal PTC involving
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3
Thyroid pathology. A, Normal thyroid parenchyma composed of colloid (CO)-rich follicles in an 8-week-old wild-type mouse (original magnification 3100). B, Intrathyroidal papillary thyroid carcinoma (PTC) encompassing the majority of the thyroid lobe, which is surrounded by a thick fibrotic capsule (CAP), in a 3-week-old BrafV600E/TPO-Cre mouse (original magnification 3100). C, Papillae lined by tumor cells with nuclear overlapping and crowding in a 3-week-old BrafV600E/TPO-Cre mouse (original magnification 3200). D, PTC and tumor-reactive stroma adjacent to skeletal muscle (SkM) and cartilage (CAR) in a 3-week-old BrafV600E/TPO-Cre mouse (original magnification 3100). E, PTC tumor composed of a large stromal component in a 12-week-old BrafV600E/Pten1/-/TPO-Cre mouse (original magnification 3100). D, High-grade PTC exhibiting central necrosis (NEC), extrathyroidal invasion, and tumor reactive stroma (TRS) in a 12-week old BrafV600E/Pten1/-/TPO-Cre mouse (original magnification 340). All sections were stained with hematoxylin-eosin [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 1
both thyroid lobes. Compared to WT (Figure 1A), the BrafV600E/Pten-/-/
identify the lymph nodes that drain the thyroid in the mouse and deter-
TPO-Cre tumor sections demonstrated an extensive stromal compo-
mine whether this technique could be used to identify and isolate
nent that was encompassed by a thick fibrotic capsule (Figure 1B-E),
tumor-draining lymph nodes.
consistent with reports that these tumors secrete tumor-derived fac-
To expose the thyroid gland, a 1.0-cm longitudinal midline cervical
tors that induce fibroblast recruitment and abundant stromal deposi-
incision was made in the anterior neck. The salivary glands were
tion of fibrillar collagen.18 BrafV600E/TPO-Cre tumor sections exhibited
retracted laterally, and the underlying superficial strap muscles were
highly cellular, florid PTC with areas of central necrosis, extrathyroidal
then elevated away from midline, revealing the trachea and thyroid
invasion, and tumor-reactive stroma (Figure 1F). None of the tumors
lobes (Figure 2A-C). The deeper strap muscles were left intact on the
progressed to poorly differentiated thyroid carcinoma or ATC during
trachea.
the observed time course (21-84 days).
Despite intrathyroidal injection and adequate time for systemic distribution (15-20 minutes), the salivary and deep cervical lymph
3.2 | Lymph node mapping
nodes could not be visualized in WT mice (Figure 1A). In contrast, the blue-labeled mandibular (located rostromedial to the mandibular and
Accurate identification of murine lymph nodes (
