The immune network in thyroid cancer

ABSTRACT

The immune system plays critical roles in tumor prevention, but also in its initiation and progression. Tumors are subjected to immunosurveillance, but cancer cells generate an immunosuppressive microenvironment that favors their escape from immune-mediated elimination. During chronic inflammation, immune cells can contribute to the formation and progression of tumors by producing mitogenic, prosurvival, proangiogenic and lymphangiogenic factors. Thyroid cancer is the most frequent type of endocrine neoplasia and is the most rapidly increasing cancer in the US. In this review, we discuss recent findings on how different immune cells and mediators can contribute to thyroid cancer development and progression.

KEYWORDS:

• Angiogenesis
• chemokines
• CXCL8/IL-8
• dendritic cells
• lymphangiogenesis
• macrophages
• mast cells
• neutrophils
• thyroid cancer
• T reg cells

Abbreviations

Ang	=	angiopoietin
ATC	=	anaplastic thyroid carcinoma
CTL	=	cytotoxic T lymphocytes
DCs	=	dendritic cells
DTC	=	differentiated thyroid carcinoma
EMT	=	epithelial-to-mesenchymal transition
γδ	=	gamma delta
GM-CSF	=	granulocyte-macrophage colony-stimulating factor
IDO	=	Indoleamine 2,3-dioxygenase
IFN	=	interferon
IL	=	interleukin
ILC	=	innate lymphoid cell
iNOS	=	inducible nitric oxide synthase
MC	=	mast cell
MDSC	=	myeloid-derived suppressor cell
MMP	=	metalloproteinase
NK	=	natural killer
NLR	=	neutrophil-to-lymphocyte ratio
PD-L1	=	Programmed Death-Ligand 1
PDTC	=	poorly differentiated thyroid cancer
PTC	=	papillary thyroid cancer
ROS	=	reactive oxygen species
SCF	=	stem cell factor
TAM	=	tumor-associated macrophage
TAMC	=	tumor-associated mast cell
TAN	=	tumor-associated neutrophil
TC	=	thyroid cancer
TCR	=	T cell receptor
TEM	=	Tie2-expressing monocyte
Th	=	T helper cell
TNF	=	tumor necrosis factor
Treg	=	regulatory T cell
VEGF	=	vascular endothelial growth factor.

Introduction

In 1863, Rudolf Virchow first observed leukocyte infiltration in neoplastic tissues and suggested a link between inflammation and cancer.¹ Epidemiological studies and animal models have demonstrated that cancer and inflammation are closely related. Several human cancers arise from inflammatory responses due to infectious agents. However, also tumors without a microbial background present signs of “smoldering inflammation”.¹ In addition, according to the cancer immunosurveillance theory, immune cells can recognize and eliminate transformed cells.² In fact, mice lacking recombination activating gene-1 (RAG-1) or RAG-2, devoid of B, T and NK lymphocytes, more efficiently developed spontaneous and carcinogen-induced tumors.³

Thyroid cancer (TC) is the most rapidly increasing cancer in the US. The association between chronic inflammation and TC has long been recognized. Indeed, a mixture of immune cells and mediators has been described in TC, associated with tumor progression and patient clinical outcome.⁴ The purpose of this review is to discuss recent findings investigating how the immune system can affect TC development and progression and its influence on patient clinical outcome.

Thyroid cancer (TC)

Epidemiology and risk factors

TC is the most frequent type of cancer of the endocrine system, accounting for approximately 90% of the endocrine malignancies and for 70% of deaths due to endocrine cancers. During the past 5 y TC incidence increased. Risk factors for TC include being female, having a history of goiter or thyroid nodule, a family history of TC, low-iodine diet, radiation exposure and obesity.⁵

Molecular pathogenesis

Thyroid follicular cells can give rise to well-differentiated papillary or follicular (DTC), poorly differentiated (PDTC) or anaplastic thyroid carcinomas (ATC). Papillary thyroid carcinoma (PTC) accounts for 80–85% of TCs, tends to invade lymphatic vessels and displays a high incidence of regional lymph node metastasis. Follicular thyroid carcinoma (FTC) comprises 5–10% of TCs. DTCs show favorable outcome with therapies. However, PDTC displays an intermediate differentiation and a more aggressive phenotype. ATC accounts for 2–5% of TCs and is one of the most aggressive and lethal human malignancies.⁵

PTC is associated with mutations in genes that encode for effectors involved in the mitogen-activated protein kinase (MAPK) and the PI3K-AKT pathway, including RET (encoding proto-oncogene tyrosine-protein kinase receptor ret), RAS genes (which encode small GTPases) and BRAF (encoding serine/threonine kinase B-raf).⁶ A comprehensive multiplatform analysis of 496 PTCs found that the most common genetic alterations were found in BRAF and RAS, suggesting their pivotal role in driving the PTC initiation.⁷ This study identified two major PTC subsets: BRAF-like PTCs are less differentiated, highly heterogeneous and display a high expression of genes downstream MAPK activation; RAS-like tumors are better differentiated, with a common follicular histology and more favorable outcome. FTC often displays the PAX8/PPARγ rearrangement, a fusion between paired domain transcription factor 8 and the peroxisome proliferator-activated receptor γ genes. Somatic mutations of PTEN and of PIK3CA have also been reported in FTC, and germline mutations of PTEN can result in FTC arising in the context of Cowden syndrome. PDTC and ATC carry activating mutations in PIK3CA, TP53 and CTNNB1 (β-catenin). AKT1 mutations have been reported in metastatic TC. In addition to these mutations, novel genetic alterations have been recently identified in TC, including ETV6/NTRK3, STRN/ALK and TERT.⁶

Soluble mediators in TC

Soluble mediators in cancer-related immune network include cytokines, chemokines, angiogenic and lymphangiogenic factors that influence several aspects of cancer growth and progression.

Cytokines

Cytokines are mainly produced by tumor-infiltrating immune cells, but are also secreted by thyroid follicular cells. Cytokines play a role in the pathogenesis of autoimmune thyroid disease and contribute to several aspects of TC initiation and growth.⁴

IL-1 promotes tumor growth through the induction of prometastatic genes (e.g., matrix metalloproteinases), angiogenic molecules (VEGF, CXCL8/IL-8, etc.) and cytokines (IL-6, TNF-α, and TGF-β).⁸ IL-1 stimulated thyroid cell proliferation and the in vitro production of IL-8.⁹ In a study performed on 115 patients affected by a variety of thyroid conditions and 30 controls, IL-1β serum concentrations allowed discrimination between individuals with atrophic thyroiditis and those with PTC, which displayed the highest and the lowest values, respectively.¹⁰

IL-4 and IL-13 are closely related cytokines that play overlapping and distinct roles in type 2 immunity. Primary sources of IL-4 include human Th2 cells, basophils and T follicular helper cells (Tfh).^11,12 Exposure to IL-4 or IL-13 induces alternative (M2) activation of macrophages and tumor-associated macrophages (TAMs) in TC displayed an M2-like phenotype.^13,14 Recently, a link between ionizing radiation (IR), IL-13 and oxydative stress-induced DNA damage in thyreocytes has been observed. Exposure of thyreocytes to IR caused IL-13 production, which stimulated ROS increase through the activation of p38 MAPK. This mechanism might be responsible for genetic instability and emergence of neoplastic clones in TC.¹⁵

IL-10 is a pleiotropic cytokine, which can favor a shift toward Th2 response in the tumor microenvironment.¹⁶ TAMs release IL-10,¹³ and tumor cells themselves also produce IL-10.¹⁷ In a study performed on peripheral blood from 30 patients with PTC and multinodular goiter (MNG) as well as 30 patients with MNG alone, IL-10 circulating levels were higher in PTC associated with goiter compared to MNG alone.¹⁸ Malignant epithelial cells purified from papillary, follicular and anaplastic human thyroid carcinomas produced in vitro IL-4 and IL-10, which increased the expression of the anti-apoptotic proteins Bcl-2, Bcl-xL, cFLIP and PED/PEA-15 in TC cells.^19,20

IL-24 belongs to the IL-10 gene family and is a potent inhibitor of cancer cell proliferation.²¹ IL-24 was induced by RET/PTC3 expression in thyreocytes of RET/PTC3 transgenic mice and it was involved in autocrine growth/survival of RET/PTC3-expressing thyroid cells supporting its role in early cellular transformation. However, its expression decreased in poorly differentiated mouse tumors, paralleling the loss of RET/PTC3 expression along with tumor progression, thus supporting a role for IL-24 as tumor suppressor factor.²²

IL-6 is a cytokine involved in the regulation of cell proliferation, survival and metabolism.²³ In an in vitro model of human TC and mast cell (MC) lines, TC-derived conditioned media (CM) induced upregulation of IL-6 in human MCs.²⁴ Moreover, IL-6 contributed to epithelial-to-mesenchymal transition (EMT) and stemness features of TC.²⁵

TGF-β is overexpressed in aggressive cancers.²⁶ Tumors induce dendritic cells (DCs) to secrete TGF-β, which promotes regulatory T cell (Treg) expansion. TGF-β-mediated suppression of antitumor T cells is due to Foxp1 overexpression in CD8⁺ T cells.²⁶ In a murine model of transgenic BRAF mouse, thyreocytes and TAMs produced TGF-β, which was responsible for the acquisition of EMT features and invasiveness of TC cells.²⁷ In addition, TGF-β overexpression in sections of human PTCs correlated with tumor invasiveness, regardless of BRAF mutations.²⁸

IL-17A is produced mainly by CD4⁺ Th17 cells but IL-17-producing CD8⁺ T cells (Tc17 cells) have also been identified in humans.²¹ Th17 and Tc17 cells have been found in many human cancers and IL-17 was associated with both protumor or antitumor responses.^21,29 The protumorigenic effects of IL-17 include the promotion of angiogenesis, survival and the recruitment of Myeloid-derived suppressor cells (MDSCs). TC patients display higher Th17 and lower Tc17 cell proportion in peripheral blood compared to controls. The serum concentration of IL-17 was increased in TC patients and correlated with the percent of circulating Th17 cells.²⁹ In addition, IL17RA polymorphisms negatively correlated with the development and the bilaterality of PTC in a study performed on 94 PTC patients and 260 controls in Korean population.³⁰

IL-21 is highly expressed, but not exclusively, by Tfh cells and Th9 cells.³¹ Th9 cells can directly induce tumor cell death or limit tumor growth by production of IL-9 and IL-21.³² In a case-control study performed on 615 TC patients and 600 controls in Chinese population, IL-21 promoter polymorphism was associated with increased risk of TC development.³³

All three classes of IFNs, type I (IFN-α/β), type II (IFNγ), and type three (IFN-λ/s), can induce apoptosis of tumor cells and control the circuits underlying cancer immunosurveillance.³⁴ IFNγ promotes Th1 cell differentiation and classical macrophage M1 polarization.¹³ Type I IFNs induce DC maturation, enhance cytotoxicity of CD8⁺ T and NK cells and decrease Treg immunosuppression.³⁵ In TC, type I and type II IFNs in vitro induced the expression of MHC-I molecules on human TC cell lines, thus impeding TC immunoevasion and potentiating TC susceptibility to immune destruction.³⁶

Chemokines

Chemokines are a family of more than 50 functionally related small molecules with chemoattractant and cytokine-like functions. In addition to inducing chemotaxis, chemokines can modulate angiogenesis and lymphangiogenesis.³⁷

Chemokines can be produced by TC cells, following the activation of the MAPK pathways by the RET/PTC, RAS and BRAF oncogenic drivers.^38,39 Thyroid cells produce several CXC chemokines, including CXCL1, CXCL8/IL-8, CXCL9, CXCL10 and CXCL11, in basal conditions and/or under the influence of specific stimuli.^{40, 41}

A wide variety of thyroid tumor cell lines derived from PTC and ATC release CXCL8/IL-8, in basal conditions as well as under inflammatory stimuli, such as IL-1 and TNF-α.^9,42 Exogenous induction of RET/PTC1 oncogene in primary normal human thyreocytes induced the expression of CCL2, CCL20 and CXCL8/IL-8 genes. Moreover, CCL20 and CXCL8/IL-8 were upregulated in clinical samples of PTC.³⁹ Muzza and co-workers compared CCL2 and CXCL8/IL-8 gene expression in tissues from PTCs, thyroiditis and normal thyroid. Interestingly, PTCs displayed the highest expression of CCL20 and CXCL8/IL-8 compared to normal tissues and thyroiditis, regardless the genetic lesion beard by the tumor or the presence/absence of thyroiditis. Moreover, CXCL8/IL-8 expression in thyroiditis was similar to normal tissues, suggesting a prominent role in cancer-related inflammation and a minor role in thyroiditis. However, no association was found between CXCL8/IL-8 expression and patient outcome.⁴³ In a model of orthotopic TC xenograft in nude mice, CXCL8/IL-8 was involved in tumor growth and progression, at least in part due to the activation of the NF-κB pathway.⁴⁴

In addition to TC themselves, also the tumor-infiltrating immune cells can be a source of chemokines. Indeed, MC-derived CXCL8/IL-8 in vitro induced EMT features and stemness in human TC cells.²⁵ Moreover, TAMs purified from PTC patients released CXCL8/IL-8, which was responsible for PTC cell invasion and metastasis in vivo.⁴⁵

PTC and ATC cell lines autocrinously produce CXCL1/GRO-α and CXCL10/IP10, in basal conditions and under inflammatory stimuli, thus self-sustaining proliferation and invasiveness.^{38,41, 46} In addition, MC-derived CXCL1/GRO-α and CXCL10/IP10 further increased TC cell proliferation through the engagement of CXCR2 and CXCR3 on TC cells.^24,38

CXCL12/SDF-1 and its novel receptor CXCR7 have been found in PTC patients.⁴⁷ Interestingly, CXCL12 expression was exclusively found in PTCs compared to other thyroid lesions and displayed high sensitivity, specificity, positive and negative predictive values. For these reasons CXCL12 has been proposed as a novel diagnostic marker for PTC.⁴⁸ From a functional point of view, the CXCL12/SDF-1-CXCR7 axis promoted in vitro TC cell line proliferation and invasion.⁴⁹ Also CCL20 was found to promote TC cell invasion and migration in vitro, presumably due to the activation of NF-κB-dependent upregulation of MMP-3.⁵⁰

We have recently found that the oncolytic adenovirus dl922-947 reduced CXCL8/IL-8 and CCL2 production by ATC cell lines.⁵¹ Interestingly, dl922-947 treatment impaired ATC-induced angiogenesis and favored the switch of tumor macrophages toward an M1 phenotype. These results indicate that dl922-947 treatment, along with its role in inducing TC cell death, impacts on ATC immune microenvironment.

Angiogenic factors

The formation of blood and lymphatic vessels is a complex process, requiring a finely tuned balance between stimulatory and inhibitory signals such as VEGFs, Angiopoietins (Angs), chemokines, oxygen sensors and many others.⁵²

Several TC-infiltrating immune cells can impact tumor angiogenesis and lymphangiogenesis (Fig. 1). For instance, MCs are a major source of proangiogenic (VEGF-A and -B) and lymphangiogenic (VEGF-C and -D) factors.⁵³ In addition, thyroid MCs modulated angiogenesis through the release of CXCL8/IL-8.²⁵ Human macrophages are also a major source of both angiogenic and lymphangiogenic factors ⁵⁴ as well as of several proangiogenic enzymes such as MMP-9, Cox-2 and iNOS.¹³ Tumor-associated NK cells produce VEGF-A and CXCL8/IL-8.⁵⁵ Tumor-associated DCs release VEGF-A, CXCL8/IL-8 and osteopontin.⁵⁶ Human eosinophils promote angiogenesis ⁵⁷ and eosinophilia can be observed in ATC patients.⁵⁸ Tregs play a role in maintaining tolerance in TC and drive tumor angiogenesis through the release of VEGF-A.⁵⁹ MDSCs promote tumor angiogenesis through the release of VEGF-A and MMP-9.⁶⁰ Tie2 TEM,⁶¹ human basophils ⁶² and human neutrophils are a source of proangiogenic factors, but their role in TC is unknown.

Figure 1. Hypothetical diagram depicting immune orchestration of tumor angiogenesis and lymphangiogenesis in TC. Immune cells infiltrating TCs can play two complementary roles at the same time. On one hand, several immune cells can modulate angiogenesis thus favoring tumor growth. On the other hand, TAMs and TAMCs, can play a dual role modulating not only angiogenesis but also lymphangiogenesis thus contributing to the formation of metastasis. The role of Tie2⁺ TEM, TAN and basophils (gray) in angiogenesis/lymphangiogenesis has been demonstrated in several human cancers and in chronic inflammatory disorders, but not yet in human TCs.

Immune cells in TC microenvironment

The tumor microenvironment, which consists of immune cells, fibroblasts, blood and lymphatic vessels, endothelial cell progenitors and extracellular matrix components (ECM), plays a critical role in tumor initiation/progression. Normal tissue microenvironment can suppress malignancy, while certain pathogenetic tissue features can induce tumor progression.^63,64

TAMs: Tumor-associated macrophages

Macrophages are the most represented leukocytes in solid tumors and are highly plastic cells.⁶⁵ Under the influence of IFNγ, macrophages undergo classical M1 polarization, characterized by an immunostimulatory phenotype. In contrast, IL-4 or IL-13 induces alternative M2 phenotype, which promotes tumor angiogenesis and suppresses immune responses.¹³

The majority of studies investigating the role of TAM in TC correlate TAM infiltration with clinico-pathological features. Indeed, TAMs are increased in TC and positively correlate with de-differentiation.⁶⁶ In PTCs, TAMs correlated with lymph-node metastasis,⁴⁵ larger tumor size ⁶⁷ and reduced survival.⁶⁸ In PDTC, TAM density correlates with capsular invasion and extrathyroid extension.⁶⁶ TAMs represent more than 50% of immune cells in ATCs forming a “microglia-like” in close contact with cancer cells.⁶⁹ In diffuse sclerosing variant of PTC, M2-like macrophages can be found in lymphatic emboli and correlated with tumor cell lymphatic invasion.⁷⁰

In a murine model of transgenic BRAF^(V600E)-induced thyroid carcinogenesis, tumors displayed a high TAM infiltration due to the increased expression of CSF-1 and CCL2 by tumor cells. In this model, TAMs displayed an M2-like phenotype, characterized by high production of Arginase-1, CCL22 and IL-10 and low levels of IL-12. TAM depletion reduced tumor growth in established tumors. In addition, Csf–/– BRAF transgenic mice displayed a reduction in tumor growth.¹⁴ Accordingly, TAMs purified from human PTC displayed a higher expression of IL-10 and CD206 compared to peripheral blood monocytes ⁷¹ and promoted invasiveness of TC cell lines in vitro through to the production of CXCL8/IL-8.⁴⁵

Dendritic cells (DCs)

Tumor-infiltrating DCs generally display an immature phenotype, with impaired antigen presentation ability.⁷² S100⁺ (mature and immature), CD1a⁺ (immature), and CD83⁺ (mature) DCs are increased in human PTC compared to normal thyroid tissue.⁷³ CM obtained from primary cultures of PTC induced chemotaxis of peripheral blood monocyte-derived DCs. Hepatocyte Growth Factor (HGF) enhanced this chemotactic activity through the engagement of the receptor Met on TC cells.⁷⁴ PTCs expressed the DC chemotactic molecule MIP-1α and DCs expressed CCR6, thus suggesting a role for TC cells in recruiting DCs.⁷⁵ PTC displayed a higher CD1a⁺ DC infiltration compared to FTC and adenomas.⁷⁵ In contrast, a reduced or absent DC infiltration in PDTC and ATC was described in comparison to DTC.⁷⁶

TAMCs: Tumor-associated mast cells

Beyond their pivotal roles in allergy, defense against parasites and immune regulation,⁷⁷ MCs are involved in tumor biology.⁷⁸ In a cohort of 96 PTC patients, tumor-associated MC density was higher than in healthy thyroids and correlated with tumor extracapsular extension.²⁴ A higher MC density was also found in FTC compared with adenomas and correlated with extracapsular extension.⁷⁹ TC cell line CM induced in vitro MC chemotaxis through the release of VEGF-A,²⁴ which activated the VEGFRs on human MCs.^53,80 In addition, TC cells activated MCs to release cytokines (IL-6, TNF-α, GM-CSF) and chemokines (CXCL10/IP10 and CXCL1/Gro-α). In turn, MCs promoted TC cell proliferation through CXCL1/GROα and CXCL10/IP10. In an in vivo model of TC xenograft, MCs were recruited at the tumor site and accelerated tumor growth, enhancing tumor vascularization and cell proliferation. These effects were inhibited by cromolyn, a MC inhibitor.²⁴ MCs also induced TC cell EMT mainly through the release of CXCL8/IL-8, thus sustaining TC invasiveness and stem cell features.²⁵

TANs: Tumor associated neutrophils

Neutrophils participate to the early phases of inflammation and resistance against extracellular pathogens.⁸¹ However, a role for neutrophils in cancer has been envisaged. Indeed, a new paradigm has been proposed, by which mouse neutrophils can be polarized toward an antitumor N1 or a protumor N2 phenotype in response to signals derived from the microenvironment.⁸² In humans, the ratio between peripheral blood neutrophil and lymphocyte count (Neutrophil-to-Lymphocyte Ratio—NLR) has been proposed as an index of systemic inflammation and has been found to be associated with tumor development.⁸³ A higher NLR was associated with a larger tumor size and higher risk of recurrence in TC patients, but failed to distinguish patients with benign or malignant nodules.⁸⁴ In contrast, an increased NLR has been found in TC compared with benign lesions and healthy controls.⁸⁵ No correlation was found with patient disease-free survival or risk of occult metastasis.⁸⁶ In a recent study, NLR failed to discriminate benign and malignant lesions.⁸⁷ Thus, the diagnostic and prognostic significance of NRL in TCs remains uncertain. No studies are so far available investigating the occurrence, roles and prognostic significance of tumor-associated neutrophils in TC.

MDSCs: Myeloid-derived suppressor cells

Cancer development is accompanied by the expansion of a heterogeneous population of myeloid cells, namely MDSCs, with immunosuppressive and cancer-promoting properties.⁸⁸ MDSCs are classically distinguished between Monocytic (Mo-MDSCs) and Granulocytic (G-MDSCs) subsets based on morphological and phenotypical aspects.⁸⁸ Peripheral blood MDSC levels were higher in patients with ATC compared to healthy controls and correlated with the serum level of IL-10, thus suggesting a correlation between MDSCs and systemic immunosuppression.⁸⁹ The only study investigating the significance of TC-infiltrating MDSCs failed to find an association between MDSC density and patient survival.⁹⁰ Indeed, in a wide cohort of histological samples from 398 TC patients (253 PTCs and 13 FTCs) MDSCs were identified as CD11b⁺ and CD33⁺ cells. Intratumoral MDSC count did not correlate with patient clinic-pathological features.⁹⁰ Unfortunately, the identification of human MDSCs is complicated by the lack of specific marker. Moreover, it has been suggested that several human MDSC phenotypes can be endowed with suppressive activity.⁹¹ Thus, further studies are required to better define the roles and functions of different MDSC phenotypes in TC.

NK: Natural killer cells

Natural killer (NK) cells are a family of innate immune cells, which play a central role in antiviral immunity and tumor immunosurveillance. Subtypes of human NK cells have been described on the basis of relative surface expression of CD16 and CD56. CD56^dim CD16⁺ NK cells display a higher cytotoxic activity, whereas CD56^bright CD16^−/low NK cells are more efficient in cytokine production.⁹² Flow cytometry analysis on tissue samples and peripheral blood from PTC and MNG patients showed that tumor-infiltrating NK cells were increased in PTCs compared to goiters and healthy thyroids, whereas no differences were found in peripheral blood NK cells. In PTC patients, NK cell infiltration negatively correlated with disease stage.⁹³ A flow cytometry analysis performed on blood and tissue samples from patients with PTC, MNG and healthy controls displayed an increased infiltration of the immunoregulatory subset of NK cells CD56^bright in PTC samples compared to MNG. CD56^bright immunoregulatory NK cells inversely correlated with the disease stage, whereas cytotoxic NK cells positively correlated with the disease stage in PTC patients. These findings suggest that a modulation of cell phenotype can occur in the TC microenvironment.⁹⁴

From the functional point of view, ATC cell lines were sensitive to lysis by PBMC-derived NK cells.⁹⁵ NK cell-mediated lysis of ATC was dependent on the expression of the activating receptor NKG2D on NK cells and its ligands ULBP2/5/6 on ATC cells. Indeed, ATC cell lines and TC derived from human fine-needle aspiration (FNA) samples from ATC patients expressed ULB2/5/6, whereas non-malignant thyroid tissue did not. Interestingly, ATC cell lines induced NK cell migration through the activation of the CXCL10-CXCR3 axis. Flow cytometry analysis of NK cells from FNA and peripheral blood of ATC patients displayed a suppressed phenotype, characterized by a lower percentage of CD56^dim cells and CXCR3⁺ cells and a reduced NKG2D expression, compared to peripheral blood NK cells. PGE₂ was identified as the main candidate responsible for ATC-mediated NK cell suppression.⁹⁵ This study indicates that ATC could be a target for NK cell-based adoptive cell therapy, paving the way to novel immune-mediated therapeutic approach to ATC.

A recent experimental study addressed the role of IL-12 immunotherapy in PTC. In a knock-in mouse model Braf oncogene was expressed under the control of the thyroid peroxidase promoter (LSL-Braf^V600E /TPO Cre mice). These mice spontaneously developed thyroid tumors at 5 weeks of age. IL-12 gene therapy and recombinant IL-12 reduced tumor growth, restored the follicular architecture of the gland and improved the survival of tumor-bearing mice. Interestingly, IL-12 improved the cytotoxic activity of CD8⁺ T cells and NK cells, and increased the infiltration of M1 macrophages compared to alternative M2 macrophages within the tumors.⁹⁶

Natural killer T cells (NKT), γδ T cells and innate lymphoid cells (ILCs)

NKT cells are a heterogeneous lymphoid population, which recognizes lipid antigens presented by CD1d. NKT are important for tumor immunosurveillance. Two NKT subsets have been identified: type I induce lysis of tumor cells directly via a perforin/granzyme B-mediated mechanism or indirectly via the activation of NK cells and DCs; type II show immunosuppressive activity by IL-13 secretion.⁹⁷ NKT subsets have not been characterized in TC.

γδ T lymphocytes, expressing a γδ TCR, are not MHC-restricted and do not recognize peptide antigens. The contribution of γδ T cells in tumor immunosurveillance is controversial. Zitvogel's group demonstrated that IL-17-producing γδ T cells play a prominent role in chemotherapy-induced anticancer immune responses.⁹⁸ The role of tumor-infiltrating γδ cells in TC is still unknown.

Innate Lymphoid Cells (ILCs) lack TCR but functionally resemble effector T cells. ILCs include three subsets of cytokine-producing helper cells: group 1 ILCs produce IFNγ and include conventional NK cells; group 2 produces Th2-type cytokines (e.g. IL-4, IL-5 and IL-13); group 3 ILCs comprises several distinct cell subsets.⁹⁹ In a mouse model of melanoma, ILCs promote tumor rejection.¹⁰⁰ Studies are required to understand the role of ILC subsets in TCs.

CD8⁺ cytotoxic T cells

CD8⁺ cytotoxic T lymphocytes (CTLs) recognize and attack tumor cells expressing tumor antigens.¹⁰¹ In a wide immunohistochemical characterization of the immune cell infiltrate in patients with chronic lymphocytic thyroiditis concurrent with DTC, a high CD8⁺ T lymphocyte infiltration was associated with improved disease free survival.⁹⁰ By contrast, in a recent study conducted in a wide cohort of DTC patients, including papillary and follicular subtypes, immunohistochemical analysis of tumor samples revealed that combined enrichment of CD8⁺ cells and Cox-2 overexpression correlated with the highest risk of disease relapse. In the majority of tumor samples analyzed (68%), CD8⁺ cells were granzyme B negative, reflecting a state of anergy.¹⁰²

A low intratumoral CD8⁺/Foxp3⁺ ratio was found in human Braf^V600E PTC, associated with increased expression of the immunosuppressive molecules Arginase-1, Indoleamine 2,3-dioxygenase (IDO) and PD-L1. The latter findings suggest a Braf-driven tumor-promoting microenvironment.¹⁰³

CD4⁺ cells

CD4⁺ T helper (Th) lymphocytes are a heterogeneous population. Th1-mediated immunity is generally considered as antitumoral, while polarized Th2 and/or Treg activity is believed protumorigenic.¹⁰⁴ This simplistic view is complicated by the plasticity of Th differentiation, which can be modulated by the microenvironment.

Increasing evidence highlights the antitumor potential of CD4⁺ Th1 cells in murine models and humans.¹⁰⁵ However, in TC, the extent of tumor-infiltrating CD4⁺ cells does not appear to predict patient outcome.^90,102 No differences were found between PTC and MNG patients, with respect to tissue or peripheral blood CD4⁺ cell frequencies.¹⁸ Interestingly, a double negative CD4⁻ CD8⁻ lymphocyte population has been recently identified in TC. This double negative lymphocyte population was identified as the dominant cell type in PTC and was more abundant in PTC than in thyroid autoimmunity.¹⁰⁶ These cells ex vivo released IFNγ and IL-17, thus resembling effector cells.

Tregs shut down antitumor immune response via the production of IL-10 or by expressing immunosuppressive molecules, including CTLA4, GITR, PD-1 and stimulate angiogenesis through the production of VEGF-A.¹⁰⁷ Moreover, increased PD-1⁺ T cells in metastatic lymph nodes correlated with a more aggressive TC.¹⁰⁸ In a small PTC patient dataset, Foxp3⁺ Tregs and VEGF, evaluated by immunohistochemistry, were found in PTC samples and Treg infiltration correlated with disease stage and lymph node metastasis.¹⁰⁹ A higher Treg density was also observed in PTC samples compared with nodular goiter, and positively correlated to the stage of the disease.⁹³ Accordingly, in a study performed on 124 tissue samples from PTC microcarcinomas, high infiltration of Foxp3⁺ Treg cells was associated with aggressive features of PTC microcarcinoma.¹¹⁰ Moreover, increased Foxp3⁺ and reduced CD3⁺ tumor-infiltrating lymphocytes correlated with IDO expression. Similarly to PD-L1, IDO is overexpressed in different tumors and is associated with activation of Foxp3⁺ Tregs and downregulation of cytotoxic cellular immunity in the tumor microenvironment.¹¹⁰

A higher percentage of Foxp3⁺ T cells and ICOS⁺ Treg cells were found in tissues, but not in peripheral blood, of PTC patients with MNG compared to MNG alone.¹⁸ In PTC plus MNG, tissue ICOS⁺ Foxp3⁺ T cells were increased in advanced stages and metastatic tumors. Tissue ICOS⁺ Foxp3⁺ T cell numbers correlated with tissue plasmocytoid DCs, which favor an immunosuppressive microenvironment.¹⁸

CD4⁺ IL17⁺ T cells (Th17) cells can exert protumor or antitumor functions depending on tissue microenvironment.²¹ Only one paper examined the prevalence and distribution of Th17 cells in TC samples. In peripheral blood and tissue samples of PTC patients, increased Th17 levels were found compared to healthy controls, whereas the percentage of CD8⁺ IL-17⁺ T cells (Tc17) in the peripheral blood was reduced. The frequency of peripheral blood Th17 cells was positively correlated with the IL-17 serum level, while no correlation between serum level of IL-17 and Tc17 cells was found. Peripheral blood Th17 cells inversely correlated with tumor size.²⁹

Tfh cells are characterized by the expression of CXCR5 and Bcl6. Tfh cells promote immunoglobulin production by B lymphocytes through the release of IL-21.³¹ Tfh cells can also be involved in the immune response to human cancer.¹¹¹ Studies investigating the presence and functions of Tfh subsets in TC are still missing.

IL-9-producing CD4⁺ helper T cells (Th9 cells) are a subset of CD4⁺ helper T cells with proinflammatory functions and anticancer properties in vivo.¹¹² The release of IL-9 has been proposed to account for the anticancer efficacy of these cells. Moreover, Th9 cells release IL-21, which promotes the production of IFNγ and tumor elimination by CD8⁺ T cells and NK cells.¹¹³ The relevance of Th9 cells in thyroid oncogenesis remains to be elucidated.

Fig. 2 schematically illustrates a hypothetical immune network in TC.

Figure 2. Hypothetical scheme of immune network in TC. The immune network in thyroid cancer is a complex and dynamic system characterized by multiple interactions between tumor cells and a wide spectrum of immune cells. Tumor-infiltrating immune cells interact each other and with tumor cells in a complex synergistic and opposite manner still largely unknown. Several cytokines, chemokines, angiogenic and lymphangiogenic factors, derived from both immune cells and tumor cells, enrich the complexity of the inflammatory tumor microenvironment. The roles of Tie2⁺ TEM, NKT cells, TAN, Tfh cells, γδ T cells and Th9 cells (gray) have been demonstrated in several other human cancers or are under investigation in human TCs. Protumor or antitumor activities of Th17 and Tc17 cells are context dependent (dashed line).

Conclusions and perspectives

During recent years the incidence of TC has increased and it now represents approximately 90% of all endocrine malignancies. While the prognosis of DTC is favorable, PDTC and ATC are among the most lethal human malignancies. The tumor microenvironment plays a critical role in tumor initiation and progression. A normal tissue microenvironment can suppress malignancy, while certain pathogenetic tissue features are critical for tumor development.^63,54 A myriad of immune cells are present in the TC microenvironment and in some cases are associated with patient outcome.^66,68 However, only studies assessing the functional role of TAMs and TAMCs in the TC microenvironment have provided evidence for their protumorigenic role.^14,24 The majority of these studies were merely qualitative and quantitative analyses of immune cells. Moreover, the presence and functional role(s) of subsets of immune cells (e.g., neutrophils, NKT and γδ T cells, ILC, Tfh, Th9), known to be relevant for tumor initiation/growth, have not yet been investigated in the microenvironment of different types of TC.

Genetic alterations in the RET/PTC-RAS-BRAF-MAPK or in the PI3K-AKT signaling pathways, which represent “driver” mutations (i.e., required for cancer cell growth/survival) have been identified in TC.⁶ Driver mutations represent prognostic biomarkers and promising new targets for the treatment of TC. Kinase inhibitors targeting RET or BRAF can induce either stable disease or partial responses in PTC and FTC metastatic patients, but are ineffective in ATC.¹¹⁴ Thus, novel therapies for these TC histotypes are needed. A possible therapeutic approach is represented by drugs targeting tumor stromal cells including immune cells and/or the interactions between immune cells and cancer cells.

Angiogenesis and lymphangiogenesis play an essential role in tumor growth and in the formation of metastasis. TC cells, TAMs, TAMCs and other immune cells are a major source of proangiogenic and lymphangiogenic molecules.^53,54 Interestingly, immune cells and tumors can produce pro- and anti-angiogenic isoforms of VEGFs.⁶² A deeper knowledge of the balance between these isoforms could lead to the discovery of novel targets that can be manipulated to block TC growth.

Most of the in vivo experimental studies of TC have been performed with athymic nude mice models. Functional studies on MCs conducted with these models have demonstrated a protumorigenic role of MCs in human TC.^24,25 These results suggest the possibility that targeting these cells and/or their mediators could be useful to interfere with TC proliferation, angiogenesis, invasive ability and stemness.^24,25 Genetically modified mouse models that recapitulate multistep TC tumorigenesis should be employed to better characterize the role of different immune cell subsets in TC. For instance, the protumorigenic role of macrophages in TC has been established by using thyroid-targeted BRAF^(V600E) mice in which macrophage deletion has been obtained by either pharmacological or genetic tools.¹⁴ Targeting macrophages has been shown effective for blocking tumor growth.¹¹⁵ This experimental approach could be also useful in TC.

TC cells and immune cells are a major source of several protumorigenic and proangiogenic cytokines/chemokines.⁵³ Targeting these mediators could be exploited to block TC growth, since this strategy has been already developed for other tumors.¹¹⁶ Moreover, boosting anticancer immune responses by blocking immunosuppressive molecules (TGF-β, IL-10, CTLA-4, PD-1 and PD-L1) expressed either by cancer cells or by tumor-infiltrating immune cells ¹¹⁷ and cytolytic viruses approaches ⁵¹ appear promising therapeutic strategies in different tumors, including TC.

In conclusion, insights into the molecular mechanisms regulating immune networks as well as angiogenesis/lymphangiogenesis in different types of TC could result in the discovery of diagnostic and prognostic markers and novel targets in this common endocrine malignancy.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors apologize to the many authors who have contributed importantly to this field and whose work has not been cited due to space and citation restrictions. We thank Fabrizio Fiorbianco for the figures.

Funding

Supported in part by grants from Regione Campania CISI-Lab Project, CRèME Project, and TIMING Project.