1) NT5E could serve as an efficient prognostic biomarker in pan-cancer.

Ecto-5′-nucleotidase (NT5E), namely cluster of differentiation 73 (CD73), is a glycosylphosphatidylinositol-anchored cell surface protein. Encoded by the NT5E gene, CD73 is widely distributed in the human body, including the central nervous system, cardiovascular system, and epithelial tissues (Thompson et al., 2004; Zimmermann et al., 2012; Jeong et al., 2020). Structurally, NT5E consists of three domains, including a glycosylated N-terminal domain and a C-terminal domain, which are responsible for metal binding and the catalytic function respectively, and an alpha helix connecting the aforementioned two domains (Buschette-Brambrink and Gutensohn, 1989; Fini et al., 2003). Functionally, NT5E possesses nucleosidase activity (Sträter, 2006), and could hydrolyze extracellular adenosine monophosphate (AMP) into adenosine (Kordas et al., 2018). Extracellular adenosine plays an important role in modulating inflammation regulation and tumor immunity (Antonioli et al., 2013; Allard et al., 2017a; Kordas et al., 2018; Boison and Yegutkin, 2019), where A_2A receptor (A_2AR)-mediated signaling pathway matters most (Colella et al., 2018). Adenosine can activate the immune suppressive effects, which is characterized by the inhibition of chemotaxis and proliferation function among T cells (Sitkovsky et al., 2004; Allard et al., 2014). Meanwhile, it has been demonstrated that adenosine promotes angiogenesis and inhibits the release of cytokines and the expression of adhesion molecules such as E-selectin (Bouma et al., 1996; Spychala, 2000), hinting that NT5E may be related to cell adhesion function (Henttinen et al., 2003). Furthermore, it has also been proved that NT5E could influence cell adhesion and migration performance by the molecular mechanism of tenascin C, one of the important factors among extracellular matrix (ECM) (Sadej and Skladanowski, 2012). Therefore, NT5E could promote tumor growth not only by accumulating adenosine to inhibit the antitumoral immune responses, but also by facilitating dissemination of cancer cells (Kordas et al., 2018).

According to previous studies, NT5E has been detected among various tumor entities, including melanoma (Sadej et al., 2006a; Sadej et al., 2006b; Wang et al., 2012), triple-negative breast cancer (Allard et al., 2014; Buisseret et al., 2018), colorectal cancer (Liu et al., 2012), and non-small cell lung cancer (Inoue et al., 2017). Furthermore, it is not only expressed on malignant cells, but also on several immune cells such as regulatory T cells (Tregs) (Alam et al., 2009), myeloid-derived suppressor cells (MDSCs) (Ryzhov et al., 2011), dendritic cells (DCs) (Berchtold et al., 1999) and natural killer (NK) cells (Neo et al., 2020), which could result in more obvious accumulation of immunosuppressive adenosine and lead to the downregulation of the T cell immune responses (Saldanha-Araujo et al., 2011), and it has been illustrated that NT5E⁺ NK cells could inhibit T cell activity by upregulating interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production (Neo et al., 2020). Moreover, adenosinergic A_2AR were also expressed on DCs, MDSCs, NK cells, and macrophages, indicating that the function of these regulatory immune cells could also be inhibited by adenosine (Allard et al., 2016; Kalekar et al., 2016; Young et al., 2016; Kalekar and Mueller, 2017).

It has been illustrated that adenosine triphosphate (ATP) concentration is about hundreds of thousands of times higher in the tumor microenvironment (TME) than the non-tumoral extracellular tissues (Zimmermann, 2000; Pellegatti et al., 2008). ATP is hydrolyzed to AMP, and lastly to adenosine by plasma membrane nucleotidases. According to previous studies, adenosine-mediated immunosuppression is a crucial part in the TME (Leone and Emens, 2018), which was constructed by vascular endothelial cells, fibroblast cells, and many types of innate and adaptive immune cells, together with ECM as well as multiple extracellular soluble molecules (cytokines, chemotactic factor, growth factors, etc.) (Binnewies et al., 2018). Furthermore, TME complexity is an important part in the differentiation of cold tumors and hot tumors. The feature of hot tumors is a high T cell infiltration level and abundant immune active molecular signatures, while cold tumors show distinctive characteristic of T cell absence (Gajewski, 2015). Thus, the immunosuppressive environment of cold tumors exhibited resistant to numerous immune checkpoint blockade therapies (Quail and Joyce, 2017).

There are several components correlated with the maintenance of an immunosuppressive environment, including some molecules like TGF-β, epidermal growth factor (EGF) and adenosine (Wei et al., 2022), and several immunosuppressive cells, including Tregs, tumor-associated macrophages (TAMs), endothelial cells, and cancer-associated fibroblasts (CAFs) (Zhu et al., 2022). In particular, CAFs may correlate with the enhancement of tumor phenotypes by regulating cancer cell proliferation and invasion and ECM remodeling (Costa et al., 2014; Gascard and Tlsty, 2016; Gentric et al., 2017). It has been validated that CAFs could secrete a vast amount of cytokines, including hepatocyte growth factor, EGF, connective tissue growth factor, insulin‐like growth factor. All these cytokines could function directly on the surrounding cells and facilitate ECM reprogramming. Meanwhile, CAFs also secrete extracellular vesicles, metabolites, ECM components and ECM‐remodeling enzymes (Jacob et al., 2012). Consequently, CAFs were considered to play a long-term role in the tumor development from tumorigenesis to cancer metastasis (Cirri and Chiarugi, 2011; Marsh et al., 2013; Kalluri, 2016).

In our previous study, we have concluded that NT5E could serve as an independent prognostic indicator for head and neck squamous cell carcinoma (HNSC) (Chen et al., 2022). Similarly, in pancreas, prostate and bladder cancer, NT5E has also been validated to correlate with tumor development and invasion (Yang et al., 2013; Mandapathil et al., 2018; Koivisto et al., 2019; Zhou et al., 2019; Chen et al., 2022). In detail, for gastric cancer patients, CD73 may serve as a regulator in RICS/RhoA-LIMK-cofilin signaling pathway by its extracellular function in adenosinergic pathway, and then promote β-catenin-induced epithelial-mesenchymal transition (EMT) process, which is correlated with metastasis property of tumor cells (Xu et al., 2020; Goulioumis and Gyftopoulos, 2022). In our study, the enrichment analyses also indicated that NT5E may be related to EMT and metastasis during HNSC progression. Furthermore, HNSC-related immune infiltration analysis and single-cell type analysis revealed that NT5E expression was positively related to CAFs infiltration in HNSC (Chen et al., 2022), which is in line with the previous conclusion that NT5E expression is related to tumor migration and invasion (Costa et al., 2014).

Although there is abundant evidence indicating that the expression level of NT5E is related to clinical outcomes and the prognosis in certain tumors, several questions still remain suspension, including the expression landscape of NT5E among various tumor types, the certain cell types expressing NT5E, and the potential signal pathways consisting NT5E in tumor growth and metastasis. To the best of our knowledge, the pan-cancer analysis of NT5E is still a virgin land. Thus, in this study, we performed NT5E expression analysis and prognosis analysis in pan-cancer, and explored the potential role of NT5E in the TME and the EMT function of CAFs, so as to provide novel clues for immunotherapy against malignant tumors.

This study is an integrated pan-cancer analysis about the potential prognostic value of NT5E. We reported that the expression level of NT5E was elevated in many tumor types, such as ovarian cancer and colorectal tumor tissues (Gaudreau et al., 2016; Wu et al., 2016). The overexpression of NT5E was also associated with poor prognosis, and was related to tumor development and invasion in pancreas, prostate, bladder and head and neck cancer (Yang et al., 2013; Mandapathil et al., 2018; Koivisto et al., 2019; Zhou et al., 2019; Chen et al., 2022). In this study, not only did we illustrate that NT5E might serve as a negative prognostic biomarker for LUAD, STAD, BRCA and UVM, but also revealed that it is positively correlated with tumor stage in several cancers. The results of immunofluorescence staining, in our study, also uncovered that the expression level of NT5E was positively correlated with T staging in the supraglottic carcinoma, which is considered to belong to the typical HNSC. These results suggested that NT5E might facilitate tumor growth and tumor metastasis.

Tumor metastasis is a complex process manipulated by multiple mechanisms (Pantel and Brakenhoff, 2004; Quail and Joyce, 2013; López-Soto et al., 2017). One of the important mechanisms is that the primary tumors cells invade through the physical barrier, namely the basement membrane, and disseminate via the circulation system. In the process of tumor invasion, the epithelial cells also break through the basement membrane and separate from neighboring cells to damage adjacent cell layers. The reason lies in the acquirement of migratory and invasive properties through EMT (Thiery et al., 2009), during which the apical-basal polarity and cell-cell adhesion of epithelial cells were weakened, and thus transited into invasive mesenchymal cells (Du and Shim, 2016). Then, mesenchymal cells could invade through ECM, one of the essential components of TME, which is composed of collagen, elastin, fibronectin, hyaluronic acid, proteoglycans and glycoproteins, undertaking the task to support tissues by encapsulating cells (Otranto et al., 2012; Pickup et al., 2014; Willumsen et al., 2018). In this case, the tumor cells would lose cell-cell adhesion and thus acquire motility. Moreover, it has been reported that the circulating tumor cells, which are important precursors of cancer metastasis, could be allowed to escape from antimetastatic checkpoints to realize distant metastasis by the mechanism of EMT process (Xiang et al., 2022). In our study, the results of enrichment analysis also demonstrated the critical role of NT5E as a regulator of cell-substrate junction, cell-substrate adherens junction, focal adhesion and external side of plasma membrane, and these functions are closely related to cell polarization and EMT (Thiery and Sleeman, 2006; Baronsky et al., 2017; Venhuizen et al., 2020), which plays an crucial role in cancer progression, especially tumor cell invasion (Son and Moon, 2010). Therefore, the pro-tumor function of NT5E may be related to facilitating the EMT of tumor cells.

Moreover, the metastatic potential of cancer cells is closely dependent on the TME. Notably, CAFs are one of the major components of the tumor stroma contributing significantly to the TME, which were differentiated from stromal fibroblast cells by the stimulation of paracrine growth factors secreted from tumor cells (Kalluri and Zeisberg, 2006; Tejada et al., 2006). Unlike normal stromal fibroblasts, CAFs could facilitate cancer cells survival (Martinez-Outschoorn et al., 2010), growth and progression (Orimo et al., 2005; Giannoni et al., 2010). It has been validated that CAFs secrete a number of cytokines, which could facilitate tumor cells invasion and metastasis by activating several signaling pathways (Ao et al., 2007; Kojima et al., 2010; Karagiannis et al., 2012; Yu et al., 2014; Shien et al., 2017; Gao et al., 2019). In addition, CAFs also play important roles in orchestrating the ECM in almost all cancers. It has been illustrated that ECM remodeling could also facilitate the invasion and migration of cancer cells, and cancer cells with the EMT phenotype make great contribution to this process by producing ECM-degrading proteases (Giannoni et al., 2010; Qiao et al., 2010). In our study, the results of enrichment analysis of NT5E may imply that it may be correlated with EMT during cancer progress. Moreover, we employed studies to gain elevated hallmark gene-sets in different cell subsets, and the results illuminated that EMT function of CAFs was elevated in BCC, SKCM, HNSC, and PAAD. Then, based on the GSEA module of TISCH database, the analysis results, also showed that the degree of EMT of CAFs was higher than that of other cell subsets. These results are consistent with the above conclusion that CAFs make great contribution to maintain tumor growth and development.

According to previous studies, a high level of CAFs infiltration was considered to make great contribute to an unfavorable clinical outcome of patients (Gieniec et al., 2019; Hosein et al., 2020; Piersma et al., 2020). Consequently, there are more and more attentions focused on the therapy targeted-CAFs, which is believed to be one of the complementary treatment strategies for cancer. The first strategy is to deplete CAFs directly by either transgenic technologies or immunotherapies (Özdemir et al., 2015). For example, the previous study revealed that a specific CAF subpopulation (referred to as CAF-S1) expressed NT5E could upregulate the power of Tregs to inhibit the proliferation of effector T cells (Costa et al., 2018; Givel et al., 2018), while this effect could be neutralize after using an anti-CD73 antibody (Magagna et al., 2021). Secondly, some molecules, including all-trans retinoic acid (ATRA) and calcipotriol, could promote CAFs to become normalized and adopt an inactive phenotype (Froeling et al., 2011). Moreover, CAFs could be used as a vehicle to deliver anticancer drugs, including TNF-related apoptosis-inducing ligand (TRAIL) or type I interferon (IFN) (Miao et al., 2017). Finally, the function of CAFs could be inhibited by regulating the activation of targeting crucial signals and effectors, such as chemokine and growth factor pathways (Giannoni et al., 2010; Albrengues et al., 2015). Interestingly, NT5E has also been considered as a novel checkpoint inhibitor target (Allard et al., 2017b). It has been illustrated that overexpression of NT5E could inhibit the immunosurveillance of immune cells, which may be correlated with immune evasion and tumor metastasis (Stagg et al., 2010; Leclerc et al., 2016). Moreover, tumor cell death could result in CAFs-NT5E overexpression via an adenosine-adenosinergic A2_B receptor (A_2BR) mediated feedforward circuit, which could furtherly exacerbate the immunosuppressive environment (Yu et al., 2020). According to the results of our study, NT5E was related positively to the infiltration stage of CAFs in most cancers (Hu et al., 2020), and single-cell analysis also showed that NT5E was mainly expressed on the CAFs in several cancers such as BCC, HNSC, PAAD, and SKCM. Furthermore, in the supraglottic carcinoma specimens, we also illustrated that NT5E was co-expressed with α-SMA, which was one of the myofibroblast markers and expressed on CAFs (Orimo and Weinberg, 2007; Sharon et al., 2013). Thus, NT5E may be a crucial signal related to the activation of CAFs. High-NT5E expressed CAFs could serve as novel targets for immunotherapy. Thus, we performed correlation analyses between immune-related genes and NT5E expression level on pan-cancer level, and uncovered that the expression levels of several immunoinhibitors were positively correlated with NT5E expression, including kinase insert domain-containing receptor (KDR), interleukin-10 receptor B (IL10RB) and transforming growth factor-beta receptor 1 (TGFBR1) (Supplementary Figures S1, S2).

Still and all, there is no doubt that our research also has some limitations. Firstly, because the data used in this study was derived from online databases, which are characterized by open and imprecise, systematic bias may become an inevitable factor. Secondly, pan-cancer analysis is of distinct heterogeneity attributing to different cancer. Thirdly, the findings of the current investigation demand clinical trial-based validation in several cancer patients receiving high-NT5E-expression CAFs-targeting immunotherapies. Finally, more functional experiments such as flow cytometry and single cell RNA-seq are needed to further elucidate CAFs and NT5E contents in specific cancer.

In summary, this study is an integrated analysis of NT5E in pan-cancer. We illuminated that NT5E could serve as an efficient prognostic biomarker in pan-cancer, its expression level was positively related to CAFs and endothelial cells infiltration in pan-cancer. Moreover, NT5E is more likely expressed in the endothelia cells and CAFs in pan-cancer, and CAFs may play an important role in EMT of various tumor species. We concluded that the underlying mechanism of the NT5E pro-tumor effect may be related to the up-regulated EMT function of CAFs, which may provide some information to study immune therapy targeted CAFs and NT5E.