Nucleoside transporters and immunosuppressive adenosine signaling in the tumor microenvironment: Potential therapeutic opportunities

Abstract

Adenosine compartmentalization has a profound impact on immune cell function by regulating adenosine localization and, therefore, extracellular signaling capabilities, which suppresses immune cell function in the tumor microenvironment. Nucleoside transporters, responsible for the translocation and cellular compartmentalization of hydrophilic adenosine, represent an understudied yet crucial component of adenosine disposition in the tumor microenvironment. In this review article, we will summarize what is known regarding nucleoside transporter’s function within the purinome in relation to currently devised points of intervention (i.e., ectonucleotidases, adenosine receptors) for cancer immunotherapy, alterations in nucleoside transporter expression reported in cancer, and potential avenues for targeting of nucleoside transporters for the desired modulation of adenosine compartmentalization and action. Further, we put forward that nucleoside transporters are an unexplored therapeutic opportunity, and modulation of nucleoside transport processes could attenuate the pathogenic buildup of immunosuppressive adenosine in solid tumors, particularly those enriched with nucleoside transport proteins.

1. Introduction

The purinergic signaling complex of a cell frequently referred to as “purinome”, comprises numerous proteins and cofactors that define purine-based cellular responses. Although the term purinome was first coined by Timothy Haystead to describe the >2000 proteins that bind purines from the perspective of screening potential drug targets (Haystead, 2006), for the scope of this review, the purinome will be described in the manner put forth by Volonté and D’Ambrosi, who elegantly reviewed the concept previously. The term was defined as “the molecular complex responsible for the biological effects of extracellular purine and pyrimidine ligands, thus consists of ectonucleotidemetabolizing enzymes hydrolyzing nucleoside phosphates, purinergic receptors classified as P1 for adenosine/AMP and P2 for nucleosides tri-/diphosphates, nucleotide channels and finally, nucleoside transporters^” (Volonte & D’Ambrosi, 2009). Nucleoside transporters play an integral role within biological membranes by transporting nucleosides, including the signaling nucleoside adenosine and various nucleoside analog drugs, across both intracellular and extracellular membranes. Although integral to the membrane purinome that could directly modulate purinergic receptor signaling, nucleoside transporters’ impact on purinergic signaling is poorly understood. This review will investigate the potential interplay between adenosine transporters and receptors in the context of immunosuppressive adenosinergic signaling within the tumor microenvironment. Understanding the therapeutic potential of the adenosine transporters in relation to other established targets within the purinome may facilitate rational development of new drug candidates not only for cancer immunotherapy but also for amelioration of other diseases such as cardiac diseases, Parkinson’s disease, psoriasis, thrombosis, dry eye, cystic fibrosis, and glaucoma where purinergic signaling plays crucial roles (Boison, 2013; Burnstock, 2006; Gendron et al., 2002; Jacobson, Balasubramanian, Deflorian, & Gao, 2012).

2. Concentrative nucleoside transporters (CNTs) and equilibrative nucleoside transporters (ENTs) are understudied components of the purinome

The two groups of nucleoside transporters are concentrative nucleoside transporters (CNTs), which are categorized within the solute carrier family 28 (SLC28), and equilibrative nucleoside transporters (ENTs), which fall within solute carrier family 29 (SLC29) (Pastor-Anglada & Perez-Torras, 2018a). Three distinct CNTs concentrate nucleosides within the cell through unidirectional transport. Each of the four ENTs instead work bidirectionally to equilibrate nucleosides between the cell and its surroundings and potentially across intracellular compartments. For CNTs to function in a single direction, CNTs use a sodium or proton gradient to help move nucleosides across membranes. CNT1 and CNT2 both have a 1:1 cation to nucleoside gradient, whereas CNT3 has a 2:1 cation to nucleoside gradient and a stronger affinity for protons over sodium ions. Alternatively, ENTs operate by facilitated diffusion (JYoung, Yao, Baldwin, Cass, & Baldwin, 2013). The multiplicity of nucleoside transporters in mammals alludes to the existing differences in their subcellular location, permeant selectivity, kinetics, and regulation.

Each of the CNT transporters has a unique role. CNT1 prefers pyrimidine substrates, CNT2 prefers purine substrates, and CNT3 transports both purines and pyrimidines (Mirabet et al., 1999). Additionally, all CNTs can transport uridine (Pastor-Anglada & Perez-Torras, 2018a). The CNTs are primarily localized to the plasma membrane of the cell and on the apical surface of polarized epithelial cells, although CNT3 is occasionally found within intracellular membranes (Young et al., 2013). CNTs also tend to have much higher affinities for their substrates as opposed to the ENT family of transporters (Pastor-Anglada & Perez-Torras, 2018a).

ENT transporters also have individualized roles. Both ENT1 and ENT2 can transport purine nucleosides and pyrimidine nucleosides with varied affinities (Pastor-Anglada & Perez-Torras, 2018a). These transporters are primarily found within the basolateral plasma membrane of the polarized cell but can be found in some intracellular membranes (e.g., nuclear membranes). Unlike the other ENTs, ENT3 is primarily found within the intracellular membranes of the cell both in lysosomal as well as mitochondrial membranes (Young et al., 2013). ENT4 is set apart in that it preferentially transports monoamines such as dopamine or serotonin and is evolutionarily distinct from the other three ENT transporters (Pastor-Anglada & Perez-Torras, 2018b).

2.1. Nucleoside transporters facilitate adenosine transport

All nucleoside transporters play at least some role in the transport of adenosine, including the pyrimidine preferring CNT1 which transports adenosine at a relatively low affinity than its counterparts (CNT2 and CNT3) (Pastor-Anglada & Perez-Torras, 2018b). While CNTs are responsible for concentrating adenosine within the cell against the concentration gradient, ENTs, in general, equilibrate adenosine concentrations across the membrane. However, the metabolism of adenosine within the cell to AMP via phosphorylation may help allow CNTs to build up intracellular adenosine concentrations (Pastor-Anglada & Perez-Torras, 2018b). Within the ENT family, ENT1 and ENT2 are the primary transporter proteins believed to be responsible for determining intracellular adenosine concentrations as ENT3 is more directly involved in intracellular rather than extracellular transport and ENT4’s role is still yet to be fully understood (Pastor-Anglada & Perez-Torras, 2018a, 2018b). In addition, ENT3 is an obligatory, acidic pH-dependent nucleoside transporter that facilitates the lysosomal exit of adenosine derived from the hydrolytic degradation of nucleic acids arising from the phagocytic and endocytic pathways. It was also reported that ENT4 transport of adenosine is optimal at acidic pH in cardiomyocytes and therefore likely to play a role in ischemic conditions (Yang & Leung, 2015). ENT transport of adenosine across the plasma membrane is reliant upon the concentration gradient as well as its intracellular metabolism, as mentioned above. It is worth mentioning that a Ph-dependent adenosine transport activity has been described for ENT4 which is optimal at acidic pH (pH 5.5) and absent in neutral conditions (pH 7.4). This pH-selective transport is adenosineselective, Na⁺-independent, mildly affected by ENT inhibitors, and does not impact the ENT4-mediated transport of other metabolites such as serotonin (Barnes et al., 2006; Tandio, Vilas, & Hammond, 2019). The equilibrative transport serves as a counter to CNT unidirectional transport and allows for excess adenosine and nucleosides to leave the cell. These roles in adenosine transport across cellular membranes influence adenosine’s ability to accumulate at the extracellular side and interact with receptors and exert regulatory effects upon biological processes (Young et al., 2013).

Studies have demonstrated that both CNT and ENT mediate the translocation of adenosine in different human tissues. There is a notable expression of different CNT isoforms on the apical surface of epithelial cells lining the pulmonary epithelium in the airway. CNT2 is mostly localized in nasal epithelia and CNT3 is expressed in both nasal and bronchial epithelial cells. CNT2 and CNT3 are responsible for the permeability of airway epithelia to airway surface liquid adenosine as well as mediating translocation of inosine generated by adenosine deaminase (ADA) (Hirsh et al., 2007; Ritzel et al., 2001a; Ritzel et al., 2001b). The role of ENTs, especially ENT1 and ENT2, in regulating apical adenosine uptake in airway epithelia is also well-defined (Lu, Gong, Monks, Zaharevitz, & Moscow, 2002; Szkotak et al., 2001). Similarly, intracellular, or extracellular adenosine formed in the brain and spinal cord is available for membrane translocation via nucleoside transporters localized to many CNS regions (Bynoe, Viret, Yan, & Kim, 2015; Jennings et al., 2001; Liu et al., 2019; Pinto-Duarte, Coelho, Cunha, Ribeiro, & Sebastiao, 2005). Such spatiotemporal and regional differences in ENT and CNT expression are prevalent in different body tissues. Quantitative studies on tissue localization of CNTs and ENTs have demonstrated a similar pattern, with a few interspecies and gender-linked variations (Gray et al., 2004; Lu, Chen, & Klaassen, 2004; Pastor-Anglada & Perez-Torras, 2018a; Young et al., 2013). A high expression of both CNT1 and CNT2 is reported in the small intestine, followed by the kidney, liver, testes, thymus, and spleen. CNT3 has elevated expression in brain tissues, salivary glands, skin, breast, placenta, urinary bladder, and lung with moderate expression in the small intestine. ENT1 and ENT2 are abundant in skeletal muscles along with some expression in the heart, small intestine, testes, lungs, and liver. ENT3 is widely expressed throughout the body tissues including the testis, uterus, ovaries, breast, pancreas, small intestine, kidney, lung, liver, and so on. Table 1 describes the tissue distribution and cellular location of CNTs and ENTs along with their affinity for adenosine.

Table 1.

Adenosine Transport Characteristics of ENTs and CNTs

Nucleoside Transporter	Solute Carrier Family	Adenosine Affinity [Km (mM)]	Tissue Distribution	Subcellular Localization	Reference
CNT1	SLC28A1	--	Small intestine, Kidney, Liver,	Plasma membrane	(Gray, et al., 2004; J. D. Young, et al., 2013)
CNT2	SLC28A2	0.008	Small intestine, Testis, skeletal muscle, liver, kidney, intestine, pancreas, placenta, brain	Plasma membrane	(Duflot, et al., 2004; Gray, et al., 2004; Pastor-Anglada & Perez-Torras, 2018a, 2018b; J. D. Young, et al., 2013)
CNT3	SLC28A3	0.0024–0.015	Mammary gland, pancreas, bone marrow, trachea, intestine	Plasma membrane, sometimes intracellular	(Pastor-Anglada & Perez-Torras, 2018a, 2018b; J. D. Young, et al., 2013)
ENT1	SLC29A1	0.04–0.78	Widely expressed with high levels in skeletal and smooth muscle, lung, thymus	Plasma, nuclear, and mitochondrial membranes	(Parkinson, et al., 2011; Ward, Sherali, Mo, & Tse, 2000)
ENT2	SLC29A2	0.14–0.78	Widely expressed, especially skeletal muscle	Plasma and nuclear membranes	(Parkinson, et al., 2011; Ward, et al., 2000)
ENT3	SLC29A3	0.78–1.86	Widely expressed in most tissues	Endosomal, lysosomal, and mitochondrial membranes	(Baldwin, et al., 2005; Kang, et al., 2010; Parkinson, et al., 2011; Pastor-Anglada & Perez-Torras, 2018a, 2018b; Rahman, Askwith, & Govindarajan, 2017)
ENT4	SLC29A4	0.78	Heart, brain, skeletal muscle	Plasma membrane	(Barnes, et al., 2006; Parkinson, et al., 2011; Pastor-Anglada & Perez-Torras, 2018a)

Despite the increasing understanding of the role of nucleoside transporters in adenosine translocation in different tissues, the extent to which the differential expression and regulation levels of different transporter families and subfamilies can affect purinergic signaling is still unresolved. More recent findings suggest a more active role for nucleoside transporters in central nervous system (CNS) beyond the default transportation abilities (Bynoe et al., 2015; Choi et al., 2004; Choudhury, Chellappan, Sengupta, Pandey, & Gorain, 2019; Liu et al., 2019; Pasquini et al., 2022). A bidirectional interaction of adenosine with nucleoside transporters as well as excitatory and inhibitory adenosine receptors suggests a dual role for nucleoside transporters.

3. Nucleoside transporters and cross talk with other members of the purinome

Nucleoside transporters are likely to impact purinergic signaling, mainly through modulating existing adenosine levels inside and outside the cell. These can also terminate downstream adenosine receptor-dependent signaling by clearing the extracellular milieu of adenosine. These effector functions highlight the possibility of regulatory feedback loops, which connect receptor signaling with transporter function. Despite the perceived critical role of nucleoside transporters as modulators of purinergic signaling in the human body, their role within the context of the purinome is not yet explored to its full potential.

The localization of ENTs and CNTs on plasma membranes of synaptic vesicles confirms their role in mediating efflux and influx of adenosine in the intracellular sites. The release of trapped adenosine from these sites via Ca⁺-dependent excitation-secretion mechanism (Melani et al., 2012; Wall & Dale, 2013), suggests the causal involvement of these transporters in both purine nucleoside uptake and release under physiological conditions. ENTs are principal transporters that facilitate intracellular to extracellular flux of the adenosine in the CNS (Latini & Pedata, 2001; Paes-de-Carvalho et al., 2005; Zamzow, Bose, & Parkinson, 2009), whereas, in the cardiovascular system (CVS), ENTs mediate the uptake of extracellular adenosine, thus terminating adenosine receptor signaling by clearing the extracellular milieu of the purine nucleoside (Loffler, Morote-Garcia, Eltzschig, Coe, & Eltzschig, 2007; Rose et al., 2010). Recent evidence also suggests a significant role of CNT2 in the modulation of adenosine levels in the CNS. CNT2 mRNA levels vary with changes in extracellular adenosine concentration (Guillen-Gomez et al., 2004; Medina-Pulido et al., 2013), while ENT1 mRNA levels remain unchanged. Additional studies have demonstrated CNT2 expression on the plasma and vesicle membranes isolated from rat striatum, supporting the relevance of this transporter as a member of the purinome (Melani et al., 2012).

3.1. Nucleoside transporters act as gatekeepers within the purinome for adenosine compartmentalization in tumor tissues

CNTs and ENTs facilitate the transmembrane flux of adenosine acting as conduits mediating the adenosine flow in a highly regulated manner. When inside, intracellular metabolism of adenosine is most readily performed by adenosine kinase (ADK), and the addition of a phosphate group to adenosine prevents efflux via nucleoside transporters. Alternatively, ADA can metabolize adenosine to inosine. Cellular stress, mechanical injury, or metabolic changes can all contribute to the release of nucleotides from intracellular storage by exocytosis or conductive release by pannexin and connexin channels or adenosine triphosphate (ATP)-conduction anion channels. Adenosine is produced from extracellular ATP by dephosphorylation reactions carried out sequentially by ectonucleotidases CD39 and CD73.

Although a key player in multiple metabolic pathways, adenosine has been recognized as an important modulator of immune cell function in the tumor microenvironment (TME) (Ohta, 2016). It serves as a constituent of ATP, the primary cellular energy source, and as a critical component of nucleic acid synthesis. In the extracellular space, adenosine is available to activate G-protein coupled adenosine receptors (ARs) on the cell surface, affecting acute inflammation, immune response, vascular tension, and neurotransmission (Eltzschig, 2013; Karmouty-Quintana et al., 2013; Karmouty-Quintana, Xia, & Blackburn, 2013). Elevated levels of extracellular adenosine have been noted in solid tumors, reaching immunosuppressive concentrations (Blay, White, & Hoskin, 1997). A multitude of processes in summation determines the concentration of extracellular adenosine. Adenosine is produced in the extracellular space by the breakdown of nucleotides that are released from intracellular stores. Intracellular compartmentalization of adenosine by nucleoside transporters facilitates the localization of adenosine, although efflux via ENTs is also possible based on the concentration gradient of extracellular and intracellular and is especially likely in scenarios where adenosine transport and metabolism are not tightly coupled.

The cumulative contribution of ATP released from intracellular stores, adenosine generated extracellularly by ectonucleotidases, combined with nucleosides available in the blood or interstitial fluid, is counter-balanced by metabolic pathways that breakdown adenosine in the extracellular space, integrated with transporter-mediated intracellular translocation and the intracellular metabolism of adenosine that traps it inside of cells preventing efflux. Therefore, it is important to keep in mind the interdependence of the various aspects of the purinome, particularly in the context of adenosine transport. Pharmacologic blockade or activation of a single aspect will impact the function of all others and the overall balance. Therefore, careful experimentation and evaluation of the nucleoside transporters within the purinome is needed to fully validate new therapies.

Due to the complexity of the purinome, it is likely that targeting only one aspect would not be sufficient to intervene in the pathogenic accumulation of extracellular adenosine. Already, the inhibition of ectonucleotide-metabolizing enzymes, CD39 and CD73 has shown efficacy in cutting down adenosine, although clinical trials failed to show any improvement over current standard-of-care treatment options. Most research efforts up to this point have been focused on understanding and targeting the ectonucleotidase enzymes CD39 and CD73, which contribute to adenosine production by the removal of phosphate groups from extracellular ATP. Yet as Boison and Yegutkin point out in their recent article, this is only one element of the complex system that regulates adenosine levels (Boison & Yegutkin, 2019; Yegutkin, 2008). Fig. 1 shows a schematic of the various elements of the purinome that work together to determine the levels of extracellular adenosine.

Fig. 1.

Adenosine transporter-receptor Interplay in Purinergic Signaling. Both intracellular and extracellular components determine the adenosine levels in the cellular milieu.

The source of extracellular adenosine which accumulates in solid tumors has been attributed to multiple sources, including ATP release from necrotic or mechanically damaged cells (Wang et al., 2013; Yin, Xu, Zhang, Kumar, & Yu, 2007), and cells undergoing inflammatory stress (Bodin & Burnstock, 1998). Additionally, the vesicular release of ATP occurs and can take part, such as that demonstrated in neurotransmission, when released from synaptic vesicles upon stimulus (Zimmermann, 2008). Further, treatment with cytotoxic chemotherapy drugs which induce apoptosis also induces tumor cell release of ATP (Martins et al., 2009). ENTs may also perform efflux of adenosine depending upon the concentration gradient of intracellular and extracellular adenosine. Besides exocytotic pathways, other conductive ATP release mechanisms include connexin and pannexin channels, and ATP-conducting anion channels, which are shown to modulate adenosine/ATP levels (Lazarowski, 2012).

Once released into the extracellular space, ATP can undergo a variety of fates. Perhaps the best-explored pathway is metabolism by ectonucleotidases CD39 and CD73 which decorate the cell surface of tumor cells and, often, infiltration of immune cells and resident stromal cells. CD39 and CD73 are upregulated in many cancers and significantly contribute to the pathogenic production of extracellular adenosine. Yet it is also possible that the reverse reaction could occur and regenerate ATP from adenosine. Adenosine itself can undergo intracellular compartmentalization by nucleoside transport processes or be further metabolized to inosine.

After intracellular translocation by ENTs or CNTs, ADK rapidly adds a phosphate group to adenosine. The addition of a phosphate group bearing a negative charge excludes AMP from efflux via ENTs whose selectivity for nucleosides does not permit the passage of AMP. Therefore, it is predicted that under normal conditions, a steady state equilibrium exists between the levels of extracellular adenosine (influenced by blood levels of adenosine, expression of ectonucleotidases, conditions that modulate ATP efflux) and the rate of intracellular adenosine metabolism, primarily performed by ADK which sequesters adenosine inside of cells which ENTs facilitating transmembrane flux according to the concentration gradient of adenosine (Antonioli et al., 2008a, 2008b; Fornai et al., 2009; Livingston, Heaney, & Ennis, 2004; Polosa & Holgate, 2006; Poulsen & Quinn, 1998). ADA inhibition is known for its neuroprotective function in pain and inflammation (Mark & Don, 2007). During metabolic stress, the release and degradation of precursor adenine nucleotides (i.e., ATP, ADP, and AMP) also contribute to adenosine accumulation (Sachdeva & Gupta, 2013). Fig. 2 is a transportercentric representation of the interplay between different members of purinome and the regulation of adenosine signaling.

Fig. 2.

Nucleoside transporters within the purinome. A transporter-centric view of the purinome describing adenosine fate within and outside a cell.

This model has optimal utility in tissue types where the expression of ENTs or CNTs predominates; however, in many epithelial cell types CNTs are primarily located on the apical surface exposed to the extracellular environment and drive inward-directed movement of nucleosides. Further, many solid tumor types are of epithelial origin and would display the asymmetric distribution of ENTs and CNTs in which apically localized CNTs would have a larger role in adenosine uptake from the extracellular environment. CNTs do not depend on the concentration gradient for adenosine flux but instead concentrate nucleosides by cotransport of sodium or hydrogen ions. However, due to their high affinities, they are likely to get saturated much earlier than ENTs. How CNTs contribute to the steady state equilibrium described when ENTs are coupled tightly to the intracellular metabolism of adenosine is yet unclear. We predict that in many early-stage epithelial tumor types, the role of CNTs is prominent and underappreciated for the sequestration of extracellular adenosine. In addition, in late-stage poorly differentiated or undifferentiated tumors, the spatial organization of ENTs and CNTs would be severely impaired to affect the adenosine disposition through transporters.

3.2. Bidirectional dialogue of adenosine with nucleoside transporters and G protein-coupled receptors

Nucleoside transporters’ transmembrane flux of adenosine is the most defining event, which is coupled to signaling responses triggered by the downstream effectors within the cells producing real-time reactions in response to the ever-changing dynamics of the extracellular environment. This downstream signaling is initiated via the binding of adenosine to G protein-coupled adenosine receptors (GPCRs). There are four main types of adenosine receptors: A₁, A_2A, A_2B, and A₃. A₁ and A₃ receptors are typically classified as inhibitory, and A_2A and A_2B receptors are typically classified as excitatory. The receptor signaling centers around the excitation and inhibition of secondary messengers to incite or prohibit different signaling cascades, based on the type of adenosine receptor activated. These receptors also show considerable variation in expression among different tissues of the body (Table 1). A₁, A_2A, and A_2B receptors are mostly conserved among mammals, however, A₃ receptors are structurally diverse. The binding of ligands leads to the dissociation of, the Gα subunit of the heterotrimeric G protein which then regulates downstream effectors. The identity of the Gα subunit determines the activity of adenylate cyclase enzymes which modulate the formation of cyclic adenosine monophosphate (cAMP) (Rosenbaum, Rasmussen, & Kobilka, 2009). The facilitatory A_2A and A_2B receptors are coupled to Gα_s protein complexes, and the binding of extracellular adenosine increases intracellular cAMP levels (Fredholm, Johansson, & Wang, 2011; Gao & Jacobson, 2019; Jacobson & Gao, 2006). In contrast, the inhibitory A₁ and A₃ receptors are paired with Gα_i proteins which block the generation of cAMP (Jacobson & Gao, 2006). βγ subunits of G proteins also act through the mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) pathways, respectively. The general cytoprotective functions modulated by extracellular adenosine are regulated by adenosine receptors. A₁ receptor regulates sleep, vasoconstriction, and inhibition of the release of neurotransmitters; A_2A receptor also modulates sleep along with angiogenesis, and immunosuppression; A_2B receptor is associated with cardiovascular function including vascular integrity and myocardial preconditioning; and A₃ receptor controls mast cell regulation and myocardial preconditioning (Fredholm et al., 2011; Jacobson & Gao, 2006). Fig. 3 lists the major pathways affected by activated adenosine receptors along with associated pro- and anti-tumoral effects.

Fig. 3.

Tumoral effects of activated adenosine receptors.

All four adenosine receptors are widely expressed in the central nervous system, along with peripheral tissues of the cardiovascular, respiratory, renal, and immune systems (Table 2), where they act with specific pharmacological features and signaling abilities. Due to the colocalization of all four receptors within a cell and the overlapping of receptor function, monitoring the overall function of adenosine is a challenging task. Although extracellular adenosine serves as the endogenous agonist for all four receptors under physiological conditions, variability in adenosine binding affinities, tissue expression, and associated G protein types give each subtype a distinct signaling profile (Cieslak, Komoszynski, & Wojtczak, 2008; Fredholm, 2014). Another notoriously challenging aspect of GPCRs is their structure determination, largely owing to their conformationally dynamic nature and poor thermostability after extraction from the plasma membrane. Despite all the challenges, researchers have been able to decipher the structure of the A_2A receptor which is currently the best structurally characterized GPCR among all four receptors (Carpenter & Tate, 2016; Carpenter & Tate, 2016; Jaakola et al., 2008; Lebon, Bennett, Jazayeri, & Tate, 2011; Lebon & Tate, 2011; Lebon et al., 2011; Xu et al., 2011), followed by some success with A₁ receptor. The other AR subtypes have proven more difficult to crystallize for structural analysis, compared to these two receptors (Cheng et al., 2017; Glukhova et al., 2017). Several selective agonists and antagonists with variable ligand affinities have been synthesized and characterized for each of the receptors (summarized in Table 3). As A_2B receptors’ threshold adenosine occupancy is achieved at higher ligand concentrations as compared to the rest of the three receptors, it is often considered a “bad copy” of the A_2A receptor owing to its low adenosine affinity.

Table 2:

Key Characteristics of Adenosine Receptors

Receptor	Structural information (Accession No.)	Chromosomal location (h)	G-protein coupling	Tissue expression	Physiological function(s)	Reference
A1	h 326 aa (P30542) m 326 aa (Q60612)	1q32.1	Gi, Go	Brain tissues, Pancreas, Heart muscle, Testis, Spleen, Kidney, Lipocytes	Bradycardia; inhibition of lipolysis; reduced glomerular filtration; tubero-glomerular feedback; antinociception; reduction of sympathetic and parasympathetic activity; presynaptic inhibition; neuronal hyperpolarization; ischemic preconditioning; sleep cycle regulation	(Babich, Vadnagara, & Di Sole, 2015; J. F. Chen, et al., 2014; Fozard, 2003; Fredholm, 2007; Fredholm, et al., 2000; Hoffman, Chang, Dall’Aglio, & Reaven, 1986; Koeppen, Eckle, & Eltzschig, 2009; Satoh, et al., 2000; Vallon, Muhlbauer, & Osswald, 2006; Yoon, Bae, & Choi, 2005; Yun, et al., 2019)
A2A	h 410 aa (P29274) m 409 aa (UO5672)	22q11.2	Gs, Golf	Basal ganglia, Thymus, Lymph node, Heart muscle, Kidney, Liver, Lung, Striatum, Nucleus accumbens, Olfactory tubercle, Immune cells, Heart, Lung, Blood vessels, Retina, Aorta	Sensorimotor integration regulation in basal ganglia; sensory nerve stimulation; inhibition of polymorphonuclear leukocytes; inhibition of platelet aggregation; vasodilatation, protection against ischemic damage	(Conti, et al., 1993; Dixon, et al., 1996; Fredholm, 2007; Martin, Ueeda, & Olsson, 1993; Salmon & Cronstein, 1990)
A2B	h 328 aa (P29275) m 332 aa (UO5673)	17p11.2–12	Gs, Gq	Urinary bladder, Esophagus, Colon, Rectum, Brain tissues, Vagina, Placenta, Skin, Caecum, Large intestine, Bladder	Smooth muscle relaxation in vasculature and intestine; stimulation of mast cell mediator release; inhibition of monocyte and macrophage function	(Dixon, et al., 1996; Fredholm, 2007)
A3	h 328 aa (P29275) m 332 aa (UO5673)	17p11.2–12	Gi	Brain, Stomach, Duodenum, Small intestine, Parathyroid gland, Liver, Gall bladder, Urinary bladder, breast, Lymph node, Thymus, eyes	Enhancement of mediator release from mast cells; preconditioning	(Borea, Gessi, Bar-Yehuda, & Fishman, 2009; J. F. Chen, et al., 2014; Fozard, 2003; Fredholm, et al., 2000; Gaytan, et al., 2006; Jacobson & Gao, 2006; Jacobson, et al., 2018; Mohamadi, Aghaei, & Panjehpour, 2018; Zhou, et al., 1992)

Table 3:

Substrate Binding Affinity of Adenosine Receptors

Receptor	Selective agonists		Selective antagonists		Radioligands		Reference
	compound	affinity	compound	affinity	compound	affinity
A1	Adenosine	70nM-0.31μM (EC50)	DPCPX	0.3–3.9nM	[3H]-CCPA	0.6 (Kd)	(J. F. Chen, et al., 2014; Fredholm, et al., 2007; Geiger, LaBella, & Nagy, 1984; Klotz, 2000; Klotz, Keil, Zimmer, & Schwabe, 1990; Kreft, Bier, Holschbach, Schulze, & Coenen, 2017; Martin, Wysocki, Barrett, May, & Linden, 1996; Stockwell, Jakova, & Cayabyab, 2017; Yun, et al., 2019)
	CPA	8.9 (pKi)	8-cyclopentyltheophylline	7.5–8.0pM (Ki)	[3H]-R-PIA	2.0 (Kd)
	CCPA	800 (pKi)	WRC0571	8.8pM (Ki)	[3H]-CHA	-
	CHA	3.6	-	-	[3H] NECA	14 (Kd)
	S-ENBA	0.3nM	-	-	125I-AB-MECA	3.4–8.5
	LUF6944	7.6 (Ki)	-	-	[3H]-DPCPX	0.3–3.9 (Kd)
	LUF6941	7.9 (Ki)	-	-	CPMMCB	3.73nM (Ki)
	-	-	-	-	CPFPX	3.49nM (Ki)
	-	-	-	-	CBCPM	8.68nM (Ki)
A2A	Adenosine	0.7μM (EC50)	Caffeine	43,000nM (Ki)	[3H]-CGS 21680	32nM (Kd)	(de Lera Ruiz, et al., 2014; Dionisotti, et al., 1997; Fernandez-Duenas, et al., 2014; Fredholm, 2007; Fredholm, et al., 2001; Grahner, Winiwarter, Lanzner, & Muller, 1994; Klotz, et al., 1998; C. E. Muller, et al., 1998; Murphree, Marshall, Rieger, MacDonald, & Linden, 2002; Nonaka, Ichimura, et al., 1994; Nonaka, Mori, et al., 1994; O’Malley, et al., 2009; Okusa, Linden, Macdonald, & Huang, 1999; Olah, Jacobson, & Stiles, 1994; Palmer, Poucher, Jacobson, & Stiles, 1995; Preti, Baraldi, Moorman, Borea, & Varani, 2015; van der Walt & Terre’Blanche, 2018)
	C2-/C8-substituted adenosine	7.19nM (Ki)	SCH 58261	0.6 nM (Ki)	[3H]-NECA, 125	20nM (Kd)
	2-((4-aryl(alkyl)piperazin-1-yl)alkylamino)-5′-N-ethylcarboxamidoadenosine (2-F, 4-Cl)	4.78nM (Ki)	ZM241385	1.4 nM (Ki)	I-ZM 241385	0.7nM (Kd)
	CGS 21680	72±10nM (Kd)	KF 17387, CSC	1± 0.057nM (Ki)	[3H]-MSX-2	8nM (Kd)
	HE-NECA	36±2nM (Kd)	5-hydroxy-2-(3-hydroxyphenyl)-4H-1-benzopyran-4-one	1.44uM (Ki)	125I-AB-MECA	25nM (Kd)
	CV-1808	76 nM (Ki)	Istradefylline	36nM (Ki)	[3H]-SCH 58261	2nM (Kd)
	CV-1674	-	-	-	-	-
	ATL146e	0.2nM (Ki)	-	-	[3H]-ZM-241385	-
A2B	Adenosine	24μM (EC50)	MRS1754 (enprofylline)	2nM (Ki)	[3H]-DPCPX)	56 nM (Ki)	(Fredholm, 2007; Fredholm, et al., 2001; Jacobson & Gao, 2006; Linden, Thai, Figler, Jin, & Robeva, 1999; Preti, et al., 2015)
	LUF5835	10nM (Ki)	MRE 2029-F20	3nM (Ki)	125 I-ABOPX	36 nM (Kd)
	2-((4-aryl(alkyl)piperazin-1-yl)alkylamino-5’-Nethylcarboxamidoadenosines scaffold	1487nM (Ki)	OSIP-339391	0.5nM (Ki)	[3H]-ZM-241385	33 nM (Kd)
A3	Adenosine	0.29μM (EC50) 6500nM	MRS 1220	0.7 0.65	[3H]-PIA	16 (Kd)	(J. F. Chen, et al., 2014; Jacobson, et al., 2012; Jacobson, et al., 2018; Y. C. Kim, Ji, & Jacobson, 1996; Klotz, et al., 1998; A. H. Li, Moro, Melman, Ji, & Jacobson, 1998; Olah, et al., 1994; Stockwell, et al., 2017; van Muijlwijk-Koezen, Timmerman, Link, van der Goot, & AP, 1998; van Muijlwijk-Koezen, Timmerman, Link, van der Goot, & Ijzerman, 1998; Varani, et al., 2000)
	2-Cl-IB-MECA	11	MRE 3008-F20	9.54 (pKi)	[3H]-NECA	6.2 (Kd)
	CCPA	18.8μM (Ki) 42	MRS 1191	31.4	125I-AB-MECA	0.6–1.5
	-	-	MRS 1523	18.93nM (Ki)	[3H]-MRE 3008-F20	0.8 (Kd)
	-	-	VUF 8504	17nM	[3H]-MRS7799	0.55nM (Kd)

3.3. Receptor classification and G-protein signaling pathways in cancer relevant functions

3.3.1. A₁ receptor

A₁ receptors inhibit cAMP production by binding and activating intracellular heterotrimeric G proteins Gi and Go. Activation of these proteins in turn inhibits adenylate cyclase (AC), which when inhibited, cannot activate cyclic AMP. Overall, this leads to an inhibition of cAMP production within the cell, thus making A₁ receptors classified as inhibitory (Chen, 2014; Chen, 2014). Additionally, A₁ receptor binding can target the inhibition of calcium and initiation of potassium signaling within the brain specifically (Kirsch, Codina, Birnbaumer, & Brown, 1990). However, the binding of the A₁ receptor to the membrane does have excitatory effects on other molecules. For example, the A₁ receptor binding to the membrane activates G protein G_α16, which in turn causes the β and γ subunits to release. This release causes another signaling cascade by activating PLC to release inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which in turn cause the release of calcium and protein kinase C (PKC), respectively (Fenton, Shea, Doddi, & Dobson Jr., 2010). The binding of the A₁ receptor is also thought to activate β-arrestin1 in a self-downregulating process. As adenosine binds to A₁ receptors on the membrane, this stimulates beta-arrestin1, which in turn activates extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 promotes the uncoupling of adenosine from the A₁ receptor, thus downregulating expression. However, this mechanism of uncoupling and downregulating is seen to simultaneously prepare the cell for increased activity by mediating long-term agonist exposure (Jajoo, Mukherjea, Watabe, & Ramkumar, 2009). Overall, A₁ receptors are classified as inhibitory due to their inhibitory effects on AC and in turn cAMP. The expression levels and function of the A₁ receptor in different cancer types are highly variable. Studies have reported variable A₁ receptor expression levels in tumor cell lines and cancer tissues in comparison to healthy controls (Gessi et al., 2011; Gessi, Merighi, Sacchetto, Simioni, & Borea, 2011; Panjehpour, Hemati, & Forghani, 2012). Despite its role in tumorigenesis, few studies have reported anti-tumor effects exerted by the A₁ receptor including increased apoptosis by activating caspases (Fishman et al., 2001; Gessi, Merighi, Fazzi, et al., 2011; Gessi, Merighi, Sacchetto, et al., 2011; Panjehpour et al., 2012; Regan et al., 2003).

3.3.2. A_2A receptor

A_2A receptor is a G-coupled protein receptor with the highest affinity for adenosine out of the four adenosine receptors. The binding affinity of this receptor for adenosine is ~0.7 μM (EC₅₀) (Fredholm, Irenius, Kull, & Schulte, 2001). A_2A receptor is present in several tissues of the human body including, but not limited to, the striatum, nucleus accumbens, olfactory tubercle, immune cells, heart, lungs, blood vessels, retina, aorta, and a variety of blood cells (Conti, Monopoli, Gamba, Borea, & Ongini, 1993; Dixon, Gubitz, Sirinathsinghji, Richardson, & Freeman, 1996; Fredholm, 2007; Lee et al., 2003; McIntosh & Blazynski, 1994). The A_2A and A_2B receptors are classified separately from A₁ and A₃ receptors because of their property of activating AC (Chen, Lee, & Chern, 2014; de Lera Ruiz, Lim, & Zheng, 2014). This activation leads to an increase in cAMP levels. Subsequently, Protein kinase A (PKA) is activated and phosphorylated to stimulate cAMP-responsive binding protein (CREB) (de Lera Ruiz et al., 2014). In monocytic THP 1 cells, the activated A_2AR uses G-coupled protein receptor kinase 2 (GRK2) to send β-arrestin to the plasma membrane of the cell to desensitize A_2A (Khoa, Postow, Danielsson, & Cronstein, 2006). Evidence shows that the activation of other MAPKs and ERKs results from the activation of A_2AR (Schulte & Fredholm, 2000). Several cancer cell lines overexpress A_2A receptor which is associated with changes in cell proliferation, apoptosis, angiogenesis, anti-tumor protective abilities, and endothelial cell tube formation (Cekic, Day, Sag, & Linden, 2014; Fishman et al., 2003; Gessi, Merighi, Sacchetto, et al., 2011; Ohta, 2016; Sun, Wang, & Hao, 2022; Yu, Zhu, Xie, & Wang, 2020).

3.3.3. A_2B receptor

A_2B receptor has the lowest binding affinity for adenosine out of the four classified adenosine receptors, with an EC₅₀ value of 24 μM (Fredholm et al., 2001). A_2B receptor primarily acts under physiopathological conditions due to its requirement for high adenosine. As an example, A_2B receptors are notably expressed during conditions of hypoxia and ischemic pre-conditioning (Aherne, Kewley, & Eltzschig, 2011; Eltzschig, 2013; Kong, Westerman, Faigle, Eltzschig, & Colgan, 2006). A_2B receptors are mainly located in the gastrointestinal tract, urinary bladder, lung tissues, immune cells, and cecum (Dixon et al., 1996; Jacobson & Gao, 2006). Like A_2A, the A_2B receptor stimulates AC thus increasing cAMP levels followed by PKA activation and CREB stimulation (Feoktistov, Murray, & Biaggioni, 1994). β-arrestin plays a role in the desensitization of A_2B like other adenosine receptors (Mundell, Loudon, & Benovic, 1999). A_2B is considered of less significance than A_2A, however recent data advocate a specific role of this receptor in pathophysiological conditions including cancer (Gessi, Merighi, Fazzi, et al., 2011; Gessi, Merighi, Sacchetto, et al., 2011). A_2B receptor is considered to play a pro-angiogenic role in cancer with demonstrated reduced tumor growth and increased survival rate (Gao & Jacobson, 2019; Sorrentino, Miele, Porta, Pinto, & Morello, 2015). The promoter region of this receptor contains a functional binding site for hypoxiainducible factor (HIF), which leads to the induction of A_2B under hypoxic conditions (Kong et al., 2006). A_2B receptor-dependent signaling in immune cells is exploited by tumor cells for their survival, and several agonists and antagonist compounds have been discovered for targeting this receptor for therapies (Allard, Turcotte, & Stagg, 2017; Gessi, Merighi, Fazzi, et al., 2011; Gessi, Merighi, Sacchetto, et al., 2011; Sitkovsky, 2009).

3.3.4. A₃ receptor

A₃ receptors follow a similar pathway to A₁ receptors and inhibit cAMP production via binding activation of Gαi. Once activated, this protein inhibits AC, resulting in a lack of production of cAMP. Therefore, the binding of adenosine to an A₃ receptor ultimately results in the inhibition of cAMP production, making it an inhibitory adenosine receptor (Chen, 2014). However, the secondary messenger cascades following activation of the A₃ receptor are arguably more complicated and less studied than those of the A₁ receptor. A₃ receptor activation also initiates the PLC pathway, which sets off several secondary messenger pathways (Chen, 2014). Activation of the PLC pathway by the A₃ receptor cascades into activating IP3 and DAG, which in turn increase intracellular calcium and activates PKC (Hammarberg, Schulte, & Fredholm, 2003). Like A1R receptors, this process activates ERK1/2. However, an additional pathway activated by A₃ receptors also results in ERK1/2 activation. When A₃ receptors bind and release the trimeric G-protein, beta and gamma G-proteins are thought to activate the phosphatidylinositol-3-kinase (PI3K) pathway (Schulte & Fredholm, 2000). Increased activity in this pathway is widely accepted as a sign of cancer, which is pertinent to investigating A₃ receptors and immunosuppression interplay (Fruman et al., 2017). PI3K activates Ras, a guanine nucleoside protein, and Akt (protein kinase B), which then together activate the rapidly accelerated fibrosarcoma 1 (RAF1) gene. This gene encodes the protein that makes mitogen-activated protein kinase kinase (MEK), which is the final step in contributing to the activation of the ERK1/2 pathway. Interactions between these two separate pathways that end in ERK1/2 activation are less studied. However, like other adenosine receptors, A₃ receptors are known to recruit β-arrestin, eventually causing receptor desensitization and internalization (Chen, 2014). Various sources have reported ligands that prefer A₃ receptor-mediated β-arrestin translocation, indicating A₃ receptor is a potential avenue for drug and therapy development (Gao & Jacobson, 2008; Gao et al., 2011). A3 receptor is also over-expressed in different types of cancer cells (Fishman, Bar-Yehuda, Madi, & Cohn, 2002; Gessi, Merighi, Fazzi, et al., 2011; Gessi, Merighi, Sacchetto, et al., 2011; Mazziotta et al., 2022) compared to their normal counterparts. The role of the A₃ receptor in mediating anti-tumor actions has been demonstrated in in vitro and in vivo models. A₃ receptor agonists are known to increase natural killer (NK) cell activity, thus promoting killing of tumor cells (Harish, Hohana, Fishman, Arnon, & Bar-Yehuda, 2003; Jacobson et al., 2018). Another study demonstrated that A₃ activation suppresses high ROS levels in prostate cancer cells via inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Jajoo et al., 2009). Further, a reduced activity of ERK1/2 has also been associated with A₃ receptor activation.

4. Adenosine receptor and transporter interplay in cancers

Although adenosine receptors and transporters share the common factor of adenosine itself, the connection is relatively under-studied. While the exact mechanisms are unknown, research suggests a connection between A₁ receptors and CNT2 transporters that is modulated by varying glucose concentrations in the cell, suggesting that extracellular conditions may bring the two together (Duflot et al., 2004). Additionally, researchers found that A₁ receptors activate ATP-sensitive potassium channels which in turn regulate CNT2 transporters, suggesting that different ion channels may potentially be the link between the receptors and transporters (Duflot et al., 2004). Although more research is needed to determine whether these observations apply to the broader spectrum of adenosine receptors and transporters, these findings show that investigating ion channels and outside cellular conditions might further support the link between the transporter and receptor. Potentially, adenosine receptors activate ATP-dependent ion channels through the adenosine exchange, which in turn encourages the flow of additional adenosine through CNTs, most likely CNT2 and CNT3 due to CNT1’s lack of high affinity for adenosine. Therefore, therapies that target the receptors themselves could in turn influence the functionality and expression of transporters, potentially resulting in less toxic and more easily developed therapies. This also begs the question of whether targeting the interplay mechanism, for instance, the potassium-dependent ATP channels, could be the key to regulating transporter levels in cancer patients.

5. Adenosine compartmentalization in immune cells within the tumor microenvironment

Nucleoside transporter proteins have been considered a static component of the purinome up to this point, facilitating the intracellular compartmentalization of adenosine at a constant rate that depends upon the flux between intracellular and extracellular adenosine levels. Notwithstanding the potential for efflux of adenosine under conditions where nucleoside transport and intracellular metabolism are uncoupled, there is evidence that the expression of nucleoside transporter proteins and therefore adenosine compartmentalization is altered in several cancer subtypes (Farre et al., 2004; Pennycooke, Chaudary, Shuralyova, Zhang, & Coe, 2001). Table 4 summarizes previously published studies that have documented alterations in nucleoside transporter expression or function in various human malignancies.

Table 4:

Nucleoside Transporter Alterations in Cancer

Nucleoside transporter	Cancer subtype	Reference
ENT1	Biliary tract	(J. Kim, et al., 2018)
	Cholangiocarcinoma	(Borbath, et al., 2012; Kawamoto, et al., 2018)
	Pancreatic	(Farrell, et al., 2009; Hagmann, Jesnowski, & Lohr, 2010; Hu, et al., 2017; M. Liu, et al., 2020; Weadick, et al., 2021)
	Leukemia	(Abraham, et al., 2015; Homminga, et al., 2011; M. Huang, et al., 2004; J. H. Kim, et al., 2016; Macanas-Pirard, et al., 2012; Molina-Arcas, et al., 2003; Takagaki, et al., 2004; Zimmerman, et al., 2009)
	Breast	(Kenny, et al., 2009)
	Ovarian	(Bock, et al., 2012)
	Gallbladder adenocarcinoma	(Espinoza, et al., 2016)
	Colorectal	(Suenaga, et al., 2017)
	Glioblastoma	(Alarcon, et al., 2020)
ENT2	Ovarian	(Bock, et al., 2012)
	Pancreatic	(Nishio, et al., 2011)
	Leukemia	(Fotoohi, et al., 2006; Homminga, et al., 2011; M. Huang, et al., 2004; Molina-Arcas, et al., 2003)
ENT3	histiocytic sarcoma	(Hsu, et al., 2012)
CNT1	Lung	(P. J. Muller, et al., 2009)
	Pancreatic	(Bhutia, et al., 2011; Garcia-Manteiga, et al., 2003; Hesler, et al., 2016)
	hepatocellular carcinoma	(Zollner, et al., 2005)
	Breast	(Gloeckner-Hofmann, et al., 2006)
CNT3	Ovarian	(Bock, et al., 2012)
	Leukemia	(Fotoohi, et al., 2006)
	Pancreatic	(Hesler, et al., 2016)

In addition to differences in nucleoside transporter expression between normal and tumor tissues, it is also noteworthy to mention that extensive interpatient variability exists in transporter expression. In hepatocellular carcinoma, a study investigating the CNT1 expression in patient samples, found an overall downward trend between normal liver and cancerous samples, although a wide range of variability existed between individual patients (Hesler et al., 2016). Likewise, a downward trend in CNT1 expression was noted in pancreatic cancer samples with distinct interpatient variability (Fotoohi, Lindqvist, Peterson, & Albertioni, 2006). In acute myeloid leukemia (AML), genetic polymorphisms in ENT1 lead to differences in sensitivity to cytarabine (Kim et al., 2016).

Although the altered purinergic signaling during immunosuppression in different cancers is well documented, knowledge of alterations in adenosine receptors on the acting immune cells is also accumulating. It is apparent that cancer cells are sometimes able to evade immunosurveillance and elimination and manage to proliferate and form malignant tumors. Immunosuppression is achieved by multiple mechanisms not limited to the expression of immune checkpoints, downregulation of antigen presentation, and accumulation of immunosuppressive factors. A continually expanding body of research aims to fully appreciate how the immune system is reprogrammed to ignore cancer cells and the mechanisms by which cancer cells evade immune detection to permit tumor growth. Studies have reported the up- and downregulation of ARs, ENTs, and CNTs specific to immune cells during inflammatory stress, as discussed in subsequent sections.

5.1. T cells

T cells express all four adenosine receptors (Gessi et al., 2004; Koshiba, Rosin, Hayashi, Linden, & Sitkovsky, 1999; Mastelic-Gavillet et al., 2019; Mirabet et al., 1999), yet A_2A receptor-mediated downstream adenosine signaling dominates functional regulation of T cells since A_2A receptor is predominantly expressed (Gessi et al., 2004). Additionally, the expression of A_2A and A_2B receptors is induced by hypoxia via HIF-α1 (Sitkovsky, 2009) and Egr-2/Egr-3 anergic stimulation (Beavis et al., 2017; Safford et al., 2005), both conditions characteristic of the TME which often has a limited blood supply and infiltration of CD8⁺ T cells rendered anergic due to the lack of proper priming by co-stimulation. The upregulation of A_2A and A_2B receptors on T cells further magnifies the impact of adenosine ligation. Increased levels of cAMP in T cells activate PKA which in turn phosphorylates and induces the activity of transcription factors CREB, cAMP responsive element modulator (CREM), and activating transcription factor-1 (ATF-1) (Linden & Cekic, 2012). CREB, CREM, and ATF-1 activation modulate the expression of several genes related to inflammation including IL-3, IL-4, IL-17, IFN-γ, and IL-2 (Vigano et al., 2019). Another downstream effector of cAMP, guanine nucleotide exchange factor EPAC1 which is also known as exchange protein activated by cAMP or EPAC, prevents the release of Rap1 from the plasma membrane which is required for TCR-mediated signal transduction via the MEK-ERK pathway (Boussiotis, Freeman, Berezovskaya, Barber, & Nadler, 1997). CD8⁺ T cells perform targeted killing of cells displaying neoantigens on MHC class I molecules. Stimulation of A_2A receptor on CD8⁺ T cells leads to suppression of proliferation and cytotoxic effector function (Mastelic-Gavillet et al., 2019; Ohta et al., 2009). Furthermore, the differentiation of T cells towards an immunosuppressive phenotype called T regulatory cell (Treg), is induced by A_2A receptor signaling (Ohta & Sitkovsky, 2014). Additionally, the secretion of cytokines is suppressed in CD4⁺ T helper (Th) cells with these cells exhibiting a decreased secretion of IFN-γ and decreased secretion of IL-4, IL-5, and IL-10 by Th2 cells (Csoka et al., 2008).

It is known that T cells predominantly express ENTs which promote T cell effector function and expansion (McCaw et al., 2019; Naes, Ab-Rahim, Mazlan, & Abdul Rahman, 2020; Pastor-Anglada & PerezTorras, 2018b). ENT promotes T cell effector function and expansion. ENT3 is abundantly expressed in peripheral T cells, while ENT1 is also expressed in peripheral T cells at lower levels. ENT3 is also suspected of playing a role in peripheral T cell homeostasis, regulation of cell sizes and organelles, and cell survival and proliferation (Hsu et al., 2012; Wei et al., 2018). In the absence of ENT3, peripheral T cells would have dysregulations such as failure to maintain cellular homeostasis, irregular quantities of organelles, failure to proliferate, and undergoing apoptosis. In addition to these factors, the absence of ENT3 in cells showed more disorganization of mitochondria and an increased number of vacuoles within the cells. The absence of ENT3 in cells also showed an increase in size compared to ENT3-proficient cells. The absence of ENT3 in cells also displayed negative effects on autophagy (Nair et al., 2019; Wei et al., 2018). Autophagy allows for the removal of unwanted mitochondria in T cells. Finally, the absence of ENT3 in cells also showed an increase in DNA damage within the affected cells (McCaw et al., 2019; Wei et al., 2018).

5.2. B cells

B cells are a component of humoral immunity and function to secrete antibodies, flagging invading cells for engulfment by circulating phagocytes, and have minor roles in antigen presentation and cytokine secretion. A subset of B cells (B reg) is identified with high expression of extracellular adenosinergic enzymes CD39 and CD73 and is capable of suppressing T cell proliferation and cytokine production (Figueiro et al., 2016; Jeske et al., 2020; Saze et al., 2013). A follow-up study demonstrated that IL-10 may be linked to CD73-mediated immune suppression, although the mechanism is yet unclear (Kaku, Cheng, Al-Abed, & Rothstein, 2014). A new role of B cells in HIV-1 pathology is reported where a skewed CD39/CD73/adenosine pathway leads to activation of the innate immune response, thus, opening new avenues for the treatment of HIV-1 patients (Song et al., 2019).

In the context of nucleoside transport, previous studies have reported significant expression of both ENTs and CNTs (mostly CNT2) transporters in human B cell lines (Raji) (Flanagan & Meckling-Gill, 1997; Soler, Felipe, Casado, Celada, & Pastor-Anglada, 2000). Further, B-cell activators such as phorbol esters (PMA) and bacterial lipopolysaccharide (LPS), were found to upregulate the level of CNTs and downregulate ENTs. A significant up-regulation of CNT3 after PMA treatment in HL-60 cells suggests its potential involvement in the immune regulation of these cells (Ritzel et al., 2001b). Significant changes in nucleoside transporter (ENT1, ENT2, and CNT2) mRNA levels have been observed in B lymphocytes isolated from diabetic rats, where transport alterations are independently and differentially regulated by glucose and insulin (Sakowicz, Szutowicz, & Pawelczyk, 2005).

5.3. Natural killer cells

NK cells are an integral component of the innate immune response and are involved in immunosurveillance by recognizing and eliminating tumorigenic cells. Adenosine has profound immunosuppressive implications for NK cell function in solid tumors. In A_2A receptor-deficient mice, the proportion of terminally mature NK cells is significantly increased compared to mice with intact A_2A receptor signaling, suggesting that adenosine suppresses the development of NK cells (Young et al., 2018). Regarding NK effector function, IL-2 stimulated NK cell cytotoxicity was impaired in both the Fas-ligand and perforinmediated pathways by Gα_s signaling via A_2A and A_2B activation (Raskovalova et al., 2005). Interestingly, Chambers and colleagues found that cytokine priming of NK cells is an important determinant of the cellular pathways activated in response to adenosine signaling, and that pre-treatment with unique cytokine signatures elicited varied NK cell responses (Chambers et al., 2018). IL-2 in presence of adenosine inhibited NK cell cytotoxic function, cytokine production (especially IFN-γ and TNF-α), and associated signaling pathways (Chambers & Matosevic, 2019; Lokshin et al., 2006; Raskovalova et al., 2005; Raskovalova, Lokshin, Huang, Jackson, & Gorelik, 2006). These cells modulate their metabolism to meet the high energy demands during cell proliferation and functionality during inflammatory stress.

An up-regulation in the expression of several solute carrier transporters in NK cells is reported during inflammation (Khan & Khan, 2021). NK cells express key transporters like ENT1, ENT2, and CNT3 which are involved in the uptake and activation of fludarabine (a nucleoside analog) to its active metabolite, fludarabine triphosphate (F-ara-ATP) (Woodahl, Wang, Heimfeld, Sandmaier, & McCune, 2009).

5.4. Dendritic cells

Responsible for antigen processing and presentation, dendritic cells (DCs) are critical for proper training of the adaptive immune system to respond to foreign antigens. There are several subsets of DCs classified mainly based on peculiar cell-surface markers (Collin, McGovern, & Haniffa, 2013; McDonald et al., 2003; Patente, Pelgrom, & Everts, 2019). The classical or conventional myeloid DCs typically express myeloid antigens CD11c, CD13, CD33, and CD11b, whereas plasmacytoid DCs lack the characteristic myeloid antigens and express CD123, CD303, and CD304 respectively. Langerhans cells are another selfrenewing DC population expressed predominantly in the brain. Monocyte-related DCs have numerous diverse subsets including inflammatory non-classical DCs with a range of CD surface markers. Expression of all four AR subtypes has been found on the surface of human DCs, although A₁ and A₃ receptors are expressed during development while the A_2A receptor subtype is most highly expressed after maturation (Panther et al., 2001; Panther et al., 2003; Schnurr et al., 2004). A₁ and A₃ receptors mediate PLC/DAG/IP3/Ca⁺ signaling in immature DCs, while A_2A acts through elevating cAMP levels implicated in the down-regulation of cytokine-producing capacity in mature DCs (Panther et al., 2001; Panther et al., 2003; Schnurr et al., 2004). Notably, the differentiation of dendritic cells in the presence of adenosine is skewed towards a tolerogenic phenotype with decreased cytokine (IL-2) secretion in response to LPS exposure and defective T-cell priming (Challier, Bruniquel, Sewell, & Laugel, 2013). A suppressive tumor-promoting DC phenotype with increased IL-10 secretion was also observed by AR signaling which could be mimicked by treatment with cAMP and PKA analogs (Kayhan, Koyas, Akdemir, Savas, & Cekic, 2019).

While there is an abundance of human nucleoside transporters in dendritic cells in general (Minuesa et al., 2008), notably, ENT1, ENT2, and CNT3 are highly expressed in monocyte-derived dendritic cells (Arimany-Nardi et al., 2014; Minuesa et al., 2008). ENT1 is also, be located on dendritic plasma membranes (Anderson et al., 1999).

5.5. Macrophages

Macrophages originate from undifferentiated stem cells in the bone marrow and proliferate, become activated, or differentiate in the presence of specific growth factors or cytokines. Within the tumor microenvironment, macrophages undergo phenotypic polarization into M1 (classically activated) and M2 (alternatively activated) phenotypes in response to tumor-derived signals (Italiani & Boraschi, 2014; Murray et al., 2014). Both tumor associated macrophage (TAM)1 and TAM2 phenotypes are involved in tumor-related inflammatory reactions, however, M2 is primarily associated with cancer progression via angiogenesis, neovascularization, stromal activation, and remolding (Afik et al., 2016; Ries et al., 2014; Ruffell & Coussens, 2015; Tiainen et al., 2015). TAMs may generate additional extracellular adenosine which can, in turn, activate immunosuppressive adenosine signaling in neighboring immune cells in a paracrine manner. A recent study demonstrated that the generation of adenosine by ovarian cancer cells favored the differentiation of myeloid cells to TAMs which also created extracellular adenosine by overexpression of CD39 and CD73 (Montalban Del Barrio et al., 2016). A separate study showed that treatment of CD39⁺ TAMs with POM-1 (a CD39 inhibitor) or ADA was both able to diminish some of the immunosuppressive functions of TAMs including the secretion of IL-10 (d’Almeida et al., 2016).

Studies have reported co-expression of CNT1, CNT2, ENT1, and ENT2 in murine bone marrow samples (Kong, Engel, & Wang, 2004; PastorAnglada et al., 2001). The findings reveal that nucleoside transporters selectively regulate macrophage proliferation and activation depending on their specific requirements for DNA and RNA synthesis (MecklingGill, Guilbert, & Cass, 1993; Pastor-Anglada et al., 2001; Soler et al., 2001; Soler et al., 2001). Further, IFN-γ differentially regulates transporter expression (CNT1, CNT2, and ENT1) in macrophages via STAT1-dependent and/or independent manner (Soler et al., 2003). ENT3 is an essential nucleoside transporter crucial for lysosomal nucleoside trafficking. A high ENT3 expression in macrophages is associated with the development of lysosomal storage disorder in ENT3 mutant mice (Hsu et al., 2012). ENT3 deficiency triggered myelopoiesis which significantly enhanced the number of splenic macrophages, with evident organ infiltration leading to histiocytic sarcoma in mice. Since ENT3 is known to coordinate lysosomal function with nucleoside availability in immune cells (Wei et al., 2018), transporter loss renders these cells with dysfunctional lysosomal abilities incapable of executing normal clearance and defense functions (Hsu et al., 2012). Abundant immunosuppressive adenosine in TME affects the phagocytic activity of TAMs, via nucleoside transporters (ENTs, CNTs) and/or adenosine receptors (A₁, A_2A, A_2B, and A₃) (Li et al., 2016). A_2A receptor activation reduces M1 polarization, whereas A_2B activation induces M2 macrophage polarization. Hypoxic conditions in TME further facilitate these processes by transcriptional induction of CD39/CD73 and transcriptional downregulation of ENTs (Eltzschig et al., 2005). The precise changes in the nucleoside transport profiles of M1 and M2 macrophages are understudied.

5.6. Myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are formed in the TME when infiltrating myeloid cells are exposed to tumor-associated factors and accumulate as an immunosuppressive cell population in tumors, lymphoid organs, and peripheral blood. They encompass immature myeloid cells from various lineages. In the context of the TME, T-cell responses are suppressed by MDSCs by their production of arginase, reactive oxidative species, and cytokines. Interestingly, MDSC expansion is promoted by A_2B receptor signaling, and the generation of adenosine by CD73 may further augment this expansion in an autocrine fashion (Ryzhov et al., 2011). Additionally, A_2B receptor signaling promotes the production of vascular endothelial growth factor (VEGF) and angiogenesis in MDSCs, whereas pharmacological blockade of the A_2B receptor resulted in decreased immunosuppression and tumor growth in a mouse model of melanoma (Iannone, Miele, Maiolino, Pinto, & Morello, 2013; Sorrentino et al., 2015). The expression of nucleoside transporters in MDSCs is not fully characterized.

6. Regulation of nucleoside transporters in cancer

The function of nucleoside transporters, both ENTs, and the less studied CNTs, as a point of regulation for adenosine compartmentalization in cancer, is less exploited. Summarized below are potential therapeutic avenues for attenuation of adenosine buildup by modulating nucleoside transport processes including mechanisms that alter nucleoside transporters in various physio-pathological states, which could be potentially exploited for the intended purpose.

6.1. Drug treatment

Many nucleoside analogs are used as anti-cancer drugs and rely on nucleoside transporters for intracellular accumulation. This includes nucleoside analog drugs that resemble both purine and pyrimidine nucleosides, such as cytarabine (pyrimidine type) and fludarabine (purine type), both used in the treatment of leukemia (Pastor-Anglada & Perez-Torras, 2015). Numerous reports have studied the relevance of nucleoside transporter expression and function for nucleoside analog chemosensitivity. Interestingly, treatment with chemotherapy can lead to the downregulation of nucleoside transporter expression, compromising the intracellular accumulation of nucleoside analog drugs. This is the case for CNT2 and ENT2 in T-lymphoblastic cell lines resistant to thiopurine nucleoside analogs (Fotoohi et al., 2006). Notably, drug treatment can modulate nucleoside transporter function as well. For instance, HeLa cells were treated with over 60 different cytotoxic drugs, and etoposide was found to significantly enhance the uptake of [18F] fluor thymidine, a positron emission tomography (PET) tracer used to monitor cancer treatment (Lee & Lee, 2013). On the other hand, treatment of K562 leukemia cells with tyrosine-kinase inhibitors can inhibit nucleoside transporter function (Huang, Wang, Mitchell, & Graves, 2004). Hence, there is potential for repurposing these drugs for targeting the nucleoside transporters to ultimately either improve drug uptake or reduce the extracellular adenosine, especially in a chemotherapy setting. Wang et al. published an extensive compilation of the currently marketed drugs as well as those under clinical trials targeting the SLC family of proteins (Wang, Gallo, Jadhav, Hawkins, & Parker, 2020). So far, there are two approved vasodilator drugs namely, dipyridamole and dilazep, that are known to directly target ENT1. Table 5 lists all the clinically approved drugs and non-drug use commercial inhibitors, that can potentially impact the nucleoside transporter function.

Table 5:

Clinical Drugs/Inhibitors Inhibiting ENT And CNT-mediated Nucleoside Transport

Drug Type	Name	Cell lines/animals	Dose	Transporter	[3H]nucleosidee uptake (%Inhibition/IC50)	Reference
Nucleoside Reverse Transcriptase inhibitor (NRTIs)	Entecavir (ETV)	HeLa	1mM	ENT1 ENT2	3.05±1.56mM ([3H] uridine) 2.07±0.29mM ([3H] uridine)	(Miller, et al., 2020)
	Abacavir (ABC)		1mM	ENT1 ENT2	0.11±0.02mM ([3H] uridine) 0.22±0.01mM ([3H] uridine)
	Zidovudine (AZT)		1mM	ENT1 ENT2	2.54±0.26mM ([3H] uridine) 1.64±0.36mM ([3H] uridine)
Pyrimidopyrimidines and Pteridines	Dipyridamole	PK15NTD	10μM	ENT1 ENT2	5.0±0.9nM ([3H]NBMPR) 356±13nM ([3H]NBMPR)	(Boswell-Casteel & Hays, 2017; Boyer, Karjian, Wahl, Pegram, & Neuteboom, 2002; Ward, et al., 2000; J. D. Young Yao, Sun, Cass, & Baldwin, 2008);
Alkyl, Cycloalkyldiamine and Piperazine compounds	Dilazep	PK15NTD (hENT1 and hENT2) H9c2 (rENT2)	10μM	ENT1 ENT2	17.5nM ([3H]-5-uridine) 8800nM ([3H]-5-uridine)	(Playa, et al., 2014)
	Draflazine	U-2 OS	10-11 to 10-6	ENT1 ENT2	5.3nM ([3H]adenosine) 15.85nM ([3H]adenosine) 16.0± 5.9nM ([3H] uridine) 2400± 400nM ([3H] uridine)	(Bohm, et al., 1994; Hammond, 2000; Noji, Karasawa, & Kusaka, 2004; Vlachodimou, Konstantinopoulou, AP, & Heitman, 2020)
Cannabinoids	Tetrahydrocannabinol (THC)	EOC-20 RAW264.7	100–1000nM	ENT1	0.17μM ([3H]thymidine) 0.27μM ([3H]adenosine)	(Carrier, et al., 2006; Stollenwerk, Pollock, & Hillard, 2021)
	Cannabidiol (CBD)		100–1000nM	ENT1	0.19μM ([3H]thymidine) 0.12μM ([3H]adenosine)
Quinolinone derivative	Cilostazol (Pletal)	Cardiac ventricular myocytes, Coronary artery smooth muscle, Endothelial cells	5–10μM	ENT1	~10μM (EC50 ([3H]adenosine)	(Y. Liu, et al., 2000)
Receptor inhibitors	Propentofylline	L1210/B23.1 cells; L1210/C2; Walker 256; L1210/MA27.1		es ei cif	9μM ([3H]adenosine) 170μM ([3H]adenosine) 6mM ([3H]adenosine)	(Parkinson, Paterson, Young, & Cass, 1993; Parkinson, Rudolphi, & Fredholm, 1994)
	Ticagrelor	MDCK	0.1–100 nM/L; 1–100 μM/L	ENT1 ENT2	6.59+0.09μM ([3H]adenosine) 4.76+0.03μM ([3H]adenosine)	(Armstrong, et al., 2014)
Thiazolidinediones	Troglitazone	HASMC	30μM	ENT1	2.35±0.35μM ([3H]adenosine) 4.38±0.34μM ([3H]uridine) 3.99±0.57μM ([3H]NBMPR)	(Leung, Man, & Tse, 2005)
	Pioglitazone		30μM	ENT1	13% ([3H]adenosine)
	Ciglitazone		30μM	ENT1	8% ([3H]adenosine)
Antiviral drugs	Remdesivir	HeLa	-	ENT1 ENT2	38±2μM ([3H]uridine) 73±14μM ([3H]uridine)	(Miller, et al., 2021)
	EIDD-1931		-	ENT1 ENT2	259±118μM ([3H]uridine) 467±101μM ([3H]uridine)
	Molnupiravir		-	ENT1 ENT2	701±294μM ([3H]uridine) 851±152μM ([3H]uridine)
Dihydrochalcones	Phloridzin	-	-	CNT3	16±0.01μM (Ki)	(Gupte & Buolamwini, 2009)
Tyrosine kinase inhibitors (TKIs)	Erlotinib	AsPc-1 A549, H292, H1975 cells, Yeast	0–100μM	CNT1 CNT3 ENT1	160±20μM ([3H]-uridine) 11±1μM ([3H]-uridine) 1.6±0.4 to 34±6μM ([3H]-uridine)	(Damaraju, et al., 2014)
	Gefitinib			CNT1 ENT1	37±11μM ([3H]-uridine) 2.0±0.6 to 14±6μM ([3H]-uridine)
	Vandetanib			CNT1 CNT2 CNT3 ENT1 ENT2	64±17μM ([3H]-uridine) 82±4μM ([3H]-uridine) 28±9μM ([3H]-uridine) 11±1 to 33±8μM ([3H]-uridine) 89±17μM ([3H]-uridine)
	Imatinib	Saccharomyces cerevisiae	0–300μM	CNT2 ENT1	2.3μM ([3H]uridine) 110μM ([3H]uridine)	(Damaraju, Weber, Kuzma, Cass, & Sawyer, 2016)
	Dasatinib			ENT1	60μM ([3H]uridine)
	Bosutinib			ENT1	13μM ([3H]uridine)
	Nilotinib			ENT1	0.7μM ([3H]uridine)
	Ponatinib			ENT1	9μM ([3H]uridine)
	Abemaciclib	HAP1-ENT2 KO	10μM	ENT1	25.0±7.9% ([3H]-uridine)	(Jouan, et al., 2021)
	Acalabrutinib		10μM	ENT1	42.6±10.4% ([3H]-uridine)
	Afatinib		10μM	ENT1	54.5±2.4% ([3H]-uridine)
	Alectinib		10μM	ENT1	54.0±4.9% ([3H]-uridine)
	Brigatinib		10μM	ENT1	52.5±11.1% ([3H]-uridine)
	Cabozantinib		10μM	ENT1	48.8±2.5% ([3H]-uridine)
	Ceritinib		10μM	ENT1	26.3±1.7% ([3H]-uridine)
	Crizotinib		10μM	ENT1	34.7±2.7% ([3H]-uridine)
	Dacomitinib		10μM	ENT1	43.9±2.3% ([3H]-uridine)
	Entrectinib		10μM	ENT1	22.9±5.2% ([3H]-uridine)
	Ibrutinib		10μM	ENT1	72.7±1.1% ([3H]-uridine)
	Itacitinib		10μM	ENT1	21.9±13.3% ([3H]-uridine)
	Lapatinib		10μM	ENT1	25.7±15.2% ([3H]-uridine)
	Lenvatinib		10μM	ENT1	63.1±5.9% ([3H]-uridine)
	Lorlatinib		10μM	ENT1	86.6±6.4% ([3H]-uridine)
	Neratinib		10μM	ENT1	61.0±8.5% ([3H]-uridine)
	Nintedanib		10μM	ENT1	46.9±9.6% ([3H]-uridine)
	Osimertinib		10μM	ENT1	32.3±11.3% ([3H]-uridine)
	Pacritinib		10μM	ENT1	61.6±8.1% ([3H]-uridine)
	Regorafenib		10μM	ENT1	21.4±2.7% ([3H]-uridine)
	Ribociclib		10μM	ENT1	17.8±10.2% ([3H]-uridine)
	Ruxolitinib		10μM	ENT1	34.1±13.2% ([3H]-uridine)
	Tofacitinib		10μM	ENT1	4.8±8.3% ([3H]-uridine)
	Vemurafenib		10μM	ENT1	27.3±10.9% ([3H]-uridine)
Inhibitors not approved for drug use	S-(4-nitrobenzyl)-6-thioinosine (NBMPR)	PK15NTD	1μM	ENT1, 2	0.4± 0.1nM ([3H]NBMPR) 2.8±0.3mM ([3H]NBMPR)	(Ward, et al., 2000)
	FPMINT		10nM-100μM	ENT1 ENT2	8.04±1.04μM ([3H]-uridine) 1.34±0.154μM ([3H]-uridine)	(R. Li, et al., 2022)
	Rapadocin		10μM	ENT1	3.3nM ([3H]-thymidine)	(Y. Wang, et al., 2021)

6.2. Epithelial-mesenchymal transition

The epithelial-mesenchymal transition (EMT) is a set of phenotypic changes that confer cancer cells with migratory and invasive properties. During EMT, cells undergo morphologic changes driven by EMT master-regulator transcription factors such as SNAI1 and TWIST that result in the rearrangement of cytoskeletal elements, changes in cell polarity, and loss of cell-cell adhesion (Nieto, Huang, Jackson, & Thiery, 2016). Zheng and colleagues demonstrated that silencing the EMT program in a spontaneous mouse model of pancreatic cancer resulted in increased expression of ENT2 and CNT3 (Zheng et al., 2015). A recent study on the loss of ENT1 in pancreatic cancer further established the role of EMT in regulating nucleoside transporter expression, identifying that altered expression of adhesion molecules, especially the downregulation of E-cadherin and EpCAM and upregulation of N-cadherin, correlated with decreased ENT1 at the plasma membrane (Weadick et al., 2021). Interestingly, mesenchymal-type pancreatic cancer mouse xenografts had a loss of ENT1 and decreased sensitivity to gemcitabine (a mainline agent for treating pancreatic cancer) treatment, resulting in larger tumors and more extensive metastatic spread (Weadick et al., 2021). In addition, the loss of cellular polarity in EMT may shift the directionality of vectorial transport of nucleosides mediated by ENTs and thereby outcomes. Elaskalani and colleagues studied the role of platelet-derived ATP and ADP and found that ADP-mediated P2Y₁₂ receptor purinergic signaling regulated the expression of EMT transcription factor SLUG, which in turn suppressed ENT1 expression in pancreatic cancer cells (Elaskalani, Falasca, Moran, Berndt, & Metharom, 2017).

6.3. Secreted factors in the TME

The TME fosters an environment that favors tumor progression, drug resistance, and metastasis. Besides tumor cells, the TME can include cancer-associated fibroblasts, stromal cells, stellate cells, and immune cells. Many of these cell types secrete soluble factors, i.e., cytokines, that modulate tumor cells, including the expression and function of nucleoside transporters. In leukemia, the TME consists of bone marrow that harbors a cancer-promoting niche that includes stromal cells which influence leukemic cells. In particular, the sensitivity of AML cells to nucleoside analog cytarabine was decreased by co-culture with stromal cells, and mechanistic studies found that ENT1 transport capacity was reduced by over 50% due to soluble factors secreted by stromal cells (Macanas-Pirard et al., 2012). In pancreatic cancer, a negative association between the mRNA expression of matricellular protein CYR61 in pancreatic stellate cells and ENT1 and CNT3 led the researchers to further investigate. They found that transforming growth factor beta (TGF-β) signaling induced the expression of CYR61 in stellate cells, which decreased gemcitabine chemosensitivity by downregulation of ENT1 and CNT3 (Hesler et al., 2016). Both studies point to the role of the TME in regulating the expression of nucleoside transporters.

It is a distinct possibility that the dysfunction of nucleoside transport processes contributes to the buildup of extracellular adenosine by failure to facilitate the uptake of adenosine. One cannot safely assume that adenosine intracellular compartmentalization, performed exclusively by nucleoside transporters, is static in the tumor microenvironment. Depending on the discrepancy between normal tissue expression of nucleoside transporters and expression level in cancer, loss of a nucleoside transporter could be a highly significant factor. This may provide further explanation to what is currently known about extracellular adenosine accumulation; at least partially influenced by ATP release from cancer cells driven by metabolic changes and stress, and the overexpression of CD39/CD73 that metabolizes ATP to adenosine in the extracellular space.

6.4. Soluble factors in the TME

Notably, some soluble factors that are characteristic of the tumor-friendly TME have demonstrated the capability to influence nucleoside transporter expression or function. Cytokines are chemical messengers secreted by immune cells and stromal cells that modulate cell behavior in both autocrine and paracrine manners. The cytokine profile in tumor tissues can vary greatly from normal tissues, and cytokine signaling is involved in the selective process that drives the evolution of cancer cells. IFN-γ is a cytokine that modulates innate and adaptive immune responses. Interestingly, IFN-γ inhibited ENT1 activity via signal transduction and activator of transcription 1 (STAT-1) while CNT1 and CNT2 were upregulated by STAT-1 independent mechanisms (Soler et al., 2003). TGF-β is commonly found in the TME at elevated concentration levels and can lead to the downregulation of CNT1 and CNT3 in pancreatic cancer by induced expression of CYR61 in stromal cells (Hesler et al., 2016). In chronic lymphocytic leukemia, however, CNT3 plasma membrane trafficking and function are enhanced by treatment with all-trans retinoic acid by activation of TFG-β whose downstream effectors promote CNT3 trafficking (Fernandez-Calotti & Pastor-Anglada, 2010).

The TME of many solid tumors is lacking in oxygenation as angiogenesis struggles to keep up with the growing demand for blood flow to cancer cells. Under hypoxic conditions, expression of the transcription factor HIF-1 is increased, reprogramming cellular metabolism and homeostasis to adapt to anaerobic conditions. HIF-1 binds to the promoter of ENT1, repressing the expression of this transporter protein, and decreasing the uptake of adenosine, ultimately leading to enhanced extracellular adenosine (Eltzschig et al., 2005). Based on these findings, the authors speculate that the hypoxia phenomenon may be a putative mechanism that drives the accumulation of extracellular adenosine.

7. Targeting nucleoside transport to curtail immunosuppressive adenosine signaling

Situated as the shuttles between intracellular and extracellular adenosine levels, nucleoside transporter proteins can play a central role in regulating adenosine signaling capabilities. Therefore, manipulation of transporter expression or function can modulate levels of extracellular adenosine and the impact of adenosine receptor signaling. Considering the buildup of extracellular adenosine in the TME and its overall immunosuppressive qualities, an ideal intervention would limit this accumulation of adenosine outside of cells. One can conceive that this could be accomplished primarily by enhancing the intracellular translocation of adenosine via CNTs and ENTs. Yet, it is possible that in scenarios where nucleoside transport and intracellular metabolism are uncoupled, significant adenosine efflux could occur via ENTs, in which case inhibiting transporter function would be beneficial.

7.1. Pharmacologic targeting of nucleoside transporters

Indeed, inhibition of nucleoside transporters is already in use in the cardiovascular system. Adenosine reuptake inhibitor drugs such as dilazep and dipyridamole block individual or multiple ENTs to raise levels of extracellular adenosine and augment the cardioprotective function of adenosine receptor signaling. While blocking ENTs could raise adenosine levels and potentiate immunosuppressive signaling, analysis of the purinome may reveal that efflux via ENTs contributes to the buildup of adenosine, and inhibition of this efflux would help contain adenosine inside cells. In this case, currently approved adenosine reuptake inhibitors may provide therapeutic benefits (Table 5). Considering the multiple genetic and epigenetic alterations in cancer cells and the abnormal conditions of the TME, it is reasonable to hypothesize that significant disruption of purine homeostasis may involve adenosine efflux.

Some of the clinical trials and lab-scale pilot studies have targeted the anti-tumor aspect of inhibitor drugs, dipyridamole, and dilazep in combination treatment with other compounds. The studies as summarized in Table 6, advocate a significant downfall in cancer risk among patients. However, there is limited clinical data available to support the in vivo applications. Moreover, the studies largely involved insufficient patient cohorts to reach any evident conclusion. Therefore, it calls for further extensive and high-throughput studies focused on implementing these treatments in patients.

Table 6:

Clinical Trials and Studies Involving Inhibitor Drugs Targeting Nucleoside Transporters

Human trials
Inhibitor	Cancer		Study Phase		Study type	Identifier/Approval		Purpose		Outcomes		Reference
Dipyridamole	Ovarian		Phase 2		Interventional	NCT00002487		Studied the effectiveness of combining methotrexate and dipyridamole		Not reported		-
	Lymphoid neoplasms		-		Interventional	Dnr 2012/795		Exploring the chemo-preventive potential of dipyridamole		Significant reduction in risk of lymphoid neoplasms (95% CI)		(W. Huang, Sundquist, Sundquist, & Ji, 2022)
	Several cancers		-		Observational	-		Antiplatelet agents aspirin and dipyridamole and the risk of different carcinoma in patients with type 2 diabetes mellitus		dipyridamole and aspirin decreased the risk of liver cancer		(H. Y. Huang, et al., 2022)
In vitro studies
Inhibitor		Cancer		Cell line			Concentration		Outcomes		Reference
Dipyridamole		Colorectal cancer		HCT-8, CD133+/CD44+ HCT-8, U937 cells			0–20μM		Decreased proliferation of cancer cells		(Abdelghany El-Mahdy, Kawabata, Goto, & Li, 2021)
		Leukemia		CFU-GEMM, BFU-E, CFU-GM, HL-60/C1, CCRF-CEM			0.1μM-10μM		reduced toxicity of tubercidin		(Cass, King, Montano, & Janowska-Wieczorek, 1992)
Dilazep		Prostate cancer		LNCaP, LNCaP-Abl, 22Rv1, LAPC4			0–50μM		inhibit proliferation and AR signaling		(Kaochar, et al., 2021)
		Leukemia		CFU-GEMM, BFU-E, CFU-GM, HL-60/C1, CCRF-CEM			0.1μM-10μM		reduced toxicity of tubercidin		(Cass, et al., 1992)

7.2. Genetic regulation of nucleoside transporters

Evidence in the literature points to epigenetic regulation of nucleoside transporter expression, which is a potential point of therapeutic intervention. CNT1 is heavily expressed in the liver, kidneys, and intestines, and its expression can be induced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4α), which acts by binding to the CNT1 promoter (Klein, Kullak-Ublick, Wagner, Trauner, & Eloranta, 2009). Further, the expression of ENT1 was reduced in a chronic myelogenous leukemia mouse model in response to chemical stress mediated by JNK activation and c-Jun binding to the mENT1 promoter (Leisewitz et al., 2011). Similarly, the expression of zinc transporter ZIP4 activated ZEB1 and induced integrin signaling causing reduced expression of ENT1 via JNK/c-Jun signaling (Liu et al., 2020). Further, loss of histone 3 lysine 27 demethylase KDM6A expression led to nucleoside analog drug resistance in acute myeloid leukemia cells by reducing the acetylation at the ENT1 locus (Stief et al., 2020). Together, these studies show that epigenetic changes modulate the expression of nucleoside transporters. Activation of the signaling pathways known to affect transporter expression, such as JNK/c-Jun and ENT1, could be an alternative to the use of pharmacologic inhibitors. In the case of HNF4α, inducing this transcription factor in tumors to increase the expression of CNT1 could increase the intracellular compartmentalization of adenosine, especially considering that CNTs work against the concentration gradient to concentrate nucleosides inside of cells. In that manner, induction of CNTs could convert tumor cells into immunosuppressive adenosine “sinks.”

Different gene editing methods have been explored to mitigate the effects caused due to transporter loss, by upregulating transporter genes via gene promotors, and at the transcriptional level, targeting epigenetic regulation. Restoring the expression of hCNT1 in NP-9 and NP29 cells via adenoviral vectors affected cell-cycle progression, cell migration, and basal phosphorylation status of selected signaling kinases and significantly reduced tumor growth in an ectopic mouse model of human adenocarcinoma (Perez-Torras et al., 2013). Garcia-Manteiga et al. reported increased gemcitabine sensitivity in pancreatic neoplasia cells (NP9, NP18, NP29, and NP31) heterologously expressing hCNT1, despite high constitutive hENT1 activity (Garcia-Manteiga, MolinaArcas, Casado, Mazo, & Pastor-Anglada, 2003). Similarly, Bhutia et al. showed that hCNT1 overexpression improved the anti-tumorigenic and chemosensitivity potential of pancreatic cancer cells (Bhutia, Hung, Patel, Lovin, & Govindarajan, 2011). Overexpression of hENT1 enhanced gemcitabine response in human pancreatic cancer, and cells lacking hENT1 expression are highly resistant to gemcitabine (Mori et al., 2007; Perez-Torras et al., 2008; Perez-Torras et al., 2013).

Micro RNAs (miRs) represent another form of epigenetic regulation that could potentially be harnessed to modulate nucleoside transporters. Although there is still much to be learned about how miRs may downregulate the expression of nucleoside transporters, a recent study evaluating gene-miR interactions identified 175 miRs that may target ENT1. Interestingly, 4 ENT1-miR hits are upregulated in pancreatic cancer (Randazzo et al., 2020). CRISPR-Cas9 gene editing represents the gold standard for precise programming of the genetic code. In addition, there is a significant difference in CNT1 mRNA and protein in human pancreatic cancers, likely due to miR dysregulation, as demonstrated by independent studies (Bhutia et al., 2011; Boces-Pascual et al., 2021). The CRISPR technology could be used to deliver nucleoside transporter genes to tumor tissues which may display loss of expression. Conversely, CRISPR-based deletion of ENTs may be strategic in situations where efflux of adenosine is observed. Table 7 is a compilation of the nucleic acid therapeutics, particularly miRs and siRNA-based silencing of nucleoside transporters with potential application in cancer therapy.

Table 7:

Nucleic Acid-Based Approaches Targeting Nucleoside Transporters

Type	Name	Disease state	Animal/Cell lines	Nucleoside transporter targeted	Outcome	Reference
Micro-RNA	MiR-26b	Cirrhotic portal hypertension	Rats	ENT1	Regulate the invasion and migration ability by targeting hENT1 depending on the RhoA/ROCK-1 pathway	(Y. Chen & Tian, 2019; Y. Gao & Yang, 2017)
		Lung cancer	A549 cells
	miR-106a	Colorectal carcinoma and Pancreatic cancer	NP9; CP15T; CaCo2; HT29	CNT1	hCNT1 inhibition could contribute towards chemoresistance to fluoropyrimidine-based treatments	(Boces-Pascual, et al., 2021)
	miR-17
	hsa-miR-221-5p	Pancreatic cancer	-	ENT1	Downregulation of hENT1 in pancreatic cancer contributes to chemoresistance	(SenGupta, et al., 2002)
	hsa-miR-23b-3p		-	ENT1		(Jaramillo, et al., 2020)
	hsa-miR-155-5p		-	ENT1		(Sundaram, et al., 1998)
	hsa-miR-196a-3p		-	ENT1		(Mantini, et al., 2020; Randazzo, et al., 2020)
Small-interfering RNA (siRNA)	-	Astrocytes	Primary astrocytes	ENT3	ENT3 downregulation abolished the release of gliotransmitter ATP via inhibition of adenosine uptake in vesicles	(D. Song, et al., 2014)
	-	Leukemia	MOLT4	CNT3, ENT1, ENT2	Inhibition of nucleoside transport resulted in increased 6-MP tolerability	(Fotoohi, et al., 2006)
	-	Prostate cancer	PC3 cells	ENT1	Impaired ENT activity imparts anti-proliferative potential to Cimicifuga racemosa extract BNO-1055	(Dueregger, et al., 2013)
Overexpression	Recombinant adenovirus (Ad-hENT1)	Pancreatic cancer	NP-9, NP-18, NP-2	ENT1	Improved the therapeutic response to gemcitabine	(Perez-Torras, et al., 2008)
Nucleic acid conjugates	Lipid conjugates	Pancreatic cancer	HEK293	CNT3	Delivery of hCNT3 cDNA using ultrasound and microbubbles reversed gemcitabine resistance	(Paproski, et al., 2013)

7.3. Nanocarriers: drug delivery via transporter-independent mode

Drug delivery via transporter-independent mode has shown promising potential in overcoming chemoresistance due to transporter loss. Nano-formulations of nucleoside analog drugs such as gemcitabine overcame chemoresistance in ENT1-deficient mouse lung cancer cells (Wonganan et al., 2013) and ovarian cancer cells with low hCNT1 expression (Hung et al., 2015). Table 8 lists the various types of nanomedicines employed for increasing gemcitabine delivery into different cancer cell lines. This strategy could be adopted when nucleoside transporters are required to be inhibited in which case the hindered chemotherapeutic uptake can be circumvented using nanocarriers.

Table 8:

Nanocarriers for Gemcitabine Delivery into Cancer Cells

Nanomedicine	Ingredient	Cell system	Reference
Nanoparticles	PLGA, Chitosan, BSA, Soy lecithin, GMS, Tween 20, PLGA-b-PEG-OH, Stearic acid, Squalene, Lipid bilayer-MSNP	Panc-1, MIA PaCa-2, AsPc-1, CEM-AraC-8C, CEM-AraC-8D, L1210R	(Arya, Vandana, Acharya, & Sahoo, 2011; Chung, Sandoval, Sloat, Lansakara, & Cui, 2012; Cosco, et al., 2012; Daman, Ostad, Amini, & Gilani, 2014; Hung, et al., 2015; Lansakara, Rodriguez, & Cui, 2012; J. Li, et al., 2013; Meng, et al., 2015; Papa, et al., 2012; Reddy, et al., 2007; Sloat, et al., 2011; Wonganan, et al., 2013)
Nanoassemblies	Ethanol, aqueous dextrose solution	Panc-1, L1210 10K cells	(Maksimenko, Caron, Mougin, Desmaele, & Couvreur, 2015)
Liposomes	DPPC, Chol, DSPE-PEG2000, DOPE, CHEMS, DSPE-mPEG2000	MDA-MB-231 breast cancer cells, MIA PaCa-2	(Papa, et al., 2013; Y. Xu & Meng, 2016)
Micelles	PEG-PLA, Stearic acid, PEG-PCC, Stearoyl-PEG	AsPc-1, MIA PaCa-2	(Chitkara, Mittal, Behrman, Kumar, & Mahato, 2013; Daman, et al., 2014; Papa, et al., 2012; Zhu, Budin, & Szostak, 2013)

7.4. Improving the efficacy of nucleoside transporters

The efficacy of nucleoside transporters in TME is jeopardized due to the overexpression of mucin proteins in certain cancer types like pancreatic cancer. This results in the formation of a mucoid barrier between cancer cells and the extracellular matrix. The disruption of mucolytic layers produced by mucins on cancerous cells enables the effective uptake of cytotoxic drugs into cells resulting in better patient survival. Therefore, several agents with mucolytic properties have been investigated for targeting mucins, especially in pancreatic cancer (Gautam et al., 2017; Spratlin et al., 2004). A variety of miRNAs, pharmacological and natural agents have also been investigated for their ability to limit pancreatic cancer progression by specifically targeting and downregulating well-known mucins such as MUC1 and MUC4 (Srivastava et al., 2011; Torres, Chakraborty, Souchek, & Batra, 2012). Four key miRNAs (miR-Let-7b, miR-150, miR-219-1-3p, and miR-200c) have been investigated and found to downregulate MUC4 expression (Arora et al., 2014; Lahdaoui et al., 2015; Seshacharyulu et al., 2015; Wang et al., 2013). Mucins can also be controlled through anti-inflammatory drugs, corticosteroids such as dexamethasone, as well as pan-EGFR family inhibitors, afatinib and canertinib (Gollub, Waksman, Goswami, & Marom, 1995; Macha et al., 2013; Torres et al., 2012). MUC4 mucin is reported to negatively regulate the CNT1 transporter expression via the NF-κB pathway and alter the drug-susceptibility of pancreatic tumor cells. MUC4-deficient pancreatic cancer cell lines (CAPAN-1 and CAPAN-1) were found to be sensitive to gemcitabine and reduced activation of NF-κB-mediated signaling pathways in MUC4-deficient cells is associated with gemcitabine sensitivity and regulation of CNT1 (Carter, Mekkawy, & Morris, 2021; Skrypek et al., 2013). Such studies emphasize the therapeutic value of strategies targeting the down-regulation of mucin proteins to enhance transporter expression and/or function as well as highlight the potential of exogenous transporter overexpression for controlling mucin production in cancer cells.

Bromelain (a cysteine proteolytic enzyme extracted from the stem of Ananas comosus) has been used for its anti-inflammatory and mucolytic properties for the treatment of ovarian and breast cancer with an evident decrease in metastasis and tumor growth. Patients treated with bromelain in combination with chemotherapeutic agents such as 5-FU resulted in greater patient outcomes with more tumor regression (Amini, Masoumi-Moghaddam, Ehteda, Liauw, & Morris, 2016; Carter et al., 2021; Gautam et al., 2017; Pillai et al., 2020; Suh, Pillai, & Morris, 2017; Zheng, 2017; Pillai et al., 2020).

7.5. Other routes to modulate nucleoside transporters

The dietary intervention of naturally occurring compounds such as Indole-3-carbinol (I3C) has shown promising chemopreventive effects for breast cancer prevention (Wong et al., 1997). I3C, in combination with clinically relevant concentrations of gemcitabine, has been reported to enhance cancer cell death through up-regulation of hENT1 in pancreatic cancer (Wang, Word, & Lyn-Cook, 2011).

8. Future directions

Nucleoside transporters are central players in dictating the levels of extracellular adenosine, which has a profound effect on tumor immunity. Adenosine recycling between various metabolic and signaling pathways would be severely limited without the transmembrane flux of adenosine conducted by nucleoside transporters. Although traditionally thought of as only facilitating the inward-directed transfer of adenosine, the possibility of efflux via ENTs highlights the versatility and dynamic nature of nucleoside transport processes within the purinome. Up to this point, most research on nucleoside transporter pharmacologic intervention has been in the cardiovascular and nervous systems. However, minimal studies have been conducted on the function of nucleoside transporters in cancer as it pertains to the regulation of adenosine concentration levels. Below we will describe some of the most important topics for further study that will hasten the development of therapies targeting nucleoside transporters in cancer.

8.1. Evaluating potentially important, yet understudied, component of the purinome: CNTs

Concentrative nucleoside transporters remain underexplored compared to ENTs, and much is still to be determined about their function in the purinome. Bidirectional ENTs play a major role in maintaining nucleoside homeostasis, whereas CNTs contribute to nucleoside sensing and signal transduction. The putative role of CNTs as transceptors is abundantly evident from recent research. The interacting proteins identified for hCNT2 suggest a link to energy metabolism. Being purine transporters, CNT2 and CNT3 also play a role in purinergic signaling by mediating adenosine transport.

As mentioned earlier, previous studies on nucleoside transporter proteins and their contribution to adenosine levels have been conducted in the central nervous system or cardiovascular system. For example, ENTs regulate vascular tension by modulation of adenosine receptor signaling in the cardiovascular system. However, these tissues specifically rely on ENTs with little or no contribution of CNTs. Both CNTs and ENTs are likely involved in regulating adenosine levels in tumors. CNTs may be especially important in early-stage tumors of epithelial origin, as epithelial-type cells classically display asymmetric distribution of ENTs and CNTs, with CNTs decorating the apical surface of cells that directly interfaces with the extracellular environment. As dedifferentiation is frequently observed in cancer progression, it will be important to study changes in CNTs that may occur during cancer cell transformations as more malignant cells gain survival advantages in response to drug treatment and other selective pressures in the TME. Additionally, CNTs may have a more pronounced role in cancers arising from tissue types with high endogenous levels of CNTs. Nevertheless, further research is warranted for cases such as the involvement of CNT3 in pancreatic cancer, as CNT3 is highly expressed in pancreas acinar cells. The development of CNT knockout mice models is also lacking, and experimentation using model animal systems will be critical for understanding the physiological relevance of these transporters. Additionally, CNT knockout mouse models could be used to differentiate between adenosine-dependent tumor cell-intrinsic and extrinsic influences on tumor growth. Such studies are likely to uncover the role of adenosine transporters in tumor cell-stromal cell and tumor cell-immune cell interplays.

All in vitro and in vivo studies have provided significant insights into the tissue-specific distribution and functionality of different nucleoside transporters. The gene knockdown/knockout investigations have been beneficial in figuring out the transporter-specific functions and crosstalk with other signaling players. The exogenous transporter expression studies demonstrated their potential in overcoming drug chemoresistance in tumor cells. The in silico computational and molecular modeling has deciphered the structural aspects of drug-transporter interactions. In nutshell, prior research has improved the understanding of nucleoside transporter-adenosine receptor interplay. However, there are critical challenges to the therapeutic success of these strategies. The application of CNT-based approaches requires a parallel inhibition of predominant ENT transporters to prevent leaching out of adenosine and counterbalance the nucleoside flux. However, specific, and complete inhibition of a transporter is not always achievable and presents a major challenge in studying the role of individual transporter in signaling pathways. Another bottleneck is the shared substrate selectivity and often tissue localization of both ENTs and CNTs, which could lead to undesirable off-target effects of inhibitor compounds and drugs targeting nucleoside transport.

Non-specific targeting by drugs could result in systemic inhibition of nucleoside transporters, disrupting the normal flow of nucleosides across cellular compartments. Increased extracellular levels of nucleosides such as adenosine might trigger the inflammatory pathways, especially affecting CNS activity, causing sleep disturbances and drowsiness (Bjorness & Greene, 2009; Sims, Wu, & Dale, 2013). However, not all nucleoside transporters cause adverse effects upon inhibition or downregulation. Studies on ENT-null mice have demonstrated that adenosine uptake is almost completely abolished in ENT1^−/− mice along with a slightly decreased body weight, increased alcohol consumption, and reduced hypnotic and ataxic responses to ethanol due to low adenosine levels (Choi et al., 2004; http://www.informatics.jax. org/marker/MGI:1927073). ENT2-null mice exhibited normal adenosine uptake in erythrocytes with protective effects in case of acute lung injury is observed (http://www.informatics.jax.org/marker/MGI:1345278). In contrast, a variety of severe health effects including lymphadenopathy, splenomegaly, histiocytic sarcoma, anemia, and premature death associated with extramedullary hematopoiesis, increased macrophage proliferation, apoptosis, and abnormal lysosome function have been reported in ENT3-null mice (http://www.informatics.jax.org/marker/MGI:1918529). Studies in mice models with knockout genotype for the key effectors involved in purinergic signaling (adenosine receptors, protein kinases) demonstrated a neutral unaltered phenotype compared to wildtype (Braz et al., 2004; Chen et al., 1999; Gimenez-Llort et al., 2002; Hua et al., 2007; Johansson et al., 2001; Ledent et al., 1997; Salvatore et al., 2000; Xiao, Liu, Jacobson, Gavrilova, & Reitman, 2019), with no significant effect on growth, metabolism, breeding, and body temperature regulation. To summarize, systemic effects are mostly observed with inhibition or knockout of predominant ENT transporters, especially ENT3 and a mild phenotype is evident with the absence of ENT1 and ENT2. CNTs are facilitatory transporters working alongside ENTs, there will likely be minimal adverse effects under conditions of their complete systemic inhibition/down-regulation.

The reversible competitive uptake of nucleoside analogs by CNTs/ENTs is another common phenomenon reported in numerous uptake studies (Carrier, Auchampach, & Hillard, 2006; Huang, Zeng, Shi, & Liu, 2017; Scholtissek, 1968). It is certainly useful for short-term assessment studies but as soon as the optimal concentration of a specific substrate is restored, the transport may come back to the default setting. CNTs and ENTs have a relatively short half-life and soon be replaced with nascent proteins via cellular protein synthesis machinery. Therefore, it is highly likely that the effects due to irreversible inhibition would not last long enough to cause any significant effect on cellular function. Lastly, the lack of in vivo data limits the therapeutic potential of most in vitro approaches. The generation of knockout mice and xenograft models, which closely mimic the human tumor microenvironment, is a very expensive and time-consuming process. Apart from ethical concerns associated with animal use in research, the huge failure rate in the accurate translation of animal data in phase I clinical trials and approval of new drugs have raised concerns about the importance of these models (Plenge, Scolnick, & Altshuler, 2013; Swaminathan, Kumar, & Kaul, 2019). Technological advances in tissue engineering, ‘omics’ approaches, and in silico modeling are enabling. With advances in high throughput cutting-edge instrumentation, ‘omics’ approaches, in silico modeling, 3D organoid development, etc., scientists can conduct their research with limited use of animal models.

8.2. Nucleoside transporters and intracellular metabolism coupling

Researchers have established a solid understanding of the metabolic pathways instrumental for the fate of adenosine in the extracellular and intracellular milieu (Boison & Yegutkin, 2019; Borbath et al., 2012; Burnstock, 2017; Fox & Kelley, 1978; Fredholm et al., 2011; Fredholm, Chern, Franco, & Sitkovsky, 2007; Goldthwait, 1957; Hasko, Antonioli, & Cronstein, 2018; Katori & Berne, 1966; Newton & Perry, 1960; Pastor-Anglada & Perez-Torras, 2018b). Extracellular adenosine levels are determined by the balance between its synthesis and degradation. In most cases adenosine is generated because of the sequential metabolic action of various ectonucleotidases (CD73, CD39) on nucleotide precursors namely, ATP, ADP, and AMP (Dos Santos-Rodrigues, GraneBoladeras, Bicket, & Coe, 2014; Nguyen, Ross, Ryals, Lee, & Venton, 2015; Pastor-Anglada & Perez-Torras, 2018b) and sometimes released from the cells (Almeida, Rodrigues, de Mendonca, Ribeiro, & Cunha, 2003). The disposal of extracellular adenosine is mediated by either metabolism, i.e., conversion to inosine via adenosine deaminase, or by its uptake into cells followed by metabolic trapping as AMP after being phosphorylated by adenosine kinase. The transport across the cell membranes occurs via selected adenosine transporter proteins, which makes transport processes key modulators of extracellular adenosine disposal.

The direct coupling of influx performed by nucleoside transporters and intracellular metabolism facilitated primarily by adenosine kinase and to a lesser extent, adenosine deaminase, is assumed to be the driving force that pulls adenosine inward. By the addition of a phosphate group by adenosine kinase or removal of the amino group by adenosine deaminase, adenosine is altered to prevent efflux via nucleoside transporters, thus trapping it inside cells. Further, once intracellular metabolism has taken place, these adenosine metabolites no longer factor into the concentration gradient. Thus, extracellular adenosine levels rapidly overtake the intracellular levels driving inward movement by ENTs. While this system maintains a homeostatic mechanism for the salvage of nucleosides, it cannot be counted on to function as normal under pathological conditions such as cancer. In the above section, we have outlined changes in the expression and function of each nucleoside transporter across multiple cancer subtypes. These alterations alone may disrupt the balance in transport and metabolism coupling.

Additionally, the expression of intracellular or extracellular adenosine metabolizing enzymes may be changed in cancer. One could reasonably project that the downregulation of adenosine kinase may have a significant influence on the sequestration and concentration of adenosine inside cells. Centelles presented a model for adenosine transport and metabolism in different steady states based on their kinetic behavior (Centelles, Cascante, Canela, & Franco, 1992). It was observed that CD73 regulates the fluxes towards intracellular adenine nucleosides in some circumstances and nucleoside transporters in others. The model predicts that a change in one independent variable such as extracellular AMP, re-routes the system towards a completely new steady state with a different control pattern. The pharmacological inhibition (50%) of CNT2, CNT3, and ENT1 only slightly alters the adenosine fluxes as the concentrations of adenosine and inosine change to counterbalance the effect. The results suggest a key role for the nucleoside carriers that deal with adenosine and inosine, which maintain the adenosine influx following the chemical gradient for the Na⁺-dependent transporter, however, an efflux of inosine against the gradient with an adenosine influx down its concentration gradient is observed in Na⁺-independent transporter. ENT inhibition alone affects the extracellular adenosine levels with minimal change in the intracellular milieu. Rose et al. reported higher levels of extracellular adenosine in ENT1-null cardiomyocytes (4360 ± 1840 pmol/mg protein) compared to wild-type cardiomyocytes (3035 ± 730 pmol/mg protein, p < 0.05) after hypoxic challenge. Furthermore, the pharmacological inhibition of ENT2 led to a further increase in extracellular adenosine in KO (6805 ± 1830 pmol/mg protein) versus WT cells (4260 ± 1225 pmol/mg protein, p < 0.05) under the same treatments revealing a significant contribution of ENT2 to adenosine uptake. No difference in intracellular adenosine levels was observed between ENT1-null and wild-type cardiomyocytes under normoxia or hypoxia, supporting an outside-to-inside gradient for adenosine. The study also ruled out the effect of metabolic enzymes such as CD39, and CD73 on extracellular levels of adenosine. However, adenosine kinase which can affect intracellular levels (Eltzschig, 2009), showed significantly lower expression compared to wild-type cardiomyocytes. Further research is required to ascertain how the alteration of both metabolic enzymes and nucleoside transporters affects the capability of cells to concentrate adenosine.

Computational modeling theories and software have been quite useful for in silico prediction and correlation analysis so far and keep on improving with advancements in technology. Petri net theory was successfully applied to study the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) regulation network (Hardy & Robillard, 2008). BioWays software has been used for the quantitative modeling and analysis of the glycogen breakdown pathway and the HIV life cycle (Chiarugi, Falaschi, Hermith, Guzman, & Olarte, 2013). All these tools could be applied to study the adenosine transporter-receptor interplay in various cancer settings. The resulting data will aid in narrowing down the focus on significant effectors by filtering out the neutral players. However, only detailed experimental testing would confirm the reliability of such approaches for therapeutic targeting and precisely study how perturbations in a single transporter component in the purinome are likely to impact the adenosine sequestration and consequent effects.

To conclude, elaborated and more affirmative outcomes are needed for applying these models to oncology settings. The current studies have been beneficial in breakdown a lot of complexities in the nucleoside transporter-mediated adenosine signaling network. However, the quantitative information is still not enough to distinguish each component’s role separately in the context of the tumor microenvironment.

8.3. Simultaneous targeting of multiple components of the purinome

Up to this point, multiple elements of the purinome have been targeted to limit the accumulation of adenosine inside cells. Yet to date, none of these therapies that have shown efficacy in in vivo models have been successfully transferred to cancer patients. Here, we suggest the consideration of modulating multiple components of the purinome. Due to the redundancy and complexity of pathways that all contribute to adenosine concentration levels, targeting one aspect may likely allow for compensatory mechanisms to overcome the therapeutic targeting applied. We suggest that further research focus on strategies that involve multiple elements of the purinome. For instance, perhaps the inhibition of ectonucleotidase enzymes combined with the induction of CNT transporters would both curtail the production of extracellular adenosine while enhancing the uptake of existing pools of adenosine.

In summary, we see nucleoside transporter research in the context of the purinome as a whole to be an important missing link in understanding how adenosine builds up in the extracellular space of cancer cells. Surprisingly, nucleoside transporter processes have not yet been targeted to counteract high levels of extracellular adenosine, although modulation of nucleoside transport has resulted in approved therapies in other disease types. Therefore, we call for a more thoughtful study of the dynamic processes that control adenosine levels and tumors to underpin the rational design of new drugs to boost anti-tumor immunity.

Abbreviations:

A₁

adenosine 1 receptor

A_2A

adenosine 2A receptor

A_2B

adenosine 2B receptor

A₃

adenosine 3 receptor

AC

adenylate cyclase

ADA

adenosine deaminase

ADK

adenosine kinase

AKT

protein kinase B

AML

acute myeloid leukemia

AMP

adenosine monophosphate

ARs

adenosine receptors

ATF-1

activating transcription factor-1

ATP

adenosine triphosphate

BSA

bovine serum albumin

cAMP

cyclic adenosine monophosphate

CaMKII

calcium/calmodulin-dependent protein kinase II

CHEMS

cholesterol hemisuccinate

CNS

central nervous system

CNT

concentrative nucleoside transporter

CREB

cAMP responsive binding protein

CREM

cAMP responsive element modulator

DAG

diacylglycerol

DC

dendritic cells

DOPE

dioleoylphosphatidylethanolamine

DPPC

dipalmitoylphosphatidylcholine

DSPE

1.2-distearoyl-sn-glycro-3-phosphorylethanolamine

EC50

half maximal effective concentration

EPAC1

exchange protein activated by cAMP 1

ENT

equilibrative nucleoside transporter

ERK1/2

extracellular signal-regulated kinases 1 and 2

GMS

glyceryl monostearate

GPCR

G-protein coupled receptor

GRK2

G-coupled protein receptor kinase 2

HASMCs

human aortic smooth muscle cell

HIF

hypoxia-inducible factor

HNF4-α

hepatocyte nuclear factor 4 alpha

IFN-γ

interferon-γ

IL

Interleukin

IP3

inositol 1,4,5-trisphosphate

MAPK

mitogen-activated protein kinase

MDSCs

myeloid-derived suppressor cells

MEK

MAPK/ERK kinase

miRs

micro RNAs

mRNA

messenger RNA

NADPH

nicotinamide adenine dinucleotide phosphate

NK

natural killer

PCC

precipitated calcium carbonate

PEG

polyethylene glycol

PET

positron emission tomography

PI3K

phosphatidylinositol-3-kinase

PLA

polylactic acid

PLC

phospholipase C

PLGA

poly (lactic-co-glycolic acid)

PKA

protein kinase A

SLC28

solute carrier family 28

SLC29

solute carrier family 29

STAT1

signal transducer and activator of transcription 1

TAMs

tumor-associated macrophages

TGF-β

transforming growth factor beta

Th

t-helper

TME

tumor microenvironment

TNF-α

tumor necrosis factor alpha

Treg

immunosuppressive regulatory T cell

VEGF

Vascular endothelial growth factor

Data availability

No data was used for the research described in the article.