Gene expression analysis was conducted using the GEPIA2 database (http://gepia2.cancer-pku.cn), which utilizes data from the TCGA and GTEx databases (22). To compare HIF-1α gene expression levels between squamous cell carcinoma of the cervix (SESC) and corresponding normal tissues, the “box plot” function for expression analysis was employed. The following statistical parameters were utilized: a Log2FC (Logarithm to the base 2-fold change) cutoff value of 1, and a p-value cutoff value of 0.01. Additionally, GEPIA2 was employed to assess the overall survival of SESC patients using the “Survival Analysis” module, with the Group Cutoff set to the Quartile. Hazard ratios (HRs) with 95% confidence intervals (CIs) and log-rank P-values were computed to ascertain survival outcomes. The representative immunohistochemistry image of HIF-1α expression was obtained from the Human Protein Atlas (HPA) database (23).

HEK293T, Jurkat, Hela, and PANC-1 cell lines were acquired from the Iranian Biological Resource Center (IBRC). HEK293T, Hela, and PANC-1 cells were maintained in D10 media, comprising DMEM (Gibco, Life Technologies), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (Gibco, Life Technologies). Jurkat cells were cultured in R10 media, containing RPMI-1640 (Gibco, Life Technologies) supplemented with 10% FBS, 25 mM HEPES (Sigma Aldrich), 2 mM glutamine (Gibco), and 1% penicillin/streptomycin. Flow cytometry was used to validate mesothelin expression in the relevant cell lines prior to experiments. Regular mycoplasma contamination checks were conducted on all cell lines.

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood using standard methods with Histopaque^®-1077 (Sigma Aldrich). Primary human T cells were negatively selected with immunomagnetic beads (Pan T Cell Isolation Kit, Miltenyi Biotec) and stored at -80°C. T cells were cultured in TM10 media, composed of TexMACS™ Medium (Miltenyi Biotec), supplemented with 10% human serum and 100 IU/mL premium-grade rhIL-2 (Miltenyi Biotec). Blood samples were obtained from healthy volunteers under approval from the Research Ethics Committees of the School of Medicine, Tehran University of Medical Sciences [IR.TUMS.BLC.1402.015].

Lentiviral vectors were produced following previously established protocols (24). HEK293T cells were transfected with lentiviral CAR and packaging plasmids using the calcium phosphate method. Lentiviral supernatants were collected at 48- and 72-hour time points post-transfection and then concentrated through high-speed centrifugation. The concentrated lentivirus batches were resuspended in cold RPMI-1640 media and stored at -80°C. Titration of lentiviral vectors was performed using Jurkat cells.

mesoCAR T cells were generated as per previous descriptions (25). Briefly, 1 × 10⁶ T cells were seeded in each well of 12-well tissue culture plates and activated using Dynabeads™ Human T-Expander CD3/CD28 (Gibco, Life Technologies, 11161D) at a 1:1 ratio in TM10 media. Activated T cells were infected with lentiviral vectors supplemented with 8 mg/mL Polybrene (Santacruz) 24 hours after activation. Centrifugation at 850g for 1 hour at 32°C was employed to enhance transduction efficiency. Two hours later, 2 mL/well of TM10 media was added to the transduced T cells. At day 4 post-transduction, Dynabeads™ were removed from transduced T cells using a DynaMag™ magnet, and GFP expression, indicative of mesoCAR expression, was assessed via flow cytometry.

PX-478 (MedChemExpress, USA) was dissolved in Dimethyl sulfoxide (DMSO). To assess the impact of PX-478 on T cell proliferation, 2 × 10⁵ CFSE-labeled T cells were seeded in 96-well tissue culture plates and exposed to varying concentrations of PX-478. Human T Cell-Expander Dynabeads™ CD3/CD28 (Gibco, Life Technologies, 11161D) were used at a 1:1 ratio in TM10 medium to activate T cells. After three days, T cells were harvested, and their proliferation was evaluated via flow cytometry.

Adherent cells were washed twice with PBS, scraped, and transferred to 1.5 ml tubes. T cells were also harvested and washed twice with PBS. After centrifugation, cells were lysed using RIPA buffer containing 1mM PMSF at a ratio of 60 μl per 10⁶ cells. Proteins were separated by 10% SDS-PAGE under reducing conditions and subsequently transferred to PVDF membrane. The membrane was then blocked for 1 hour using a 5% BSA blocking reagent in Tris-Buffered Saline (pH=7.5) containing 0.05% Tween-20 (v/v) (TBST) and incubated with Rabbit anti-HIF-1α antibody diluted at 1:2,000 (Novus Biologicals NB100-449, Centennial, Colorado, USA) or Rabbit anti-β-actin antibody (Sigma-Aldrich) diluted at 1:2,000, overnight at 4˚C. The blots were further incubated with anti-Rabbit horseradish peroxidase-conjugated antibodies for 1 hour. Protein bands were detected using ECL method and X-ray film was used for visualization. Quantification was conducted using image J (imagej.org).

Culture plates were incubated either under normoxic conditions (37°C in humidified air, 5% CO2) or under hypoxic conditions (1% O2, 5% CO2, 94% N2). Hypoxia was induced using a hypoxia incubator chamber (StemCell Technologies, Inc.) purged at 25L/min for 4 minutes with a gas mixture containing 1% O2, 5% CO2, and 94% nitrogen as a balance before sealing the chamber.

For in vitro cytotoxicity assays, 1x10⁴ target cells were seeded in 96-well U‐bottomed tissue culture plates and pretreated with 25µM PX-478 for 24 hours under both hypoxic and normoxic conditions. Transduced or non-transduced T cells were then added to the wells at effector-to-target ratios of 1:1, 10:1, and 20:1 for 4 hours in TM10 media, with a final volume of 200 ml/well. To distinguish between effector and target cells, effector cells were stained with CFSE. Prior to flow cytometry analysis, 7-AAD (Miltenyi Biotec) was added to stain dead cells. Flow cytometry analysis utilized CFSE and 7-AAD staining to differentiate T cells from dead tumor cells. The frequency of lysed target cells (CFSE-/7-AAD+ cells) was calculated by subtracting the percentage of spontaneous lysis of target cells from the percentage of lysis of target cells in coculture with mesoCAR T cells. Normalized lysis of target cells (Specific lysis) was reported based on mesothelin expression on target cells.

Target cells were treated with 50 mg/ml of mitomycin C (Sigma Aldrich) for 30 minutes at 37°C and subsequently washed. 2x10⁵ target cells were seeded in 48-well tissue culture plates and pretreated with 25µM PX-478 for 24 hours under both hypoxic and normoxic conditions before removal of the media. For cell proliferation analysis, mesoCAR T cells and untransduced T cells were stained with 5 mM CFSE at room temperature for 8 minutes. An equal amount of FBS was added to halt the reaction. After washing three times with complete RPMI 1640 medium, CFSE-labeled cells (0.2 × 10⁶/well) were cocultured with either target cells or media, in the absence of exogenous IL‐2, in 48‐well plates, with a final volume of 800 µl/well. After 24 hours, 200 µl of the supernatants were harvested and stored at −80°C. The subsequent cytokine analysis was carried out by enzyme-linked immunosorbent assay (ELISA) to quantify IFN-γ and IL-2. After 72 hours, cells were stained with PerCP-conjugated anti-human CD3 antibody (Clone: HIT3a, BioLegend), and CFSE dilution of CD3+ cells was determined by flow cytometry, as an indicator of proliferation.

The purity of isolated T cells was confirmed using APC-conjugated anti-human CD3 (Clone: UCHT1, BioLegend). PE-conjugated anti-human mesothelin (Clone: #420411, R&D Systems) was used to detect mesothelin expression. FITC-conjugated anti-human CD3 (Clone: HIT3a, BioLegend), PE-conjugated anti-human CD279 (PD-1) (Clone: EH12.2H7, BioLegend), and APC-conjugated anti-human CD366 (Tim-3) (Clone: F38-2E2, BioLegend) antibodies were used to measure the expression of exhaustion markers. For proliferation assays, cells were loaded with CellTrace™ CFSE (Life Technologies, #C34554) according to manufacturer’s instructions, and T cells were detected using PerCP-conjugated anti-human CD3 antibody (Clone: HIT3a, BioLegend). Data were collected using a BD FACSCalibur (BD Biosciences) and analyzed with FlowJo software (v10.6). All assays were performed in duplicate and repeated two to three times.

Normality tests and one-way/two-way analysis of variance (ANOVA) were performed using GraphPad Prism software (v9) to identify differences among various treatment groups. p-values below 0.05 were considered statistically significant.

To assess the significance of HIF-1α expression in cervical cancer, we utilized the GEPIA2 database to visualize the mRNA expression levels of HIF-1α in cervical cancer. The analysis involved 13 normal tissue samples and 306 samples from cervical cancer patients. Our data unequivocally demonstrate an upregulation of HIF-1α in cervical cancer patients ( Figure 1A ). Complementing this, the immunohistochemistry image of HIF-1α protein levels in tissue samples from the Human Protein Atlas (HPA) dataset confirmed similar findings ( Figure 1B ).

We further conducted a Kaplan-Meier survival analysis using GEPIA2 to investigate the prognostic value of HIF-1α. Our results reveal a statistically significant association between high expression of the HIF1A gene and shorter overall survival in cervical cancer patients ( Figure 1C ).

PX-478, previously identified as a compound that decreases cellular HIF-1α levels, was evaluated in our study. To validate the increase of HIF-1α under hypoxic conditions and assess the inhibitory effect of PX-478, HeLa and T cells were cultured under normoxic and hypoxic (1% O2) conditions while exposed to varying doses of PX-478 for 24 hours. Western blot analysis confirmed the efficient stabilization of HIF1α in hypoxic conditions and demonstrated that PX-478 inhibits the hypoxia-induced increase in HIF-1α protein levels in a dose-dependent manner ( Figures 2A-D ).

We generated and characterized second-generation mesoCAR T cells, as described previously (25). Briefly, human CD3+ T cells were efficiently infected with lentiviral particles encoding the second-generation mesoCAR transgene ( Figure 3A ). We subsequently assessed the in vitro antitumor capacity of these cells. We used PANC-1 and HeLa cells, which represent mesothelin-negative and positive tumor cells respectively ( Figures 3B, C ). T cells expressing the mesoCAR transgene exhibited specific cytotoxicity against mesothelin-positive HeLa cells, while no cytotoxicity was observed against mesothelin-negative PANC-1 cells ( Figure 3D ). To test the effectiveness of mesoCAR T cells against HeLa cells, we investigated their proliferation and their capacity to produce IL-2 and IFN-γ cytokines in vitro. mesoCAR T cells demonstrated a high mesothelin-specific proliferation rate comparable to untransduced T cells after being stimulated with Hela and PANC-1 cells ( Figure 3E ). After CAR T cell stimulation with HeLa, mesoCAR T cells showed high mesothelin-specific proliferation rates and produced large amounts of IFN-γ and IL-2, comparable to untransduced T cells ( Figures 3E-G ). No IL-2 and IFN-γ secretion was detected in cultures of T cells alone, tumor cells alone, or when irrelevant target cells like PANC-1 were involved ( Figures 3F, G ).

The efficacy of CAR T cell immunotherapies against solid tumors hinges on T cell proliferation, persistence, and accumulation (26). To investigate the influence of PX-478 on mesoCAR T cell proliferation, we performed a series of experiments. Initially, T cells activated with anti-CD3/CD28-coated beads were exposed to varying concentrations of orally available PX-478, revealing that PX-478 can dose-dependently reduce antigen-nonspecific T cell proliferation ( Figure 4A ), while concurrently maintaining T cell viability unchanged ( Figure 4B ).

Consistent with the data obtained from T cells, we observed that the antigen-nonspecific proliferation of mesoCAR T cells in an IL-2-containing medium was also impeded by PX-478 ( Figure 4C ). Given the observed direct inhibitory effect of PX-478 on T and mesoCAR T cell proliferation, we conducted experiments in which tumor target cells were pretreated with PX-478 for 24 hours under both hypoxia and normoxia conditions. After supernatant removal, mesoCAR T cells were introduced. Interestingly, pretreatment of tumor cells with PX-478 did not significantly impact mesoCAR cell proliferation ( Figure 4D ). This result was supported by the analysis of cytokine production, where no significant changes in the levels of IL-2 and IFN-γ were observed ( Figures 4E, F ). Collectively, our findings indicate that PX-478 directly inhibits antigen-nonspecific T and mesoCAR T cell proliferation, while pretreatment of tumor cells with PX-478 has no influence on mesoCAR T cell proliferation and cytokine production.

Having established the effect of PX-478 on mesoCAR T cell proliferation, we sought to examine its impact on the cytotoxicity of these cells against tumor target cells. HeLa cervical cancer cells were cultured, pre-exposed to 25µM PX-478 for 24 hours, and then co-incubated with mesoCAR T cells under normoxic and hypoxic (1% O2) conditions. mesoCAR T cells demonstrated effective killing of HeLa cells under both hypoxic and normoxic conditions ( Figure 4G ). However, PX-478 significantly reduced the cytotoxicity of mesoCAR T cells under both hypoxic and normoxic conditions ( Figure 4G ).

In an effort to understand the underlying reason for the impairment of mesoCAR T cell cytotoxic function, we considered the possibility that these cells become exhausted in the presence of PX-478. Exhausted T cells can be categorized into progenitor-exhausted T cells (T_pex) and terminally exhausted T cells (T_tex) based on their function and phenotype (24, 27, 28). T_pex cells express PD-1 but not TIM3, retain stem-like characteristics, and remain polyfunctional. In contrast, T_tex cells express both PD-1 and TIM3 at high levels, have a limited lifespan, and cannot effectively suppress tumor growth (29). Immune checkpoint blockade can rejuvenate T_pex but not T_tex cells (30). To explore this, cells from our cocultures were analyzed via flow cytometry for the expression of PD-1 and TIM3. Our results revealed that pretreatment of tumor cells with PX-478 did not significantly alter the expression of PD-1 but led to an increased expression of TIM3 ( Supplementary Figure S1A, B ). Furthermore, the abundance of T_tex cells (PD-1+TIM3+) increased, while the abundance of T_pex cells (PD-1+TIM3-) decreased under both normoxic and hypoxic conditions ( Supplementary Figure S1C ).