The role of Chemokines in Thyroid Carcinoma

: The global incidence of thyroid cancer is increasing and metastatic spread to lymph nodes is common in papillary thyroid carcinoma. The metastatic course of thyroid carcinoma is an intricate process involving invasion, angiogenesis, cell trafficking, extravasation, organ specific homing and growth. A key aspect in this process involves a multitude of interactions between chemokines and their receptors. Chemokines are a group of small proteins, which act to elicit normal physiological and immune responses principally through recruitment of specific cell populations to the site of infection or malignancy. Thyroid cancer cells, like other tumors, possess the ability to corrupt the chemokine system to their advantage by altering cell movement into the tumor microenvironment and affecting all aspects of thyroid cancer progression.


Thyroid
The Role of Chemokines in Thyroid Carcinoma (DOI: 10.1089/thy.2016.0660) This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. cells which circulate amongst the tumor cells and supporting stroma (19). These leucocytes can account for up to 50% of the tumor mass with the predominant subset being macrophages (20). Chemokines shown to exist at relatively high levels within the microenvironment are CXCL8 (8,21-24) and CXCL20 (25,26,27); these are produced by both tumor cells and immune cells ( Table 1). Production of inflammatory mediators within the thyroid tumor mass also results in de novo chemokine receptor expression on the malignant cells. As expected a number of receptors have been shown to be up-regulated (Table 1)  This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Method:
A comprehensive literature search was performed using Pubmed, Google Scholar and MEDLINE databases between the years 1996 to 2016 with the following key words: 1.) Chemokine and thyroid carcinoma; 2.) leukocyte and thyroid carcinoma; 3.) chemokine and metastasis in thyroid carcinoma; and 4.) inflammation and thyroid carcinoma. Four separate searches were performed in each database. A total of 26,200 studies were found, with 141 being relevant to thyroid carcinoma. No restrictions on language were used.
Unpublished reports, including dissertations and conference abstracts were not considered.

Inclusion and exclusion criteria:
All titles and abstracts of the retrieved articles were reviewed. Studies were included if they met the following criteria: 1. Studies investigating chemokines and oncogenic changes in thyroid carcinoma; 2. Studies looking at chemokines, inflammation and tumor associated leukocytes; 3. CK induced metastatic potential of thyroid carcinoma; and 4. The therapeutic potential of chemokines in thyroid carcinoma. The major reasons for exclusion were due to the studies not being specific to chemokines and thyroid carcinoma.

Tumor progression and migration: CXCL8 and CCL20
One of the more abundantly expressed chemokines by thyroid carcinoma cells is CXCL8.
This was the first chemokine shown to be secreted by thyrocytes (24) and there is evidence to support a role for CXCL8 and its interaction with receptors CXCR1 and CXCR2 in regulating cell migration, tumor growth, progression, metastatic spread and tumor-

Tumor neovascularization and growth: Endothelial cells and CXC chemokines
It is well known that neovascularization is crucial for tumor growth, progression and metastasis and some chemokines are known to be involved in this process (39 This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. suppress the angiogenic effects of the ELR+ CXC chemokines in thyroid cancer as it can sequester these without eliciting any intracellular signals (27), thus the balance between pro-and anti-angiogenic CK determines the degree of tumor neovascularization.

Tumor associated macrophages (TAM):
The This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. it with anti-CXCL8 neutralizing antibody significantly decreased PTC metastases and exogenous CXCL8 enhanced PTC metastases in vivo (41). It was also noted that CXCR1/2 receptors (which CXCL8 binds to) were only present in tumor tissue, and not found in normal healthy thyroid tissue. Therefore TAM may facilitate PTC cell metastases through CXCL8 and its paracrine interaction with CXCR1/2 (41).
Furthermore, a recent study by Cho et al. in 2016 (42) found that CXCR6 expression positively correlated with lymph node metastasis and CXCL16 signaling mediated macrophage enhanced PTC tumor cell invasion and also changed the macrophage phenotypes into M2-macrophages in the PTC environment.
A study of 398 patients with DTC and 132 patients with benign thyroid tissue indicated that TAM were found more frequently in aggressive cases with metastasis at diagnosis, but conversely macrophage infiltration was also associated with improved disease free survival (48). This paradoxical result was attributed to three reasons; i.) Varying microenvironments in different histological subsets ii.) TAM modulation of the TME by secretion of a rich repertoire of chemokines and growth factors that exert paracrine effects on tumor cells to facilitate progression, and iii.) TAM phagocytic activity, which could also limit tumor progression (49). This was further confirmed by Fiumara et al.
(50), in a study of 121 well differentiated thyroid carcinoma specimens demonstrating that tumors with TAM in situ showed evidence of active neoplastic cell phagocytosis which was in turn positively correlated with infiltration of lymphocytes and dendritic cells, and negatively correlated with vascular invasion. It is possible that these different results are also related to different populations (50).

Mast cells:
Mast cells are an important constituent of the thyroid carcinoma microenvironment.
Recent studies indicate that PTC induces mast cell activation and chemoattraction via secretion of CXCL1, CXCL10 and CXCL8 (51). Mast cell numbers are elevated in PTC compared to normal thyroid tissue, and their increased density correlates with a poor prognosis (51). Further experiments using mast cell lines showed that PTC culture medium This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
induced mast cell activation and chemo-attraction, thereby increasing vascularity, proliferation, survival, invasiveness and DNA synthesis of cancer cells and inhibiting apoptosis. These actions were mediated by histamine, CXCL1 and CXCL10; therefore, targeting these pathways may be of therapeutic benefit in future.
It binds to CCL21 which causes proliferation and migration of tumor cells (30), (33) and also plays a critical role in lymphocyte and dendritic cell trafficking into lymph nodes, which are the preferential sites of metastasis of PTC and medullary thyroid carcinoma It is well established that poorly differentiated thyroid carcinoma preferentially metastasize to bone, lung, liver and brain whereas DTC metastasize to lymph nodes. PTC has two variants; follicular variant of PTC and classic PTC of which the latter has a higher propensity for lymph node metastasis. Sancho et. al. (33) analyzed CCR7 expression in 46 normal thyroid tissue and thyroid carcinoma specimens. The expression of CCR7 was higher in classic PTC and MTC, which are both prone to lymph node metastasis compared to follicular variants and was absent in healthy thyroid tissue. As only a subset of PTC develop lymph node metastasis, this would support the concept that CCR7 signaling is not only involved in proliferation and migration but also may play a key role during the tumor initiation phase. CCR7 expression is regulated in vitro by extracellular conditions, namely nutrient deficiency, which may mimic areas inside tumors with high proliferation rates (33) and its activation is significantly increased after 48 and 72hr stimulation with CCL21 suggesting that the lymph nodes, where CCL21 is abundant, provide favorable conditions for the proliferation of metastatic thyroid cells once they migrate and arrest at these secondary sites. This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

CXCR7/CXCL12:
CXCR7 is a receptor for CXCL12, which is expressed in many cancer cell lines. It is produced by human thyrocytes (29) and is involved in cell migration and metastasis (62). Zhen et. al.
(62) showed, by immunohistochemistry, higher expressions of both CXCL12 and CXCR7 in PTC compared to benign tissue (non-malignant tissue 2 cm from the edge of the cancer specimen; n=79) and these expression patterns correlated significantly with lymph node metastasis. Additional work by others showed that down-regulation of CXCR7 in PTC suppressed cell growth, invasion and induced S-phase arrest as well as promoting apoptosis, indicating that it could be a promising target for therapeutic intervention (2).

CXCR1:
CXCR1 is one of two high affinity CXC chemokine receptors for CXCL8 and it is a major This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. infiltrate. There was no significant link between DARC and metastasis probably due to the propensity of PTC to metastasize via lymphatics rather than blood. It was concluded that larger tumors with decreased DARC expression may present more chemokines to the TME; this in turn could foster increased vascularization and tumor growth. This study clearly indicates that expression of these atypical CK receptors may be a key step in arresting tumor development however further research needs to be done to confirm this.

Decreased cell growth and increased apoptosis
Lymphocytes are frequently found within and surrounding thyroid tumors (82-83).
Their recruitment and trafficking is controlled by CC and CXC chemokines, particularly

Oncogenic changes inducing a pro-inflammatory response in thyroid carcinoma:
In PTC, genomic rearrangement and activating point mutations create dominantly transforming oncogenes the most common, and best studied being, RET/ PTC1 (89-90) and  This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. associated with local aggressiveness of neoplastic cells. This could induce and promote a more aggressive phenotype in PTC thereby increasing tumor spread outside the thyroid gland.

Chemokines and their receptors as potential screening tools and therapeutic targets in thyroid carcinoma
Enhancing anti-tumorigenic chemokine pathways and tumor-mediated immunity: As TAM are the most predominant leucocytes in tumors (20)

Conclusion
It is evident that research during the last three decades has found multiple links between This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.  This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.