Adenosine Concentration Determination for in Vitro Tumor Microenvironment Simulation

Abstract

Objectives

We aimed to investigate the concentration of adenosine (ADO) in the tumor microenvironment (TME), focusing on its potential to modulate tumor cells and natural killer (NK) cells, thereby facilitating tumor immune escape.

Methods

In this study, an in vitro simulation system was developed to systematically evaluate the effects of ADO (0-500 μM) on the colony formation and migration capability of A549 (lung carcinoma) and A375 (melanoma) cell lines, as well as its action on NK92 cell activity, cytokine secretion, and cytotoxicity against tumor cells.

Results

The results showed that 50 μM ADO significantly promoted tumor cell proliferation (increasing the colony formation rate by 60%-80%) and migration (increasing the migration rate by 30%-40%), whereas high concentrations (>200 μM) exhibited an inhibitory effect. ADO suppressed NK92 cell activity in a dose-dependent manner, reducing the relative proliferation rate by 14.5% at 50 μM, significantly decreasing IFN-γ secretion (by 24% at 50 μM), and impairing the killing efficiency of A549, A375, and HepG2 cells (reducing their respective cytotoxicity by 20.3%, 22.4%, and 31.5%).

Conclusion

This study provides biological evidence that 50 μM represents a critical threshold concentration for TME simulation, elucidates the concentration-dependent bidirectional regulation of ADO.

Graphical Abstract

Introduction

Cancer is a leading cause of mortality globally, and its incidence continues to rise. ¹ The tumor microenvironment (TME) is a dynamic and complex system composed of tumor cells, immune cells, microvessels, and the extracellular matrix, all of which contribute to tumor growth and progression.^2,3 Key characteristics of the TME include hypoxia, abnormal metabolite accumulation, an acidic pH, and the presence of immunosuppressive factors and cytokines.^4-6 Among these, adenosine (ADO), an immunosuppressive metabolite, plays a crucial role in establishing an immunosuppressive network within the TME.^7,8

In the TME, adenosine promotes tumor angiogenesis and growth.^9,10 It can also directly influence tumor cell proliferation, survival, adhesion, and migration by activating the cAMP/PKA signaling pathway. ¹¹ Additionally, adenosine significantly impairs the functions of key immune effector cells and contributes to immune exhaustion. ¹² In the TME, adenosine primarily suppresses macrophage activation and TNF-α release through A2AR (one of G protein-coupled receptors). ¹³ Additionally, A2AR signaling impairs the infiltration and effector functions of CD8 + T cells ¹⁴ and NK cells, ¹⁵ promoting tumor progression. Notably, Young et al demonstrated that targeted ablation of A2AR in NK cells enhances tumor suppression and delays tumorigenesis by increasing the fraction of terminally mature NK cells. ¹⁶ Furthermore, adenosine activates the mTOR signaling pathway, suppressing NK cell proliferation, ¹⁵ while promoting immune evasion through the stimulation of regulatory T cells (Tregs) ¹⁷ and myeloid-derived suppressor cells (MDSCs).^18,19

Extracellular adenosine (eADO) is primarily generated through 2 pathways. ²⁰ The classical pathway involves ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and 5′-nucleotidase (CD73), where CD39 hydrolyzes ATP into ADP and AMP, and CD73 subsequently converts AMP into eADO.^21,22 In the alternative pathway, nicotinamide adenine dinucleotide (NAD+) serves as a substrate, producing AMP via CD38 and CD203a, which is then converted into eADO by CD73. ²³ Under normal physiological conditions, eADO concentrations are maintained at nanomolar levels (30-200 nmol/L) due to the activity of nucleoside transporters (ENT1/2, CNT1/2) and rapid metabolism by adenosine deaminase (ADA).^24,25 However, in solid tumors, this balance is disrupted, and eADO levels can increase up to 100-fold compared to normal tissues, primarily due to hypoxia-induced upregulation of CD39/CD73 expression and inhibition of adenosine kinase (AK) activity.^17,26

Based on the critical role of elevated eADO concentrations in immunosuppression, developing an in vitro model that simulates TME adenosine levels is essential. This study aims to establish adenosine concentration gradients to systematically investigate their biological effects on tumor cells and NK cells in TME simulation.

Material and Methods

Cell Lines

The IL-2-dependent NK cell line NK-92, human lung carcinoma cell line A549, human melanoma cell line A375 and human liver cancer cell line HepG2 were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China). NK-92 cells were cultured in complete medium (MEM-α supplemented with 12.5% fetal bovine serum and 12.5% horse serum) (Gibco, USA). A549 cells were maintained in 10% Ham’s F-12K medium (Gibco), A375 and HepG2 were maintained in 10% DMEM medium (Gibco).

Effects of Gradient Adenosine Concentrations on Tumor Cell

Colony Formation Assay

The colony formation assay was conducted using a modified crystal violet staining method, as previously described. ²⁷ This technique measures cell adherence, where crystal violet binds to cellular proteins and DNA to indicate colony formation. Briefly, tumor cells (A549 cells or A375 cells) in the logarithmic growth phase were digested with 0.25% phenol red-free trypsin (Sangon Biotech, Shanghai), centrifuged, and counted for assay. 200 cells were seeded into 6-well culture plates per well and incubated at 37°C with 5% CO₂ (HealForce, China) for 24 h before replacing the medium with adenosine-containing culture medium. Adenosine (Sigma-Aldrich, No. A9251) were diluted with culture medium (10% Ham’s F-12K medium for A549 cells or 10% DMEM medium for A375 cells) to obtain the gradient concentrations as follows: 25, 50, 100, 200, 400 μM. An equal volume of culture medium was considered as 0 μM.

The medium was refreshed every 3 days. After 14 days, colony formation was observed using an inverted microscope (Nikon Ti-S, Japan). Cells were then washed 1-2 times with 1× PBS and fixed with 1 mL of 4% paraformaldehyde (HaoKe Biotech, Hangzhou) for 30 min. After discarding the paraformaldehyde, wells were washed twice with 1× PBS, followed by staining with 1 mL of crystal violet solution (Sangon Biotech, Shanghai) for 15 min. The wells were then rinsed with 1× PBS, air-dried, and imaged with camera. The colony formation rate was analyzed using ImageJ software.

Scratch Wound Healing Assay

The scratch wound healing assay was used to evaluate two-dimensional cell migration, as previously described. ²⁸ A uniform artificial wound was introduced into a confluent cell monolayer, and cell movement was monitored through microscopy. Briefly, A549 or A375 cells was assigned the same adenosine concentration groups (same as described in the colony formation assay). Cells in the logarithmic growth phase were digested with 0.25% phenol red-free trypsin, resuspended as a single-cell suspension, and seeded into 6-well culture plates with 5 horizontal guide marks on the underside of each well. Each well was seeded with 4 × 10⁵ cells in a final volume of 2 mL of culture medium. Cells were incubated at 37°C with 5% CO₂ for 24 h. Once the monolayer reached 100% confluence, a scratch was introduced vertically using a 200 μL pipette tip. The cells were gently washed 3 times with 1× PBS before adding serum-free medium containing different concentrations of adenosine. Images were captured using a 4× objective lens at 0, 12, 24, and 48 h. The wound closure was quantified using ImageJ software by randomly selecting 6-8 horizontal measurement points. The cell migration rate (wound healing rate) was calculated as follows: $\text{Cell} \text{migration} \text{rate}\%=\frac{\left(A_{\text{Initial}}-A_{\mathrm{t}}\right)\times 100\%}{A_{\text{Initial}}}$ , where $A_{\text{Initial}}$ represents the initial scratch area, $A_{\mathrm{t}}$ represents the scratch area at time t (0, 12, 24, and 48 h, respectively).

Effects of Gradient Adenosine Concentrations on NK92 Cells

NK92 Cell Activity Assessment and Morphological Observation

The activity of NK92 cell was determined using the CCK-8 assay kit (Beyotime, China) following the manufacturer’s instructions. NK92 cells (2 × 10⁵ cells/mL) were seeded into 96-well plates (100 μL/well) with complete medium. Cells treated with increasing concentrations of adenosine (5, 50, 500 μM) diluted with MEM-α medium were considered as experimental group. Cells treated with an equal volume of MEM-α medium and complete medium were considered as blank control group (0 μM adenosine) and positive control group, respectively. Notably, due to the rapid metabolism of adenosine by adenosine deaminase (ADA), the ADA-inhibitor EHNA (Sigma-Aldrich) was added at a final concentration of 10 μM, along with the nucleoside transport inhibitor dipyridamole (Sigma-Aldrich) at a final concentration of 0.5 μM.

After incubation for 12 h, 10 μL of CCK-8 solution was added to each well, followed by incubation at 37°C for 2 h. Absorbance (A) was measured at 450 nm using a FlexA-200 microplate reader (Aosheng Biotech, China). The relative proliferation rate was calculated using the formula: $\text{Relative} \text{proliferation} \text{rate}\%=\frac{(A_{\text{experiment}}-A_{\text{zero}})\times 100\%}{A_{\text{positive}}-A_{\text{zero}}}$ , where $A_{\text{experiment}}$ represents the absorbance of the experimental group, $A_{\text{positive}}$ represents the absorbance of the positive control group, and $A_{\text{zero}}$ represents the absorbance of the blank control group. NK92 cells treated with 50 μM and 0 μM adenosine was examined on day 8 using a Ti-S inverted microscope (Nikon) for morphological observation.

NK92 Cell Apoptosis

NK92 cells seeded in 6-well plates (3 mL/well) were incubated for 12 h with 0, 5, 50, and 500 μM adenosine. Cells were washed twice with 1× PBS (centrifuged at 400 × g for 5 min) and harvested. The cell pellet was resuspended in 500 μL of 1× binding buffer, followed by the addition of 5 μL Annexin V-FITC and 10 μL PI. The mixture was gently vortexed and incubated at 4°C in the dark for 5 min. Apoptosis was analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter). The apoptosis rate was calculated using the formula: $\text{Apoptosis} \text{rate}=\text{Late} \text{apoptosis}\left(\mathrm{Q}1-\text{UR}\right)+\text{Early} \text{apoptosis}\left(\mathrm{Q}1-\text{LR}\right)$ .

NK92 Cell Cytokine Secretion

Prior to NK92 cells were incubated with different adenosine concentrations, HepG2 cells (1 × 10⁵ cells/mL) were prepared for adhesion in 96-well plates at 37°C. Once adhesion of HepG2 cells was complete in 12 h, NK92 cells, pre-incubated with adenosine for 30 min, were added at an effector-to-target ratio of 2:1. The final NK92 cell density was set as 1 × 10⁵ cells/mL (100 μL/well). After 12 h of incubation, the culture supernatant was harvested by centrifuging at 400 × g for 5 min and stored in −20°C. All experiments were repeated 3 times.

Cytokine secretion was assessed using the Human Th1/Th2 Cytokine Kit (Catalog No. 550749, BD Bioscience) following the manufacturer’s instructions. Briefly, 50 μL of detection beads, 50 μL of detection reagent, and 50 μL of the study sample or standard were sequentially added to each sample tube and incubated at room temperature in the dark for 3 h. The samples were then washed with 1 mL of wash buffer, centrifuged, and the supernatant was discarded. The remaining pellet was resuspended in 300 μL of buffer and analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter) on the same day. Data analysis was conducted using FCAP Array v2.0 software (Soft Flow, Hungary). Prior to sample analysis, the flow cytometer was calibrated using Set-up Beads according to the manufacturer’s instructions.

Effect of Gradient Adenosine Concentration on the Cytotoxicity of NK92 Cells Against Tumor Cells

A549, A375, and HepG2 cells in the logarithmic growth phase were prepared in a density of 1 × 10⁵ cells/mL and incubated for adhesion in 96-well plates at 37°C for 12 h. NK92 cells pre-incubated with different concentrations of adenosine (0, 5, 50, and 500 μM) for 30 min were added into 96-well plates at an effector-to-target ratio of 2:1. After 6 h of co-culture, 10 μL of CCK8 solution was added to each well, followed by incubation at 37°C for an additional 2 h. The absorbance (A) was measured at 450 nm using a microplate reader. The cytotoxicity percentage of NK92 cells at different adenosine concentrations was calculated using the formula: $\text{Cytotoxicity}\%=100\%-\left[\frac{A_{\left(E+T\right)}-A_{\mathrm{E}}}{A_T-A_B}\right]\times 100\%$ , where $A_T$ represents the absorbance of the tumor target cell control group, $A_{E+T}$ is the absorbance of wells co-cultured with tumor target cells and NK92 effector cells, $A_E$ is the absorbance of the NK92 cell control group at the corresponding concentration, and $A_B$ is the absorbance of the blank well. Each group was tested in triplicate, with a total volume of 100 μL per well.

Statistical Analysis

All data were analyzed using the DPS statistical software system. ²⁹ For comparisons between 2 groups, an unpaired t test was applied, while one-way analysis of variance (ANOVA) was used for multiple-group comparisons. A P-value of <0.05 was considered statistically significant. Data are presented as mean ± standard deviation (SD).

Results

Effect of Adenosine on Tumor Cell Colony Formation

To investigate the effect of adenosine on tumor cell colony formation, A549 and A375 cells were treated with varying concentrations of adenosine (0, 25, 50, 100, 200, and 400 μM) (Figure 1A and B). The results indicated that at lower concentrations, colony formation rates increased with rising adenosine concentrations, reaching a peak at 50 μM (Figure 1C and D). Under 50 μM treatment, the colony formation rates for A549 and A375 cells were 70.17 ± 4.31% and 69.67 ± 3.82%, respectively—160% and 180% higher than those of the control group (P < 0.01). However, as the adenosine concentration increased beyond 50 μM, colony formation declined significantly. At concentrations exceeding 200 μM, a notable inhibitory effect was observed, with colony formation rates falling below control levels (P < 0.01). These results suggest that 50 μM adenosine significantly promotes tumor cell proliferation, while higher concentrations inhibit proliferation. This inhibitory effect may be attributed to the suppression of A549 and A375 cell activity at elevated adenosine levels.

Figure 1.

Colony Formation of Tumor Cell Treated With Different Adenosine Concentrations. (A), (B) Represent the Colony Photos of A549 and A375 Tumor Cells, Respectively. Scale bar = 10 mm. (C), (D) Represent the Colony Formation Rates of A549 and A375 Tumor Cells, Respectively. Note: ns = Not Significant (P ≥ 0.05); P < 0.05 (*), P < 0.01 (**), P < 0.0001 (***)

Effect of Adenosine on Tumor Cell Migration

To investigate the impact of adenosine on tumor cell migration, scratch assays were performed on A549 and A375 cells treated with varying concentrations of adenosine (0, 25, 50, 100, 200, 400 μM) (Figure 2A and B). At lower concentrations, the migration rate of both cell lines increased with rising adenosine concentrations, reaching a peak at 50 μM. The cell migration rates at this concentration were 32.99 ± 6.15% for A549 cells and 28.59 ± 3.49% for A375 cells, which represented 140% and 130% increases compared to the control, respectively (Figure 2C and D). However, at concentrations exceeding 100 μM, cell migration decreased significantly, with a significant inhibitory effect (ie, migration rate lower than the control) observed at P < 0.01. These results suggest that within the TME, 50 μM adenosine may optimally promote tumor cell growth and migration.

Figure 2.

Scratch Migration of Tumor Cells Treated With Different Adenosine Concentrations. (A), (B) Represent Scratch Migration Images of A549 and A375 Tumor Cells, Respectively. Scale bar = 1 mm. (C), (D) Represent the Migration Rates of A549 and A375 Tumor Cells, Respectively. Note: ns = Not Significant (P ≥ 0.05); P < 0.05 (*), P < 0.01 (**), P < 0.0001 (***)

Effect of Adenosine on NK92 Cell Activity

The CCK8 assay was used to evaluate the effect of adenosine on the proliferation of NK92 cells, as shown in Figure 3A. After the addition of the adenosine deaminase inhibitor EHNA and the equilibrative nucleoside transporter 1 inhibitor dipyridamole, the relative proliferation rates of NK92 cells at adenosine concentrations of 5, 50, and 500 μM after 12 h of treatment were 95.9%, 85.5%, and 73.9%, respectively. Moreover, the inhibitory effect became more significant with increasing adenosine concentration, demonstrating a clear dose-dependent effect. These results indicate that prolonged exposure to 50 μM adenosine inhibits NK92 cell activity. Observations under a microscope on the eighth day revealed a significant increase in cell fragments in the experimental group (Figure 3C) compared to the control group (Figure 3B). Additionally, a higher number of apoptotic, non-clustered single cells were present in the experimental group, suggesting that adenosine affects the survival and morphology of NK92 cells. To investigate whether adenosine influences the apoptosis cycle of NK92 cells, apoptosis was assessed after 24 h of adenosine treatment (Figure 3D). Compared with the control group, no significant differences were observed in apoptosis rates among the 5, 50, and 500 μM adenosine-treated groups (P > 0.05). This suggests that adenosine primarily suppresses NK92 cell function by activating adenosine receptors and inducing immunosuppressive effects rather than directly affecting the apoptosis cycle of NK92 cells.

Figure 3.

Effects of Different Concentrations of Adenosine on NK92 Cells. (A) Represents Effect of Adenosine on the Relative Proliferation Rate of NK92 Cell; (B) Represents Morphology of NK92 Cells Without Adenosine Treatment; (C) Represents Morphology of NK92 Cells Treated With 50 μM Adenosine, Where Red Circles Indicate Cell Debris; (D) Represents Effect of Adenosine on NK92 Cell Apoptosis Rate. Note: ns = Not Significant (P ≥ 0.05); P < 0.05 (*), P < 0.01 (**)

Effect of Adenosine on IFN-γ and TNF-α Secretion in NK92 Cells

NK92 cells were pre-treated with different concentrations of adenosine for 30 min, followed by stimulation with HepG2 cells for 12 h. Flow cytometry was then used to measure IFN-γ and TNF-α secretion (Figure 4A and B). When the adenosine concentrations were 0, 5, 50, and 500 μM, IFN-γ secretion levels were 8.10 ± 1.36 pg/mL, 7.43 ± 0.96 pg/mL,6.16 ± 0.46 pg/mL, and 4.05 ± 0.14 pg/mL, respectively, while TNF-α secretion levels were 19.90 ± 1.20 pg/mL, 19.43 ± 0.58 pg/mL, 19.37 ± 0.94 pg/mL, and 17.88 ± 0.56 pg/mL, respectively. Adenosine treatment significantly inhibited IFN-γ secretion in NK92 cells induced by tumor cells. Their reduction of 5, 50, and 500 μM adenosine treatments were 8%, 24% and 50%, respectively. Although TNF-α secretion did not show a statistically significant difference across adenosine concentrations, a downward trend was observed.

Figure 4.

Adenosine Inhibits Cytokine Secretion in NK92 Cells. IFN-γ Secretion (A) and TNF-α Secretion (B) by NK92 Cells Following Treatment With Different Concentrations of Adenosine. Note: ns = Not Significant (P ≥ 0.05); P < 0.05 (*), P < 0.01 (**)

Effect of Adenosine on the Cytotoxicity of NK92 Cells

When adenosine concentrations were 0, 5, 50, and 500 μM, the cytotoxicity of NK92 cells against A549 cells was 24.5%, 22.1%, 20.3%, and 11.1%, respectively (Figure 5A). Under the same conditions, the cytotoxicity of NK92 cells against A375 cells were 27.3%, 26.7%, 22.4%, and 13.1%, respectively (Figure 5B). At 50 μM adenosine, the cytotoxicity of NK92 cells against both tumor cell lines were significantly reduced compared to the control group, and this inhibition became highly significant at 500 μM (P < 0.05). These results indicate that adenosine suppresses NK92 cell cytotoxicity against human melanoma (A375) and human lung carcinoma (A549) cells in a concentration-dependent manner.

Figure 5.

Cytotoxicity of NK92 Cells on Tumor Cells (A: A549; B: A375; C: HepG2) at Different Adenosine Concentrations. Note: ns = Not Significant (P ≥ 0.05); P < 0.05 (*), P < 0.01 (**)

Additionally, the effect of adenosine on NK92 cell-mediated cytotoxicity against HepG2 cells is presented in Figure 5C. When the adenosine concentration was 0, 5, 50, and 500 μM, the corresponding average cytotoxicity were 48.6%, 44.6%, 31.5%, and 38.1%, respectively. The data suggest that adenosine inhibits the cytotoxic effect of NK92 cells against HepG2 liver cancer cells. Notably, the cytotoxicity at 500 μM was higher than that at 50 μM, which may be attributed to the excessive concentration of adenosine (500 μM) suppressing HepG2 cell activity. These results imply that adenosine may inhibit the proliferation of tumor cells directly at ultra-high concentrations. Consequently, the killing activity of NK92 cells increased.

Discussion

Extracellular adenosine contributes to tumor progression not only through immunosuppression but also via direct stimulation of malignant cell growth and migration. ⁸ While reported adenosine concentrations in human TMEs vary considerably—from 9-13 μM in HT-29 xenografts ²⁴ to approximately 12 μg/g/ml in tumor tissue supernatants ²⁵ —the selection of a biologically relevant concentration for in vitro TME modeling requires empirical validation. ³⁰ Our investigation establishes 50 μM adenosine as a functionally significant reference concentration, corroborating measurements of 50-100 μM in rat sarcoma tissue. ³¹ A colony formation assay demonstrated that 50 μM adenosine significantly increased the colony formation rate of both cell lines, with an observed increase of 60%-80% (P < 0.01). Within the 0-50 μM range, cell proliferation increased proportionally with adenosine concentration. Notably, these stimulatory effects diminished at higher concentrations, with complete suppression of proliferation at 500 μM. Furthermore, a scratch wound healing assay indicated that 50 μM adenosine significantly enhanced cell migration, increasing the migration rate by 30%-40%, whereas concentrations between 100 and 500 μM exhibited inhibitory effects. The results of these 2 functional assays suggest that maintaining extracellular adenosine at 50 μM within the TME may be crucial for tumor development.

A significant inhibitory effect on both colony formation and cell migration was observed at elevated eADO concentrations (above 200 μM). This effect may be attributed to the activation of the A3 adenosine receptor (A3R, one of G protein-coupled receptors), which has a relatively low affinity for adenosine and requires higher concentrations for effective activation. In various cancer cell types, A3R activation has been linked to the inhibition of cell proliferation, induction of apoptosis, and suppression of angiogenesis. ³²

Moreover, natural killer (NK) cells, as key anti-tumor immune effector cells, are significantly regulated by adenosine in the TME.^6,7 It has been reported that a high adenosine concentration of 1 mM significantly suppresses IL-2/IL-15-induced NK cell cytotoxicity. ¹⁵ In this study, we observed that after 12 h of adenosine treatment, NK92 cell activity was significantly inhibited in a dose-dependent manner (Figure 3A), reducing the relative proliferation rate by 14.5% at 50 μM and 26.1% at 500 μM. Zhang et al. ³³ reported that adenosine suppressed the proliferation of IL-2-stimulated CTLL-2 T cells. Similarly, Young et al. ¹⁶ demonstrated that adenosine reduces the proportion of terminally mature NK cells in steady-state conditions via A2AR signaling. Our results further show that prolonged exposure of NK92 cells to 50 μM adenosine impaired cell viability (Figure 3B). Functional assays demonstrated that IFN-γ secretion was significantly inhibited after NK92 cells were pretreated with 5, 50, and 500 μM adenosine, with a 24% reduction observed in the 50 μM group. The result aligns with previous studies reporting decreased IFN-γ secretion by NK cells in tumors such as liver cancer ³⁴ and lung cancer. ³⁵ These results suggest that adenosine may contribute to the inhibition of IFN-γ secretion by NK cells. Although TNF-α secretion by NK92 cells exhibited a slight decline with increasing adenosine concentrations, the difference was not statistically significant (P > 0.05), possibly due to the limited duration of adenosine treatment.

Cytotoxicity assays further revealed that adenosine inhibited the tumor-killing capacity of NK cells in a dose-dependent manner. The cytotoxicity of NK92 cells against A549, A375, and HepG2 tumor cells decreased in response to increasing adenosine concentrations, with reductions of 20.3%, 22.4%, and 31.5% in the 50 μM group, respectively. These results likely reflect the combined effects of adenosine on NK92 cells. ³⁶ Notably, 50 μM adenosine exerts a bidirectional regulatory effect on tumor and immune cells. It not only maximally enhances tumor cell proliferation and migration but also significantly suppresses NK92 cell activity, IFN-γ secretion, and tumor cell cytotoxicity. This concentration-dependent dual effect provides an experimental basis for selecting 50 μM as a reference concentration for simulating the TME in vitro.

Although this study demonstrates that a 50 μM concentration of adenosine exerts bidirectional regulatory effects on tumor cells and NK cells in a simulated in vitro TME, several limitations remain. First, the actual concentration of adenosine in the TME has not yet been verified through in vivo experiments in mice, which restricts a comprehensive understanding of adenosine’s role. Second, while 50 μM adenosine promotes tumor cell growth, higher concentrations have the opposite, inhibitory effect. This phenomenon may be attributed either to the cytotoxicity of high adenosine concentrations or to inhibitory pathways activated via other adenosine receptors. Finally, although the activity and cytotoxic effects of NK92 cells are significantly diminished at 50 μM, this observation may reflect specific characteristics of the NK92 cell line. Future studies should include large-scale validation with other tumor cells and immune cells and the potential mechanisms into the signal transduction pathways associated with adenosine receptors.

Conclusion

This study established an in vitro adenosine concentration system to simulate the tumor microenvironment and systematically examined the regulatory effects of varying adenosine concentrations on tumor cells and NK92 cells. The results demonstrated that 50 μM adenosine significantly promoted tumor cell proliferation and migration while suppressing NK92 cell activity, cytokine secretion, and cytotoxicity against tumor cells. Therefore, 50 μM adenosine should be selected as a critical threshold concentration for simulating the tumor microenvironment.