Activated T cells induce apoptosis in A549 lung adenocarcinoma cells via TRPV4-mediated calcium influx.

Introduction
Cancer is a major global health challenge, with its prevalence and mortality projected to reach 19.3 million cases across 185 countries in 38 regions, influenced by demographic factors such as age and sex. Lung carcinoma, responsible for approximately 2.5 million new cases and 1.8 million deaths in 2024, is the second most commonly diagnosed malignancy and the leading cause of cancer-related mortality worldwide1–4. In 2022 alone, lung cancer accounted for 2,480,675 new cases globally, including 234,580 in the United States (116,310 men and 118,270 women), and was responsible for roughly 125,070 deaths (65,790 men and 59,280 women)4. Current treatments for lung cancer, including surgery, chemotherapy, radiotherapy, and targeted therapies, are often limited by treatment resistance and disease recurrence. Immunotherapy has emerged as a promising approach, offering durable responses by inducing immunological memory. Among immune effector cells, cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells play pivotal roles in eliminating cancer cells. T cell activation is a complex process initiated by antigen recognition through the highly variable region of the T cell receptor (TCR), which transduces signals intracellularly via the CD3 complex5. Jurkat T cells, an immortalized human T lymphocyte line, are widely employed to investigate TCR signaling, acute T cell leukemia, and immune-related disorders, including immuno-oncology research6. A critical early event in T cell activation is the release of intracellular Ca²⁺ ions, which triggers a second messenger cascade that sustains oscillatory Ca²⁺ influx through plasma membrane channels. This Ca²⁺ signaling is essential for regulating T cell functions, including proliferation, cytokine production, and cytotoxic activity7,8. Intracellular Ca²⁺ levels also influence cancer cell apoptosis, highlighting the dual role of calcium in modulating both immune and tumor cell behavior9,10. Functional Ca²⁺ channels govern cytosolic calcium dynamics, which are critical for various cellular processes, including proliferation, apoptosis, and migration. CTLs and NK cells, upon reaching tumor sites, form immune synapses with cancer cells, leading to targeted cytotoxicity11.
T cell activation involves multiple intracellular signaling pathways, including the phosphatidylinositol cascade, which promotes the release of Ca²⁺ from intracellular stores via inositol 1,4,5-trisphosphate, and the activation of transcription factors required for interleukin-2 production12. Among calcium-permeable channels, the transient receptor potential vanilloid 4 (TRPV4) channel is highly expressed in various tissues, including the lung, and is responsive to both physical and chemical stimuli13,14. TRPV4 activation results in increased intracellular Ca²⁺ concentrations, which have been linked to apoptosis induction. Overexpression of TRPV4 in A549 lung cancer cells has been shown to enhance apoptosis and suppress proliferation, largely through the p38 MAPK signaling pathway. Calcium modulation not only affects cancer cell viability but also regulates T lymphocyte and NK cell function, emphasizing its relevance in anti-tumor immunity. Pharmacological targeting of calcium channels is emerging as a strategy to influence cancer cell proliferation and apoptosis. Despite the recognized importance of calcium signaling, the interplay between activated T lymphocytes and calcium channels, particularly TRPV4, remains underexplored. This study aims to investigate how activated T cells induce apoptosis in A549 lung cancer cells via TRPV4-mediated calcium signaling, providing insights into a novel immunotherapeutic mechanism.

Materials and methods

Drugs & reagents
Dulbecco’s modified Eagle’s medium (DMEM) culture medium (BIOSERA company, USA), fetal bovine serum (FBS) (GIBCO company, USA), trypsin (Sigma company, Germany), ethylenediaminetetraacetic acid (EDTA) (Merck company, Germany), 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) (Carl Roth company, Germany), Coomassie blue (Merck company, Germany), Sodium dodecyl-sulfate (SDS) (Sigma-aldrich company, Germany), Na2Co3 (Sigma-Aldrich company, Germany), (NH4)2S2O8 (Sigma- Aldrich company, Germany), Bis acrylamide (Sigma- Aldrich company, Germany), Skim Milk (Sigma-aldrich company, Germany), Tris-base (MERK company, Germany), Tween20 (Sigma-aldrich company, Germany), Tetramethylethylenediamine (TEMED) (MERK company, Germany), Glycerol (Dr.Mojalali chemical industries complex, Iran), Acrylamide (MERK company, Germany), NaOH (Merck company, Germany), Polyvinylidene fluoride (PVDF) (Sigma-aldrich company, USA), Diaminobenzidine (DAB) (Merck company, Germany), H2O2 (Dr.Mojalali chemical industries complex, Iran), Methanol (Dr.Mojalali chemical industries complex, Iran), NaCl (Merck company, Germany), Protease inhibitor (Kiazist company, Iran), Radioimmunoprecipitation assay buffer (RIPA) (Cytomatingene company, Iran), Glycine (MERK company, Germany), Sodium potassium tartrate (MERK company, Germany), Tris-HCL (MERK company, Germany), CuSO4.5H2O (MERK company, Germany), Radio Immuno Precipitation Assay (RIPA) (MERK company, Germany).

Ethical approval
This study was reviewed and approved by the Ethics Committee of the Islamic Azad University, Tehran, Iran (Approval No. IR.IAU.SRB.REC.1402.405). All procedures were conducted in accordance with relevant institutional and national guidelines and regulations.

Cell culture and cell viability assay
Human lung adenocarcinoma cells (A549) were obtained from the National Center for Genetic Resources, Iran, and all experiments were performed in high-glucose DMEM (Gibco, Cat. No. 11965-092) containing 1.8 mM CaCl₂, 44 mM NaHCO₃, and 25 mM HEPES buffer. The medium was supplemented with 10% FBS (Gibco, Lot No. 42G0586K; Ca²⁺ content 0.35 ± 0.05 mM) and 1% gentamicin. The final baseline total calcium concentration in complete medium was ~ 1.9 mM, corresponding to an ionized Ca²⁺ level of 1.7 ± 0.1 mM as measured by a Ca²⁺-selective electrode. Cultures were maintained at 37 °C in a humidified 5% CO₂ incubator, where the CO₂–bicarbonate buffering system maintained a stable pH of 7.4 ± 0.1 throughout the experiments. For each experiment, A549 monolayers were prepared as described above. Calcium chloride (CaCl₂) was freshly diluted from a 1 M sterile stock into complete DMEM to reach the desired final concentrations immediately before T-cell addition.
Activated or inactivated Jurkat T cells were added simultaneously with CaCl₂ to A549 cultures (effector-to-target ratios of 1:1, 1:0.5, or 1:0.25). Co-cultures were then incubated for 24–72 h at 37 °C in 5% CO₂ before performing MTT and Annexin V/PI assays. A sterile 1 M CaCl₂ stock solution was prepared in Milli-Q water, filter sterilized (0.22 μm), aliquoted, and stored at 4 °C. For treatments, aliquots were diluted into complete DMEM (containing 10% FBS) to yield final added CaCl₂ concentrations of 0, 0.5, 1.0, 1.8, 2.5 and 3.0 mM (final total Ca²⁺ = baseline DMEM 1.8 mM + added Ca²⁺, i.e., 1.8, 2.3, 2.8, 3.6, 4.3, and 4.8 mM respectively). For example, to add 3.0 mM CaCl₂ to 1 mL medium, 3.0 µL of 1 M CaCl₂ stock was added. Prepared media were mixed gently and used immediately. Because serum and buffer components can bind a fraction of Ca²⁺, ionized Ca²⁺ in the final media was measured directly using a [specify instruand reported (mean ± SD, n = X). Treatments were applied for 24 and 72 h and endpoints (MTT viability and Annexin V/PI flow cytometry) were performed as described above15.
Experimental groups included:

A549 (3 × 10⁶ cells/mL).

A549 + activated T cells (1:1, 1:0.5, 1:0.25).

A549 + inactivated T cells.

A549 + Ca²⁺.

A549 + activated T cells + Ca²⁺ (same ratios).

A549 + inactivated T cells + Ca²⁺.

Monolayer formation and morphology were confirmed using an inverted phase-contrast microscope. Jurkat T lymphocytes were cultured in RPMI 1640 with 10% FBS, with medium changes every three days. Cells were seeded at 4 × 10⁵ cells/mL, incubated overnight, and treated with 10 µg/mL Brefeldin A for one hour to inhibit protein secretion. Activated Jurkat T cells were generated as described above using PMA (10 ng/mL) and ionomycin (250 ng/mL) for 4 h, followed by extensive washing to remove stimulants. Activated and inactivated Jurkat cells were then added directly to A549 monolayers at effector-to-target ratios of 1:1, 1:0.5, or 1:0.25 in fresh complete DMEM, without irradiation or mitomycin-C treatment. Cells were then harvested, washed, and stained for CD3 expression prior to flow cytometry analysis.

Cytotoxic activity by MTT assay
For the MTT assay (Catalog Number: M5655), each well contained a consistent cell population, achieving 80–90% confluency, with 3 × 10⁵ cells per well. Cell suspensions were prepared using trypsin digestion. Based on in vitro calculations, 11.5 mL of suspension containing 1 × 10⁶ cells was seeded into 96-well plates and incubated at 37 °C with 5% CO₂ for 24 and 72 h.
Cell viability was assessed following the manufacturer’s protocol. Briefly, 10 µL of a 5 mg/mL MTT solution (Sigma) was added to each well and incubated for 4 h at 37 °C. Subsequently, 70 µL of dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals, followed by a 20-minute incubation in a shaker incubator. Optical density was measured at 595 nm using a microplate reader, and the IC₅₀ was calculated. Cell viability (CV) was determined using the formula16,17:

Apoptosis and necrosis analysis by Annexin V-FITC/PI staining
Apoptosis and necrosis were quantitatively assessed using Annexin V-FITC/propidium iodide (PI) dual staining followed by flow cytometry. Briefly, 1 × 10⁵ A549 cells from each treatment group were harvested, washed twice with cold phosphate-buffered saline (PBS), and resuspended in 500 µL of 1× binding buffer (BD Biosciences). An unstained control sample was included for cytometer calibration and compensation adjustment. Cells were incubated with 5 µL FITC-conjugated Annexin V for 15 min at 4 °C in the dark. After incubation, cells were washed once with binding buffer and resuspended in 500 µL of fresh 1× binding buffer, followed by immediate addition of 3 µL PI (50 µg/mL) prior to analysis. Samples were analyzed within one hour using a BD FACSCalibur flow cytometer equipped with a 488-nm excitation laser and standard FL1 (FITC) and FL2 (PI) emission filters. Compensation controls and fluorescence-minus-one (FMO) controls were included to correct for spectral overlap and to accurately define gating boundaries. During data acquisition and analysis using CellQuest Pro and FlowJo software (version 10.8), gating strategies excluded debris and doublets based on forward scatter (FSC) versus side scatter (SSC) profiles. Events were displayed as Annexin V-FITC (x-axis) versus PI (y-axis) dot plots, with four distinct quadrants defined as follows:

Q4 (Annexin V⁻/PI⁻): Viable cells.

Q3 (Annexin V⁺/PI⁻): Early apoptotic cells.

Q2 (Annexin V⁺/PI⁺): Late apoptotic or secondary necrotic cells.

Q1 (Annexin V⁻/PI⁺): Primary necrotic cells.

The percentage of cells in each quadrant was calculated, and data are presented as mean ± standard deviation (SD) from three independent experiments (n = 3).

Immunophenotyping protocol
Fixed Jurkat cells were resuspended in 500 µL PBS and centrifuged at 1,500 rpm for 5 min at 4 °C. The supernatant was discarded, and cells were incubated overnight at 4 °C with 500 µL of primary antibody against CD3 diluted 1:100. After incubation, cells were thoroughly washed with culture medium and centrifuged again to completely remove any residual PMA, ionomycin, or Brefeldin A, preventing carryover into subsequent co-culture experiments. Next, 50 µL of fluorescently labeled secondary antibody (1:100) was added, and samples were incubated in the dark at room temperature for 30 min. Following a final wash and centrifugation, 50 µL of cell suspension was retained for analysis. Flow cytometry data acquisition was performed using a BD FACSCalibur flow cytometer. Compensation controls, gating strategies, and fluorescence-minus-one (FMO) controls were employed to ensure accurate identification of CD3-positive populations and to minimize spectral overlap18.

Measurement of oxidative stress biomarkers
Frozen samples (−20 °C) were used to determine total antioxidant capacity (TAC) and total oxidant status (TOS). For TAC measurement, approximately 100 mg of cells was lysed in 1 mL RIPA buffer (Kiazist Company, Iran), and the homogenate was centrifuged at 4,000 × g for 20 min at 4 °C. The supernatant was collected, and TAC levels were measured using a commercial kit (ZB-TAC) according to the manufacturer’s instructions. Absorbance was read at 490 nm using a microplate reader (Biotek, Refelx800, USA). For TOS assessment, 100 mg of cells was lysed in 1 mL RIPA buffer and homogenized on ice. The homogenate was centrifuged at 3,000 × g for 30 min at 4 °C, and the supernatant was collected. TOS levels were measured using a commercial kit (Navand Salamat Company, Iran) according to the manufacturer’s protocol, and absorbance was recorded at 560 nm with the same ELISA reader.

Western blotting
Samples were homogenized and centrifuged at 10,000 rpm for 15 min at 4 °C in RIPA buffer containing protease inhibitors. The supernatant was collected, and total protein concentration was determined using the Lowry assay. For electrophoresis, 50 µg of protein from each sample was loaded onto a 12.5% SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA, USA), followed by transfer to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% nonfat milk at room temperature for 60 min, then incubated with primary rabbit polyclonal anti-TRPV4 antibody (orb337420) on a shaker for 120 min. After three washes with Tris-buffered saline containing 0.1% Tween 20 (TBST), membranes were incubated with horseradish peroxidase-conjugated rabbit monoclonal secondary antibody (BA1054-2) for 90 min at room temperature. Protein expression was normalized to GAPDH (GTX100118), and band intensities were quantified using ImageJ software (NIH, USA)19.

Statistical analysis
Statistical analyses were conducted using GraphPad Prism software. Data were subjected to one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Comparisons were initially made between each experimental group and the A549 control group, followed by pairwise comparisons among experimental groups to assess relative differences.

Results

Cell viability
To assess the cytotoxic effects of activated T cells and extracellular calcium (Ca²⁺) on A549 lung adenocarcinoma cells, the cells were treated for 24 and 72 h, and viability was determined using the MTT assay (Fig. 1). After 24 h, A549 cells treated with activated T cells at effector-to-target (E: T) ratios of 1:1 (37.9 ± 2.02%), 1:0.5 (54.1 ± 3.19%), and 1:0.25 (79.48 ± 1.84%) showed a significant, dose-dependent decrease in viability compared with untreated controls (p < 0.0001, versus control). Cells exposed to inactive T cells (108.3 ± 8.85%) exhibited no significant change relative to control (p = 0.1917). Treatment with Ca²⁺ alone (130.8 ± 1.23%) significantly increased viability (p < 0.0001, versus control). Co-treatment with activated T cells and Ca²⁺ further enhanced cytotoxicity, reducing viability to 32.29 ± 2.94% (1:1), 36.72 ± 2.21% (1:0.5), and 46.98 ± 2.65% (1:0.25) (p < 0.0001, versus control). In contrast, cells treated with inactive T cells plus Ca²⁺ (132.4 ± 3.25%) showed a significant increase in viability (p < 0.0001, versus control). After 72 h, a further decline in cell viability was observed in activated T cell–treated groups at E: T ratios of 1:1 (25.83 ± 1.08%), 1:0.5 (35.99 ± 1.47%), and 1:0.25 (39.42 ± 0.88%), all significantly lower than control (p < 0.0001, versus control). Inactive T cells (99.94 ± 5.06%) showed no significant effect (p = 0.99), while Ca²⁺ alone (129.4 ± 4.04%) significantly increased viability (p < 0.0001, versus control). Co-treatment of activated T cells and Ca²⁺ produced the most pronounced cytotoxicity, with viability reduced to 16.12 ± 1.02% (1:1), 23.56 ± 1.75% (1:0.5), and 26.86 ± 0.59% (1:0.25) (p < 0.0001, versus control). Conversely, the inactive T cell + Ca²⁺ group (128.6 ± 7.71%) showed increased viability (p < 0.0001, versus control). Collectively, these findings demonstrate that activated T cells induce dose- and time-dependent cytotoxicity in A549 cells, and that this effect is significantly potentiated by extracellular Ca²⁺, whereas inactive T cells have no cytotoxic effect (Table 1).

Apoptosis and necrosis analysis (Annexin V/PI)
Flow cytometry was used to distinguish viable (Annexin V⁻ PI⁻), early apoptotic (Annexin V⁺ PI⁻), late apoptotic/necrotic (Annexin V⁺ PI⁺), and necrotic (Annexin V⁻ PI⁺) A549 cells. Representative dot plots and gating strategies are shown in Fig. 2A. Quantitative analysis (Fig. 2B) revealed that treatment with activated T cells significantly increased both early apoptosis (Annexin V⁺ PI⁻, 21.3 ± 3.2%) and late apoptosis/necrosis (Annexin V⁺ PI⁺, 44.4 ± 8.3%) compared to the untreated control (3.8 ± 0.9% and 5.1 ± 1.4%, respectively; ****p < 0.0001). Co-treatment with Ca²⁺ further enhanced late apoptosis/necrosis (49.9 ± 0.8%) while maintaining high early apoptotic levels (24.6 ± 2.7%). Inactivated T cells and Ca²⁺ alone did not significantly alter apoptotic fractions relative to control. These results confirm that activated T cells induce both early and late apoptosis in A549 cells, with Ca²⁺ co-treatment amplifying the late apoptotic/necrotic population.

Immunophenotyping
To verify the activation status of Jurkat T cells, CD3 surface expression was analyzed by flow cytometry (Fig. 3). The flow cytometric histograms clearly show a marked rightward shift in fluorescence intensity for activated Jurkat cells compared with the inactivated population, indicating a higher proportion of CD3⁺ cells. Quantitative analysis revealed that activated Jurkat T cells exhibited significantly elevated CD3 expression (72.6 ± 3.84%), whereas inactivated Jurkat cells displayed markedly reduced expression (6.72 ± 4.20%). These findings confirm the successful activation of Jurkat T cells, validating their use as effector cells in subsequent co-culture cytotoxicity and signaling assays.

TAC and TOS concentration
To assess oxidative stress and antioxidant capacity in A549 cells following co-culture with T cells and/or calcium (Ca²⁺), total antioxidant capacity (TAC) and total oxidant status (TOS) were measured (Fig. 4). As shown in Fig. 4, A549 cells co-cultured with activated T cells exhibited a markedly reduced TAC concentration (0.23 ± 0.03 mM) compared with the untreated control group (****p < 0.0001). In contrast, cells treated with inactivated T cells (0.77 ± 0.12 mM) showed no significant difference from the control group (p = 0.9849). Treatment with Ca²⁺ alone (0.96 ± 0.02 mM) also did not significantly alter TAC levels (p = 0.0884). Notably, A549 cells co-treated with activated T cells and Ca²⁺ displayed a further reduction in TAC (0.20 ± 0.04 mM), significantly lower than the untreated control (p < 0.0001). Conversely, A549 cells exposed to inactivated T cells with Ca²⁺ (0.87 ± 0.01 mM) showed no significant difference compared with the control group (p = 0.7918). In parallel, TOS levels were substantially elevated in A549 cells co-cultured with activated T cells (17.26 ± 1.85 mM) compared with controls (****p < 0.0001). No significant changes were observed in cells treated with inactivated T cells (6.36 ± 0.43 mM; p = 0.8002) or Ca²⁺ alone (3.49 ± 0.95 mM; p = 0.8733). Importantly, A549 cells treated with activated T cells and Ca²⁺ exhibited the highest TOS concentration (22.47 ± 2.16 mM), indicating enhanced oxidative stress (p < 0.0001), while those treated with inactivated T cells plus Ca²⁺ (4.08 ± 1.66 mM) showed no significant difference from the control group (p = 0.9881). Collectively, these findings suggest that activated T cells induce oxidative stress in A549 lung cancer cells, which is further intensified by extracellular calcium. This oxidative imbalance, characterized by elevated TOS and suppressed TAC, may contribute to TRPV4-mediated calcium influx and subsequent cytotoxic signaling, highlighting a potential mechanistic link between immune cell activation, oxidative stress, and TRPV4 pathway activation.

Western blotting
As shown in Fig. 5, TRPV4 protein levels in A549 cells treated with activated T cells (0.18 ± 0.04%) were not significantly different from the untreated control group (p = 0.734). Similarly, treatment with inactivated T cells (0.16 ± 0.12%) did not alter TRPV4 levels compared to the control (p = 0.9971). In contrast, A549 cells exposed to calcium alone (0.80 ± 0.06%) exhibited a significant increase in TRPV4 protein levels relative to the untreated group (p < 0.0001). Cells treated with activated T cells plus calcium (0.90 ± 0.08%) also showed a marked elevation compared to the control (p < 0.0001). Likewise, cells treated with inactivated T cells plus calcium (0.78 ± 0.06%) displayed a significant rise in TRPV4 expression relative to the untreated group (p < 0.0001) (Figure S1).

Discussion
Lung cancer remains the second most common malignancy and the leading cause of cancer-related mortality worldwide20. Despite the emergence of targeted therapies and immune checkpoint inhibitors, treatment resistance and tumor immune evasion remain major obstacles to durable clinical outcomes. The present study investigated the apoptotic effects of activated T lymphocytes in cooperation with extracellular calcium on A549 lung adenocarcinoma cells, emphasizing the potential involvement of the calcium-permeable TRPV4 channel. Our findings demonstrate that activated T lymphocytes significantly reduced A549 cell viability in a dose-dependent manner, and this cytotoxic effect was further potentiated in the presence of extracellular calcium. This suggests a synergistic interaction between T-cell activation and calcium signaling in inducing apoptosis, with TRPV4 potentially serving as a key mediator. Indeed, TRPV4 protein expression was markedly upregulated in the A549 + activated T cell + Ca²⁺ group, indicating its potential role in linking immune activation to calcium-dependent tumor cell death. However, TRPV4 upregulation may be necessary but not sufficient for T-cell-mediated killing, as expression alone does not confirm functional involvement. Interestingly, TRPV4 expression also increased in response to Ca²⁺ alone or in combination with inactivated T cells, indicating that calcium exposure itself is a primary driver of TRPV4 upregulation. Consequently, while TRPV4 may contribute to the enhanced cytotoxicity observed with activated T cells, functional studies (e.g., TRPV4 inhibition or knockdown) are required to confirm whether TRPV4 activity is essential for T-cell-mediated killing.
Ca²⁺ alone increased A549 cell viability, likely through pro-proliferative calcium signaling pathways, including CaMK, calcineurin/NFAT, and ERK1/2, which promote cell cycle progression and survival. However, in the presence of activated T cells, this pro-survival effect was counteracted. Cytotoxic factors released by T cells—such as perforin, granzyme B, and pro-inflammatory cytokines (IFN-γ, TNF-α)—appear to dominate, reducing cell viability despite elevated Ca²⁺. These findings indicate that Ca²⁺-induced proliferative signaling can be modulated or overridden under immune-mediated cytotoxic conditions.
We intentionally used supraphysiological extracellular CaCl₂ to increase the electrochemical driving force for Ca²⁺ influx and probe TRPV4-dependent cytotoxic mechanisms; final total Ca²⁺ in complete medium after 3.0 mM added CaCl₂ was ~ 4.8 mM, and dose–response experiments (0.5–3 mM added; total 2.3–4.8 mM) showed a concentration-dependent potentiation of T cell–mediated cytotoxicity. The selected 3 mM CaCl₂ concentration corresponds to a final ionized Ca²⁺ level of approximately 4.4 mM in complete medium—sufficient to activate TRPV4 channels without causing nonspecific toxicity—thereby serving as an effective supra-physiological model to evaluate calcium-dependent apoptotic signaling.
Activated T lymphocytes are known to secrete cytokines including IFN-γ, TNF-α, and IL-6, which can modulate calcium signaling and TRP channel expression in epithelial and cancer cells (Fallah et al., 2022; Neuberger and Sobolevsky, 2023). These cytokines may activate transcriptional regulators through intracellular pathways such as NF-κB, JAK/STAT, and MAPK, leading to TRPV4 upregulation. Additionally, direct T cell–tumor cell interactions mediated by membrane-bound molecules, including CD40L and FasL, may further enhance TRPV4 activation or alter calcium homeostasis (Wang et al., 2024). Together, both paracrine signaling and contact-dependent mechanisms likely contribute to T-cell-mediated modulation of TRPV4 in cancer cells.
Furthermore, the observed increase in oxidative stress parameters (elevated TOS and reduced TAC) coinciding with TRPV4 upregulation suggests the involvement of a TRPV4–Ca²⁺–oxidative stress axis (NEW). Activation of TRPV4 can increase calcium influx, stimulating mitochondrial respiration and NADPH oxidase activity, which together elevate reactive oxygen species (ROS) levels21,22. The resulting oxidative imbalance can enhance apoptosis through mitochondrial membrane depolarization and activation of calcium-dependent proteases and caspases. This mechanistic link aligns with our findings and suggests that oxidative stress may act as a downstream effector of TRPV4-mediated calcium entry, amplifying T-cell-induced cytotoxicity.
Classically, T-cell cytotoxicity is mediated by perforin–granzyme or Fas–FasL pathways, which lead to caspase activation and apoptosis23. The present findings suggest an additional, noncanonical pathway whereby T-cell-derived cytokines or contact-dependent signals may sensitize tumor cells to apoptosis by upregulating TRPV4 and enhancing calcium-dependent oxidative stress. This mechanism complements the established cytotoxic functions of T cells and highlights a novel immunoregulatory axis linking TRPV4 signaling to T-cell effector activity. The potential therapeutic implications of this mechanism are significant. Targeting TRPV4 or modulating calcium influx could enhance the efficacy of T-cell-based immunotherapies by promoting tumor cell susceptibility to immune-mediated apoptosis. Conversely, pharmacologic inhibition of TRPV4 might prevent excessive calcium overload in normal tissues, offering a dual strategy for improving safety and precision in immunotherapy. Our findings suggest an additional, noncanonical mechanism, wherein T-cell-derived cytokines sensitize tumor cells by upregulating TRPV4, thereby enhancing calcium-dependent oxidative stress and apoptosis. This indirect route complements classical immune killing mechanisms, highlighting a potentially novel immunoregulatory layer that couples ion channel signaling to T-cell effector function.

Future directions
Building on the current findings, several research avenues should be pursued to deepen mechanistic insight and enhance translational impact. First, functional studies using TRPV4-specific inhibitors, siRNA knockdown, or CRISPR-mediated gene editing, as well as calcium chelation (e.g., EGTA) and TRPV4 agonists, will be essential to delineate the causal role of TRPV4 in T cell–mediated cytotoxicity and calcium influx. These experiments will clarify whether TRPV4 functions as a direct mediator or as a secondary amplifier of immune-induced apoptosis. Second, detailed cytokine profiling of activated T cells (e.g., IFN-γ, TNF-α, IL-6) combined with analysis of downstream signaling pathways such as NF-κB, JAK/STAT, and MAPK will help define the molecular mechanisms regulating TRPV4 activity in tumor cells. Ca²⁺ imaging using Fluo-4/AM or Fura-2 could further visualize TRPV4-linked calcium dynamics during co-culture with or without inhibitors. Third, in vivo tumor models and advanced 3D co-culture systems should be employed to validate the TRPV4–Ca²⁺–oxidative stress axis under physiologically relevant conditions, including immune–tumor interactions and microenvironmental calcium dynamics. Fourth, combinatorial therapeutic strategies integrating T-cell-based immunotherapy with TRPV4 modulators or calcium channel-targeting agents could enhance antitumor efficacy and overcome immune resistance. Finally, the development of calcium-based nanoparticles or TRPV4-targeted delivery systems may provide novel avenues for targeted, synergistic cancer therapy.
While the Annexin V/PI data clearly distinguish early and late apoptosis from necrosis, the current study did not include molecular analyses of apoptosis or necroptosis effectors (e.g., cleaved caspase-3/9, PARP, or RIPK1/RIPK3/MLKL). Future investigations employing caspase inhibition (z-VAD-fmk), detection of cleaved caspase and PARP, and evaluation of phosphorylated MLKL will be necessary to confirm the precise mode of cell death and to determine whether TRPV4-mediated Ca²⁺ influx triggers classical apoptosis or contributes to mixed apoptotic–necrotic processes.

Limitations
Although this study provides novel insights into the potential role of TRPV4 in mediating T-cell–induced apoptosis in lung cancer cells, several limitations should be acknowledged. First, the experiments were conducted exclusively in vitro using the A549 cell line, which may not fully replicate the complexity of the tumor microenvironment in vivo, including stromal and immune interactions. Second, the mechanistic link between activated T cells and TRPV4 upregulation was inferred from correlative evidence; direct validation through TRPV4 silencing, pharmacological inhibition, or pathway-specific assays was not performed. Third, T-cell activation was assessed using CD3 as a marker, which does not directly indicate activation status; functional activation markers such as CD69, CD25, or p-ERK/NFAT were not measured. Fourth, specific cytokine secretion patterns from activated T cells and their relative contribution to TRPV4 modulation were not fully characterized. Fifth, oxidative stress markers (TOS/TAC) were measured at the cellular level but were not directly linked to calcium influx or TRPV4 activity through molecular assays. Finally, functional assays distinguishing apoptosis from necrosis were limited, which may have led to partial overlap in interpreting cell death mechanisms.

Conclusion
This study demonstrates that activated T lymphocytes, in conjunction with extracellular calcium, markedly reduce the viability of A549 lung adenocarcinoma cells by enhancing apoptosis. The observed upregulation of TRPV4 protein in co-cultured cells suggests that T-cell-derived signals—potentially mediated by cytokines such as IFN-γ, TNF-α, and IL-6—may transcriptionally regulate TRPV4 expression, thereby amplifying calcium influx and oxidative stress within tumor cells. These findings reveal a previously underexplored interaction between immune activation and calcium signaling in lung cancer, proposing a novel TRPV4–Ca²⁺–oxidative stress axis as a contributing mechanism to T-cell-mediated tumor cytotoxicity. Future investigations using mechanistic inhibitors, gene silencing, and in vivo models are warranted to establish causality and assess the therapeutic potential of targeting TRPV4 in combination with immunotherapy. Collectively, this work highlights the promise of modulating calcium signaling to potentiate T-cell-based cancer treatments and overcome tumor immune resistance.

Supplementary Information
Below is the link to the electronic supplementary material.