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 [43]. Activation of ADO receptors on cancer cells affects proliferation, apoptosis, cytoprotection, and migration [44]. Moreover, by inhibiting the antitumor function of both lymphocytes and antigen-presenting cells, ADO promotes tumor growth [45,46]. In addition, ADO can suppress T cell priming by acting directly on DC and macrophages [47]. A2AR signaling has also been shown to block the formation of Th1 and Th17 cells and induce the development of Treg cells [48,49].

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 [42]. 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 [50].

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 [51]. 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 [52]. 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) [53]. 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 [54]. 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 [55]. 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 [56]. 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 in this study [57]. In addition to migration, eATP has been shown to promote epithelial-to-mesenchymal transition following P2RX7 activation in A549 cells [58]. 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, we were unable to detect P2RX7 expression on A549 cells when using the monoclonal antibody characterized by Buell [59], whereas the use of a commercial polyclonal antibody by other authors detected its expression on various NSCLC (including A549) and SCLC cancer cell lines [22]. 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 [60]. 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 [60,61].