Once released into the extracellular space, the fate of eATP depends on ectonucleotidases. Four enzyme families have been discovered, cloned, and characterized: ecto-nucleoside triphosphate diphosphohydrolase (NTPDases), alkaline phosphatases, ecto-nucleotide pyrophosphatase/phosphodiesterases of NPP type (NPP-type), and ecto-5′-nucleotidase ^[3]. In combination with the intracellular NAD-degrading enzyme CD38, enzymes for the nucleotide hydrolysis are particularly important in cancer, but also in aging ^[4]. CD39 (NTPD-1) and CD73 (NT5E) play an important role in calibrating the specificity, duration, and strength of purinergic signals by converting ATP/ADP to AMP, and AMP to adenosine, respectively.

CD39, encoded by NTPDase-1, is the rate-limiting enzyme that hydrolyzes ATP to ADP and ADP to AMP ^[3]. CD39 is expressed by B cells, innate cells, regulatory T cells, and activated CD4 and CD8 T cells, and has been identified as a marker of exhausted T cells in patients with chronic viral infections ^[5][6]. In addition to immune cells, CD39 is also expressed on the tumor-associated endothelium and tumor cells ^[7]. The expression of CD39 was examined in 12 patients after lung surgery, and CD39 expression was found to be increased in the immune infiltrate of the tumor compared with the adjacent non-tumor tissue ^[8]. Specifically, TCD4+, TCD8+, FoxP3+ regulatory T cells, CD16+ NK cells, B cells, and macrophages expressed CD39 at higher levels. Remarkably, these cells also expressed higher levels of PD-1. The limited number of patients in this entry did not allow for the formal conclusion that expression of CD39 and PD-1 is a marker of poor prognosis, but the three out of five patients who relapsed had a high frequency of double-positive CD39+/PD-1+ CD8+ and CD4+ TILs, suggesting that expression of CD39 in cytotoxic T cells may be an important mechanism for tumor-induced immunosuppression in NSCLC. Interestingly, quantification of the proportion of total CD8+ and CD39+ lymphocytes by immunochemistry (IHC) of patients with NSCLC was not predictive of response to ICIs. However, the double positive CD39+ CD8+ fraction appears to be a strong predictive biomarker ^[9]. If confirmed, this finding will be of great importance as there is an urgent need to identify patients with LC who will benefit from ICIs alone or in combination with other treatments, including anti-CD39 antibodies.

CD73, encoded by NT5E, is essential for the generation of extracellular adenosine from AMP. Adenosine could also be formed via the non-canonical pathway of CD38-CD203a-CD73, which is independent of CD39 ^[10]. CD73 is expressed on the surface of endothelial, stromal, and immune cells, as well as on tumor cells of various origin ^[11]. In patients with NSCLC, CD73 is expressed on cancer cells, cancer-associated fibroblasts (CAFs), and tumor-infiltrating lymphocytes (TILs), and its expression correlates with the expression of hypoxia- inducible factor-1, a trend confirmed in in vitro studies using the lung cancer cell lines H1299 and A549 ^[12]. In this entry, the authors also characterized the expression of CD39 and showed that it is predominantly expressed by CAFs and TILs, in contrast to CD73, which is expressed by both cancer cells and CAFs. Overall, their results suggest that expression of CD73 and CD39 in the tumor stroma regulates immunosuppressive pathways by promoting the prevalence of FoxP3+ and PD-1+ lymphocytes as well as PD-L1 expression by cancer cells, all suggestive of an immunosuppressive microenvironment. However, no association was found between expression of ectonucleotidase and histopathological variables or survival analysis, in contrast to a previous study showing that high CD73 expression was an independent indicator of poor prognosis for overall survival and recurrence-free survival ^[13].

While it is widely accepted that lung cancer immune escape, tumorigenesis, and tumor progression are associated with high levels of adenosine, PD-1, and PD-L1 within the TME, it is clear that additional work is needed to fully unravel the relationship between all of these players and to fully understand whether their expression can be considered as powerful biomarkers that could guide the choice of treatments.

Another level of complexity lies with patients whose cancer has oncogenic mutations, as highlighted in a recent study that pooled three cohorts of NSCLC patients (n = 4189 total) with oncogenic alterations, including KRAS, MET, RET, BRAF-V600E and non BRAF-V600E, ROS1, ALK, EGFR exon 20, HER2, and classical EGFR (exon 19 deletion and exon 21 L858R) ^[14]. With regard to KRAS mutations, corresponding to the largest subgroup of oncogenic lung adenocarcinoma, it has been shown that the co-occurrence of genomic alterations in the STK11 and KEAP1 genes leads to a worse outcome in KRAS-mutated patients treated with immunotherapy ^[15]. In addition, the impact of the tumor mutation burden (TMB) and PD-L1 expression on the clinical outcome of ICI therapies has been demonstrated for NSCLC with BRAF mutations, while EGFR and HER2 mutations and ALK, ROS1, RET and MET fusions define NSCLC subgroups with minimal benefit from ICI, despite a high level of expression of PD-L1 in NSCLC with oncogene fusions. The mechanisms underlying the lack of efficacy of ICI in EGFR-mutated NSCLC patients appear to be related to an immune-influenced phenotype characterized by a low level of expression of PD-L1, low TMB, lower cytotoxic T cell numbers, and low T cell receptor clonality. The analysis of 75 immune checkpoint genes, NTE5 (CD73), and adenosine A1 receptor (A1R) were the most upregulated genes in EGFR-mutated tumors. A single-cell analysis revealed that the tumor cell population expressed CD73, in both treatment-naïve and resistant tumors ^[16]. Therefore, the CD73/adenosine pathway was identified as a potential therapeutic target for EGFR-mutated NSCLC, and there is no doubt that the information from profiling the TME and antitumor immune response can be used to tailor immunotherapy in selected patients with LC.

The purinergic receptor family is composed of two subfamilies; the G-protein-coupled adenosine receptors (A1R, A2RA1, A2RA2, A3R), which belong to the P1 family, and the ATP receptors, which form the P2 family. The P2 family includes the ligand-gated ion channel family receptors (P2RX1–7) and the G protein-coupled receptor (GPCR) family (P2RY1-2, P2RY4, P2RY6, and P2RY11–14) ^[17]. A decade ago, Geoffrey Burnstock, who was one of the first to substantiate the importance of purinergic signaling in tumor progression, hypothesized that purinergic signaling might contribute to respiratory diseases ^[18].

Adenosine (ADO) receptors (A1, A2A, A2B, and A3) are expressed in various cells and tissues throughout the body and are activated by ADO in the nanomolar range, with the exception of A2B, which is a low-affinity receptor (micromolar range) and therefore activated mainly under pathophysiological conditions ^[19]. Activation of ADO receptors on cancer cells affects proliferation, apoptosis, cytoprotection, and migration ^[20]. Moreover, by inhibiting the antitumor function of both lymphocytes and antigen-presenting cells, ADO promotes tumor growth ^[21][22]. In addition, ADO can suppress T cell priming by acting directly on DC and macrophages ^[23]. A2AR signaling has also been shown to block the formation of Th1 and Th17 cells and induce the development of Treg cells ^[24][25].

The involvement of ADO receptors in progression of LC has been poorly described. A1, A2A, A2B, and A3 receptors are expressed by type I and II alveolar epithelial cells, smooth muscle cells, endothelial cells, and immune cells ^[18]. Particular attention has been paid to A2AR, which is expressed by T cells in hypoxia and was initially described as a nonredundant immunosuppressive mechanism to protect normal tissues from inflammatory damage and autoimmunity. However, it quickly became clear that tumor cells exploit this immunosuppressive mechanism to their own advantage ^[26].

The P2Y receptor family in NSCLC. The subtypes P2RY1, P2RY2, P2RY4, P2RY6, and P2RY11–14 are expressed in mammals. These G protein-coupled receptors, that stimulate Gq- or Gi-dependent cell signaling, are activated by ligands such as extracellular ATP, ADP, UTP, UDP, UDP-glucose, and also NAD. All these ligands have different affinities for each subtype ^[27]. In lung cancer cells, especially in the most studied A549 cell line, the expression of P2RY-2, -6, -12 and -14 has been described. A mitogenic effect through ATP/UTP-mediated activation of P2RY2 and the UDP-activated P2RY6 was reported first ^[28]. Two years later, P2RY14 expression was detected in primary human type II alveolar epithelial cells, in normal bronchial epithelial cells (Beas-2b cell line), and in A549 cells.

The P2X receptor family in NSCLC. This family consists of seven members (P2RX1–7). These ATP-gated ion channel receptors are formed by three homomeric or heteromeric P2RX subunits. Their activation directly leads to Na⁺ and Ca²⁺ influx and K⁺ efflux across the plasma membrane of the cell, which in turn activates downstream signaling pathways and triggers action potential in excitable cells (e.g., neurons), but also cell proliferation, differentiation, and apoptosis in non-excitable cells (e.g., epithelial cells) ^[29]. The expression of P2RX receptors mRNA in normal (Beas-2b) and tumor cell lines (H23 and A549) was analyzed by RT-qPCR. The results showed that P2RX-3 to 7 were expressed. P2RX3, 4 and 5 are overexpressed in cancer cells compared to normal cell lines, while P2RX6 and 7 are downregulated ^[30]. The observation that P2RX7 expression is downregulated in cancer cells is in contradiction with other studies reporting its expression. For example, TGF-β1-induced A549 migration, but not proliferation, is dependent on vesicular exocytosis of ATP, which in turn causes P2RX7 activation through actin fiber formation ^[31]. This result was confirmed in an independent report using 2 other lung cancer cell lines (lung adenocarcinoma PC-9 cells and mucoepidermoid carcinoma H292 cells), in which the authors also showed that P2RX7 was overexpressed in cancer cells compared with normal bronchial epithelial Beas-2B cells. Furthermore, the authors showed that in PC-9 cells, which have the highest expression of P2RX7, ATP is constitutively released and induced cell proliferation in an autocrine manner. Of note, the expression of P2RX7 in PC-9 cells is dependent on EGFR signaling, which is constitutively activated in this cell line ^[32]. It has been also shown that increased RNA expression of P2RX4 and P2RX7 correlated with the presence of distant metastases in NSCLC patients, although the status of EGFR mutations was not reported ^[33]. In addition to migration, eATP has been shown to promote epithelial-to-mesenchymal transition following P2RX7 activation in A549 cells ^[34]. As mentioned previously, P2RX4 is also expressed by lung tumor cell lines, but its precise role in cancer progression is still unknown.

Despite many conflicting findings in the literature, it seems clear that extracellular nucleotides are actively involved in tumorigenesis by stimulating purinergic receptors expressed directly on tumor cells or on their neighboring stromal and immune cells. The published discrepancies regarding P2RX7 expression may be related to the tools used to characterize its expression. Indeed, there are several oligonucleotides on the market, some of which recognize both the full-length and truncated mRNA of P2RX7, whereas others are specific for one or the other form. The same conclusion can be drawn for antibodies. Some of them recognize the extracellular loop present in both the full-length protein and the truncated form, while others are specific for the C-terminal domain lost in the truncated P2RX7B isoform. For example, researchers were unable to detect P2RX7 expression on A549 cells when using the monoclonal antibody characterized by Buell ^[35], whereas the use of a commercial polyclonal antibody by other authors detected its expression on various NSCLC (including A549) and SCLC cancer cell lines ^[36]. In the same way, tumor cells from NSCLC patients were not stained with the conformational antibody, while immune cells from the same patients showed strong staining ^[37]. To add complexity, some tools can also detect the truncated isoform of P2RX7 encoded by the splice variant P2RX7B, which has been shown to behave like a protumor factor ^[37][38].