Thyroid carcinoma (TC) is the most common endocrine malignancy worldwide, and its global incidence rate has been growing over the last three decades. Before the 1990s, the incidence of TC in the United States was relatively stable at approximately 5/100,000. However, its incidence had tripled (15/100,000) by 2014 (1). The incidence of TC in Canada from 2012–2016 was reportedly 17.4/100,000, a figure closely similar to that of the United States (2). Such dramatic increases in the incidence of TC have also been observed in many other countries, including 29 European countries and China (3, 4). According to Global Cancer Statistics, 586,202 new TC cases were reported worldwide in 2020, amounting to an incidence of 13.2/100,000 (5). However, the mortality rate of TC has remained relatively low and stable (<1/100,000); (1–4, 6). The etiology of TC remains unclear. Exposure to ionizing radiation during childhood is considered to be risk factor that is most and closely associated with TC (7). Moreover, other factors, such as chromosomal (genetic) alterations and obesity, are reportedly associated with the occurrence and development of TC (8, 9).

Based on histological features, TC is mainly divided into the following four types: papillary thyroid carcinoma (PTC); follicular thyroid carcinoma (FTC); anaplastic thyroid carcinoma (ATC); and medullary thyroid carcinoma (MTC); (10). PTC represents the most common differentiated subtype of TC, and its incidence has reportedly increased over the past decade (11). Most PTC patients receive favorable prognoses involving 10-year survival rates ranging from 93–97% (12, 13). The 10-year survival rates for FTC and MTC are 85% and 75%, respectively, and thus worse than that for PTC (12). ATC begets the worst prognosis, with a 10-year survival rate of 14% and a median survival of six months (12, 14). Although the majority of TCs, which remain indolent, are associated with an innocuous clinical course, some cases manifest aggressive behavioral patterns, such as metastasis and recurrence, resulting in poorer prognoses. The recurrence rate of PTC following conventional treatment is reportedly as high as 28% (15). Metastasis in TC, which mostly involves the cervical lymph system, acts as an unfavorable factor which leads to poor prognoses (16). The 8th edition of the American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging system identifies sex, age, N classification, pathological subtype, and radioactive iodine avidity as some of the major factors affecting the prognosis of PTC (17). In addition, the 10-year cancer-specific survival rate for IVb stage PTC patients over 55 years old presenting with extensive extrathyroidal extension is reportedly 33.3% (18). The limited number of alternative therapeutic strategies that have been used against surgically inoperable and radioiodine-refractory TC have not been successful at improving the survival of TC patients. Therefore, development of new intervention strategies against TC are felt to be warranted.

The tumor microenvironment (TME), which is composed of the extracellular matrix, stromal cells, immune cells, and some secreted factors, plays an important role in TC (19). Of these, tumor immune cell infiltration has been demonstrated to be closely associated with TC progression and prognosis (20, 21). Tumors may be categorized as immunologically hot or cold types, which definitions are based on the degree of immune infiltration in the TME (22). The hot type is characterized by high tumor immunity, indicating association with a stronger immune response and a better survival outcome. By contrast, the TME of a cold tumor is much more immunosuppressive, responding poorly to treatment (23). Various immune cells that infiltrate tumors may either accelerate or decelerate tumor progression, depending on population and activation status (24). Tumor-promoting immune cells, including dendritic cells (DCs), macrophages, and monocytes, may promote tumor growth, metastasis, and drug resistance in the TME, whereas antitumor immune cells, namely B cells, natural killer (NK) cells, and CD8⁺ T cells, suppress tumor cell proliferation, invasion, adhesion, and metastasis. DCs, which play a key role in antigen presentation and cytokine secretion, are increased in TC (25). In TC, infiltrating DCs activate T cells and NK cells via tumor antigen presentation. Moreover, these infiltrating DCs are known to produce some immunosuppressive cytokines that inhibit immune responses (26). Tumor-associated macrophages (TAMs), which originate from monocytic precursors, infiltrate into the tumor stroma, and facilitate macrophage polarization from the antitumor M1 phenotype to the tumor-promoting M2 phenotype, thereby aggravating TC growth and metastasis (27–29). Monocytes that differentiate in the bone marrow are mainly responsible for inflammation. Increased monocyte infiltration in a mouse TC model promotes tumor progression by elevating immune-related gene and cytokine expression (30). These results show that immunosuppressive cells in the TME of TC can strengthen the ability of tumor cells to fight immune response, thereby enhancing immune escape. Therefore, investigating immune cell infiltration in relation to regulatory mechanisms of TC may be vital for developing new immunotherapeutic strategies that improve TC patient outcomes.

ATP-binding cassette (ABC) transporters are a large superfamily of membrane proteins that acquire energy from ATP hydrolysis to facilitate the translocation of heterogeneous substrates (31). A total of 49 human ABC transporters are grouped into seven distinct subfamilies as follows: ABCA; ABCB; ABCC; ABCD; ABCE; ABCF; and ABCG (32). All ABC transporters are composed of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs); (33). ATP hydrolysis which occurs at the NBDs, induces conformational changes in TMDs, which, in turn, facilitate inward or outward transportation of specific substrates across the membrane (34). ABC transporters, which are ubiquitous, have been found to be associated with diverse biochemical and physiological processes, such as maintenance of cellular environments, protection from harmful materials, and modulation of drug kinetics (35–37). ABC transporters reportedly play vital roles in numerous carcinomas (38). For example, ABCG1, which is overexpressed in clear cell renal cell carcinoma, has been found to be associated with overall patient survival, indicating its potential as a diagnostic and prognostic biomarker in clear cell renal cell carcinoma (39). ABCB1 and ABCG2 reportedly play a critical role in the prevention of chemo-resistant liver cancer stem cell death in hepatocellular carcinoma (40). Differentially expressed ABCC2 and ABCC5 are considered as diagnostic biomarkers of lung adenocarcinoma, while ABCC2, ABCC6, and ABCC8 are reportedly associated with its prognosis (40). Many ABC transporters which are differentially expressed between colorectal cancer (CRC) and non-neoplastic control tissues, may be linked to both the onset and treatment outcomes of CRC (41). The functions of ABC transporters known to be involved in immunity against infection and cancer have been summarized and reviewed, providing a broader understanding of the effects of ABC transporters on immunity to viruses and tumors (42). However, the prognostic roles and immune-related mechanisms of ABC transporters in TC remain unclear.

In this study, we aim to predict the prognostic implications and immune cell infiltration related features of ABC transporters by performing comprehensive analyses followed by validation via RT-PCR. Here, we attempt to provide a deeper insight into the immune cell infiltration patterns seen in TC as well as to identify some potential prognostic and immunotherapeutic targets in TC.

It has become increasingly evident that ABC transporters play important roles in the immunomodulation of tumors. For example, multidrug resistance protein 1 (MDR1), encoded by ABCB1, is expressed in cytotoxic T lymphocytes and NK cells, and mediates immune responses (56). A high level of the MDR1⁺ immune cell infiltrate, mostly comprising M2 macrophages, was confirmed as an independent prognostic factor associated with poor prognoses for epithelial ovarian cancer (57). ABCA1 regulates the immune sensitivity of osteosarcoma cells. Moreover, the ABCB1: ABCA1 ratio was reportedly upregulated in osteosarcoma cells and positively correlated with a higher probability of relapse (58). ABCA8 expression was downregulated in stomach adenocarcinoma and was positively associated with six types of infiltrated immune cells, particularly M2 macrophages (59). Major histocompatibility complex class I (MHC-I) molecules play a vital role in immune surveillance as well as in the presentation of antigen peptides on the cell surface (60). The heterodimer of transporter associated with antigen processing (TAP) transports antigenic peptides and provides peptides to MHC-I molecules (61, 62). TAP blockade in DCs reportedly impaired classic MHC-I presentation for CD8⁺ T cell priming (63). Moreover, low TAP1/TAP2 expression led to the overexpression and efficient presentation of the antigen preprocalcitonin in lung carcinoma, as recognized via tumor-specific cytotoxic T lymphocytes (64). ABCC8 is considered a prognostic risk factor which is closely related to the LUAD microenvironment (65). Single-nucleotide polymorphisms (SNPs) in TAP1 and TAP2 affected their expression and were associated with cervical cancer in the Chinese Han population (66). ABCD3 expression, which is considered as an independent prognostic factor of CRC, was decreased in CRC tissues and associated with the overall survival of CRC patients (67).

Previous studies have demonstrated that altered expression levels of some ABC transporters were associated with their molecular roles in TC. ABCA9 expression was upregulated by hsa_circ_0039411 sponging miR-1179, leading to enhanced oncogenic properties in PTC (68). ABCB5 expression was reported to be associated with larger tumor size in PTC (69). Moreover, in the solid variant of PTC, high ABCC1 expression was associated with larger tumor size, while high ABCG2 expression was correlated with lymphovascular invasion (70). ABCG2 expression was also found to be closely related to the induction of EMT in PTC (71). High ABCC2 expression was observed in advanced stage MTC (72). In ATC, high expression levels of ABCB1, ABCC1 and ABCG2 observed in several cell lines and tissues, were associated with cancer drug resistance (73). However, the prognostic and immunotherapeutic value of ABC transporters in TC has not been well characterized. Therefore, the current study aimed to explore the prognostic and immune related value of ABC transporters in TC, leading to the provision of new directions and targets for its treatment.

In this study, data of TC and normal thyroid tissues from TCGA were analyzed to screen out hallmark ABC transporters, which are differentially expressed and associated with the prognosis for TC. Based on the results, ABCA8, ABCA12, ABCB6, ABCB8, and ABCC10 were selected as hallmark ABC transporters in TC. Of these, the expression of ABCA8, ABCB6, and ABCB8 was down-regulated in TC, compared with those in normal thyroid tissues, while that of ABCA12 and ABCC10 was up-regulated. Usually, up-regulation of oncogenes and down-regulation of anti-oncogenes affect many behavior patterns of malignant tumor cells, including metastasis and immune resistance. Our results revealed that ABCA8, ABCB6, and ABCB8 may inhibit the malignant progression of TC. Conversely, ABCA12 and ABCC10 may promote the occurrence and growth of TC. Since these are differentially expressed in TC and closely associated with the prognoses for TC, we selected these five as hallmark ABC transporters in TC for subsequent analyses.

It is increasingly becoming evident that immunotherapies play a vital role in tumor treatment, wherein the efficacy of immunomodulation depends mainly on immune cell infiltration. Based on our results, the five hallmark ABC transporters exert a variety of effects on immune cell infiltration in TC. Firstly, the expression of five hallmark ABC transporters was associated with two different TC subsets (hot or cold), indicating that the five hallmark ABC transporters may be useful for converting immunosuppressive (cold) TCs into immunosensitive (hot) ones. Moreover, our results showed that all five hallmark ABC transporters were associated with Th2 cells, Tcm cells, Treg cells, monocytes, and DCs, which revealed that their roles in immune cell infiltration-related immunoregulation were similar. In addition, the specificity of the transporters in regulating Th1 cells, Tfh cells, CD8⁺ T cells, NK cells, M1 macrophages, and B cells demonstrated the diversity of their roles. Our results also indicated that multiple immunomodulators, such as KDR, MHC molecules, VTCN1, CD274, TGFBR1, TNFSF18, and TNFSF15, were closely associated with these five hallmark ABC transporters. KDR, also known as vascular endothelial growth factor receptor 2 (VEGFR2), is the main receptor of VEGF signaling (74). Activation of KDR, which promotes endothelial cell mitogenesis and vascular permeability, plays a vital role in the induction of tumor angiogenesis (75). The VEGF-KDR signaling pathway was found to play an immunosuppressive role in TME and immune effector cell activity (76). MHC molecule expression is known to mediate immune escape mechanisms in tumors (77). VTCN1, also termed B7-H4, reportedly inhibits T cell proliferation and cytokine secretion, thereby negatively regulating T cell immune response, and positively regulating immune escape (78). CD274, also commonly referred to as programmed cell death 1 ligand 1 (PD-L1), is a ligand of PD-1, which is expressed on many immune cells. The PD-1/PD-L1 axis, which is exploited by tumor cells, may inhibit immune response and block immune cell activation (78). Cancer cells generate multiple factors, including TGF-β1, to create an immune inhibitory environment and evade T cell surveillance (79, 80). As the irreplaceable receptor of TGF-β1, TGFBR1, which is observed in different tumor types, participates in tumor immunological reactions (81). Tumor necrosis factor (TNF) superfamily ligands, such as TNFSF15 and TNFSF18, exert diverse modulatory effects by influencing immune responses and impacting immune cells (82–84). Moreover, chemokine–chemokine receptor interactions regulate immune cell recruitment into tumors and the stimulation of immune response (85). Our results demonstrated that the immune regulation by hallmark ABC transporters was partly mediated via chemokine ligands and receptors.

The presence of complex regulatory networks that affect almost all molecular processes at both intracellular as well as TME levels, is well known. The results of the analysis conducted on correlated genes, miRNAs, and TF targets showed that each hallmark ABC transporters possessed a unique feature and a distinctive regulatory pattern. The constructed regulation network and enrichment of these five transporters showed that the genes, which were most correlated with the five hallmark ABC transporters, were closely associated with transmembrane transporter activity and ABC family protein-mediated transport. In addition, multiple cancer processes and cancer-related pathways, including RTK, RAS/MAPK, PI3K/AKT, and EMT, were partly activated or suppressed by these five hallmark ABC transporters. These findings indicated that these five hallmark ABC transporters may modulate immune cell infiltration patterns by altering the expression levels of correlated genes, regulating transmembrane transporter activity, and influencing cancer-related pathways. However, the exact regulatory mechanisms have not yet been fully elucidated and further discovery and validation are felt to be required.

The known risk factors for TC include female sex, obesity, smoking status, radioactive iodine exposure history, and family genetic history (86–88). However, these recognized causative factors do not fully explain the increasing incidence of TC. Recent studies have demonstrated the influence of anthropogenic environmental chemical factors on TC (89). For example, individuals with higher cadmium exposure were observed to be more susceptible to TC (90). Environmental radiation exposure, such as that due to the Chernobyl accident, led to radiation dose-associated DNA double-strand breaks, subsequently resulting in PTC growth (91). Artificial light at night has also been found to be positively associated with TC incidence (92). Exposure to multiple essential microelements, including manganese and strontium, were positively associated with capsular invasion, multifocality, and tumor stage of PTC (93). Bisphenol A altered endocrine function and partly facilitated EMT in PTC (94). In this study, we focused on elucidating the associations between TC-associated chemicals and the five hallmark ABC transporters. We indicated that these chemicals exerted significant effects on the expression of these five hallmark ABC transporters in TC. The methylation and mRNA expression levels of these five hallmark ABC transporters were mostly affected by bisphenol A and vincristine. The findings of the present study are consistent with those of previous studies. Therefore, more attention should be paid to regulating the impact of chemical factors on TC.

Moreover, to verify the results of bioinformatic analyses, we performed qRT-PCR to detect the mRNA expression of these five hallmark ABC transporters in collected TC and para-cancerous thyroid tissues. The qRT-PCR results were entirely consistent with our TCGA analysis. In the future, we expect to directly confirm probable mechanisms underlying the regulation of immune cell infiltration patterns in TC by these five hallmark ABC transporters, via in vitro and in vivo experiments.

Our study was affected by several limitations. Firstly, in our effort to elucidate the specific roles of hallmark ABC transporters in modulating TC prognosis and progression, we analyzed the data of all TCs obtained from the TCGA database, without differentiating between its pathological subtypes. Secondly, compared with PTC, the incidence rates of MTC and ATC are very low. As such, it is relatively difficult to collect ATC and MTC case data for qRT-PCR analysis. Therefore, only PTC and para-cancerous thyroid tissues could be collected and used for experimental validation.

The findings of this study indicate that five hallmark ABC transporters (ABCA8, ABCA12, ABCB6, ABCB8, and ABCC10) are strongly associated with immunomodulation in TC, as well as with the prognoses for TC. In addition, factors, as well as chemicals and regulatory networks, that are significantly correlated with these five hallmark ABC transporters in TC, are elucidated. These findings may help better understand the molecular mechanisms underlying the role played by these hallmark ABC transporters in TC. The findings of this study also indicate that these hallmark ABC transporters may help enhance prognostic prediction and enable the development of effective immunotherapies against TC.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Ethics Committee of Shengjing Hospital of China Medical University. The patients/participants provided their written informed consent to participate in this study.

Conceptualization, LW and ZL; Methodology, LW, XS and JH; Investigation, LW and XS; Resources, LW, XS and JH; Writing – Original Draft Preparation, LW; Writing – Review & Editing, XS, JH and ZL; Supervision, ZL. All authors read and approved the final manuscript.

This work was supported by the National Natural Science Foundation of China (Grant number: 81672644), the Young Scholar Support Program 2018 of China Medical University (Grant number: QGZD2018061), and the 345 Talent Project of Shengjing Hospital of China Medical University (50A; 30).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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