Targeting PD-L1 with BMS-202 Enhances Antitumor Cytokine and Cytotoxic T-Lymphocyte Responses in C57BLx/6 Mouse Lung Carcinogenesis.

Introduction
Lung cancer remains one of the leading causes of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all cases.1 Despite advances in early detection and therapeutic strategies, the prognosis for patients with advanced-stage lung cancer remains poor, underscoring the urgent need for novel treatment approaches.2 In recent years, significant progress has been made in developing immune checkpoint inhibitors, which have revolutionized cancer therapy by targeting key immune system regulators.3 Among these, PD-1 (programmed death-1; CD279)/PD-L1 (programmed cell death-ligand 1) blockade has emerged as a cornerstone in the treatment of NSCLC, demonstrating durable responses in a subset of patients.4 PD-L1, a key member of the B7 protein family, binds to PD-1 to help tumor cells evade the immune system.5 PD-L1 is a transmembrane protein consisting of extracellular IgV and IgC domains, a transmembrane domain, and a short intracellular domain (ICD).6 While the extracellular domain inhibits T cell activity by binding PD-1,7 research on the ICD is limited. High PD-L1 expression is linked to shorter overall survival and worse prognosis in NSCLC patients.8 Various cancers, including NSCLC, renal cell carcinoma (RCC), breast cancer, colorectal cancer (CRC), stomach cancer, papillary thyroid cancer, and testicular cancer, show high PD-L1 expression, which correlates with poor prognosis.9–14 Antibodies targeting the PD-1/PD-L1 axis are approved for cancers like melanoma, NSCLC, RCC, Hodgkin’s lymphoma, bladder cancer, head and neck squamous cell carcinoma (HNSCC), and microsatellite unstable-high (MSI-H) or mismatch repair-deficient (dMMR) tumors.15 Although most studies on PD-L1 in tumors have centered on its role as an immune checkpoint, resistance to immune checkpoint inhibitors and limited efficacy in some patient populations highlight the necessity for additional therapeutic options. On the other hand, PD-L1’s non-immune functions include regulating tumor growth, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs), metabolism, genome stability, and drug resistance.16,17
BMS-202, a small-molecule inhibitor of the PD-1/PD-L1 interaction, has garnered attention as a promising candidate in immuno-oncology.18 To date, BMS-202 has been explored predominantly in pre-clinical settings: in vitro tumour cell lines and in vivo murine models, including humanised mouse systems, demonstrating blockade of PD-1/PD-L1 interaction and tumour growth suppression.19 Recent experimental investigations have provided valuable insights into the pre-clinical development of BMS-202. Structural and mechanistic analyses have demonstrated that BMS-202 binds directly to PD-L1, inducing PD-L1 dimerization and thereby preventing its interaction with PD-1, effectively restoring T-cell activation.20 Subsequent in vitro and in vivo studies have shown that BMS-202 exerts significant antitumor activity in various cancer models, including melanoma and glioblastoma, through the promotion of apoptosis and enhancement of cytotoxic T-lymphocyte responses.18 Moreover, comprehensive reviews of small-molecule PD-(L)1 inhibitors have positioned BMS-202 as a benchmark compound, underlining its role in immune checkpoint therapy development despite the fact that it remains in the pre-clinical domain.21 However, no human clinical trial of BMS-202 has been publicly registered or reported, indicating that the compound remains at the experimental stage.
Unlike monoclonal antibodies targeting PD-1 or PD-L1, BMS-202 offers distinct pharmacokinetic properties and potential advantages regarding tissue penetration and dosing flexibility. Preclinical studies have demonstrated the ability of BMS-202 to disrupt PD-1/PD-L1 binding, thereby restoring T-cell activity and promoting anti-tumor immune responses.22 Furthermore, its efficacy has been evaluated in various tumor models, showing encouraging tumor growth inhibition and immune activation results. Recent findings indicate that BMS-202 enhances T-cell proliferation and modulates the tumor microenvironment by reducing immunosuppressive cell populations, such as regulatory T (Treg) cells and myeloid-derived suppressor cells.23
The use of murine models, such as C57BL/6 mice, has been instrumental in advancing our understanding of tumor biology and assessing the therapeutic potential of novel agents.24 Subcutaneous tumor models, in particular, provide a controlled platform to evaluate the impact of experimental treatments on tumor growth and immune modulation.24,25 In the context of lung carcinogenesis, these models enable researchers to investigate the interplay between tumor cells and the immune microenvironment, offering valuable insights into the mechanisms underlying therapeutic responses.26,27 Moreover, recent studies have highlighted the relevance of the C57BL/6 mouse strain in studying lung cancer, as it closely mimics human disease pathology and response to therapy.
This study aims to assess the impact of BMS-202 on lung carcinogenesis using a subcutaneous tumor model in C57BL/6 mice. By leveraging this preclinical model, we seek to elucidate the therapeutic potential of BMS-202 and its effects on tumor progression and immune dynamics. The findings from this investigation may contribute to the growing body of evidence supporting small-molecule inhibitors as viable alternatives or complements to existing immunotherapies in lung cancer treatment. Ultimately, this research could pave the way for more effective strategies to overcome resistance mechanisms and improve outcomes for patients suffering from this devastating disease.

Materials and Methods

CMT167 Cell Culture
CMT167 cells (mouse lung cancer cell line) were purchased from Cell Bank of Iran (Pasteur Institute, Tehran, Iran). Then, cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO2. Cultures were routinely monitored for morphology and growth, and media were changed every 2–3 days to ensure optimal growth conditions. On passage 3, upon reaching 80% confluence, the cells were detached using 0.25% trypsin-EDTA, neutralized with complete media, and centrifuged at 300 x g for 5 minutes. The cell pellet was resuspended in Hank’s Balanced Salt Solution (HBSS), and the cell concentration was adjusted to 1×106 cells/mL for subsequent experiments.

Cytotoxicity Assay
CMT167 cells and human dermal fibroblast (HDF) cells at passage three were trypsinized, resuspended at 1×105 cells/mL, and seeded into 96-well plates for 24 hours. After attachment, varying concentrations of BMS-202 (5, 10, 20, and 40 μM) were added, with control wells receiving only media. After 48 hours, 10 μL of MTT solution was added, followed by a 4-hour incubation. Media was then replaced with 100 μL of DMSO to dissolve formazan crystals, and absorbance was measured at 570 nm. Cell viability was calculated using Cell Viability (%) = (Absorbance of treated wells/absorbance of control wells) × 100. A dose-response curve was plotted, and the IC50 value was determined.

Acridine Orange/Propidium Iodide (AO/PI) Staining
To assess apoptosis in cultured CMT167 cells that were treated with BMS-202, we utilized AO/PI staining. Cells from CMT167 were seeded at a density of 1 × 104 cells per well in 6-well plates and subjected to 10, 20, and 40 μM doses of BMS-202 for 48 hours. Following the treatment, the cells underwent trypsinization, were washed with PBS, and resuspended in PBS containing acridine orange (1 μg/mL) and propidium iodide (5 μg/mL) for 10 minutes at room temperature. They were subsequently rewashed and analyzed using a fluorescence microscope (Carl Zeiss).

In vivo Tumor Model
C57BL/6 mice (8-week-old, 20 ± 2 grams) were purchased from the Pasteur Institute, Tehran, Iran. To induce lung carcinogenesis using the CMT167 cell line in a subcutaneous tumor model with C57BL/6 mice (8-week-old), acclimatize the mice for a week, then anesthetize them using a ketamine/xylazine combination. Following sterilization of the inoculation site on the flank, using a 1 mL syringe with a 27-gauge needle, inject 100 µL (1 × 105 cells) of the cell suspension in HBSS/Matrigel subcutaneously into the right flanks of each mouse.28 All C57BL/6 mice were humanely euthanized at the endpoint using an approved method. Specifically, euthanasia was carried out using intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (10 mg/kg) cocktail, followed by cervical dislocation to ensure death, in line with AVMA Guidelines for the Euthanasia of Animals (2020). This method is widely accepted for minimizing animal suffering and ensuring rapid loss of consciousness. The average tumor volume ranged from 100 to 500 mm³, with tumor diameters approximately between 6 mm and 10 mm, depending on the experimental group. The inhibition rate of tumor growth was calculated as (%) = [(mean tumor weight of the control group − mean tumor weight of the treatment group)/ mean tumor weight of the control group] × 100.

BMS-202 Treatment
One week after cell injection, the animals developed subcutaneous tumors. They were divided into two groups of 6 mice each. The treatment protocol consisted of administering daily oral gavage of BMS-202 at doses of 30 mg/kg or 60 mg/kg. At the same time, the control group of mice was given saline, commencing one week following tumor cell inoculation. This dosage and protocols were chosen based on preliminary studies suggesting they effectively inhibit PD-1/PD-L1 interaction and promote anti-tumor immune responses without significant toxicity.18 Mice were monitored for toxicity, weight changes, and overall health throughout the treatment. Mice were sacrificed by cervical dislocation on day 21 post-infection of CMT167 cells. Tumor tissues were dissected and weighed for analysis. All research procedures were conducted by the ethical guidelines for the use of animals established by the University of Mohaghegh Ardabili. All animal experiments were approved by Animal Research Ethics Committee (AREC) (Approval No. 09876/March 18, 2024) which comply with the ARRIVE guidelines and carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.

Annexin V-FITC/PI Assay
To evaluate the apoptotic effects of BMS-202 on CMT167-induced subcutaneous tumors in mice, the Annexin V-FITC/propidium iodide (PI) dual staining assay was performed. Tumor samples were minced and enzymatically digested using a combination of collagenase (1 mg/mL) and DNase I (0.1 mg/mL) at 37°C for 60 minutes to obtain single-cell suspensions. Following digestion, the cell suspension was centrifuged, washed with PBS, and resuspended in binding buffer at 1×106 cells/mL concentration. Subsequently, Annexin V-FITC (5 µL) and PI (5 µL) were added, and the cells were incubated for 15 minutes in a dark environment. The stained cells were subsequently analyzed through flow cytometry to determine the percentage of early apoptotic (Annexin V+, PI-), late apoptotic (Annexin V+, PI+), and necrotic (Annexin V-, PI+) cells.

Flow Cytometry
We utilized flow cytometry to evaluate CD3+CD8+ T cells and CD8+PD-1+ T lymphocytes in tumor tissue. Tumor samples were harvested from C57BL/6 mice and mechanically dissociated. Then, collagenase (1 mg/mL) was added to tumor tissue homogenates and incubated at 37 °C for 15 min. The sample was centrifuged, and a single-cell suspension was obtained by washing with PBS and filtering through a 70 µm cell strainer. Cells were counted and incubated with Fc receptor-blocking antibody to prevent non-specific binding. For lymphocyte quantification, 1×106 cells were stained with a cocktail of 0.5 μL (0.5 mg/mL) of FITC-CD3, PE-CD8, and APC-PD-1 antibodies and incubated for 30 min at 4°C in the dark. Following staining, cells were washed with PBS, centrifuged at 1,500 rpm for 3 m, and resuspended in PBS. Flow cytometry was performed using a calibrated flow cytometer, and data were analyzed using FlowJo software to quantify the percentage of CD3+CD8+ T cells and CD8+PD-1+ T lymphocytes infiltrating the tumor microenvironment.

Enzyme-Linked Immunosorbent (ELISA)
The ELISA method was used to measure pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ) in the plasma of C57BL/6 mice. First, the mice were anesthetized with an appropriate anesthetic agent to minimize stress and discomfort. Blood samples were then collected by carefully puncturing the orbital venous sinus with a capillary tube containing heparin. A volume of 100 μL of blood was collected and transferred into sterile tubes containing EDTA to prevent coagulation. The samples were centrifuged at 1,500 rpm for 10 minutes at 4°C to separate the plasma, which was subsequently stored at −80°C until analysis. The plasma samples were diluted according to the manufacturer’s instructions for the ELISA procedure, and cytokine concentrations were quantified using specific ELISA kits designed for interleukin 6 (IL-6), Tumor necrosis factor alpha (TNF-α), and interferon‐gamma (IFN‐γ), following the provided protocols. Absorbance was measured using a microplate reader, and cytokine levels were calculated based on standard curves for each cytokine.

Caspase 3 Activity Assay
Caspase 3 activity in tumor tissue was assessed using a colorimetric caspase 3 assay kit (Abcam, ab39401) following the manufacturer’s instructions. Tumor samples were homogenized in 50 μL of chilled Lysis Buffer and incubated on ice for 10 minutes. The homogenate was centrifuged at 10,000 g for 1 minute to collect the supernatant. The protein concentration was determined and adjusted for each assay to 100 μg/50 μL. Subsequently, 50 μL of the caspase reaction mix and 5 μL of 4 mM DEVD-pNA substrate (final concentration 200 μM) were added to each sample well. The samples were incubated for 2 hours at 37°C, and absorbance was measured at 405 nm using a microplate reader to quantify caspase activity.

Real-Time Polymerase Chain Reaction (Real Time-PCR)
To quantify Bad, Bax, Apaf1, Bcl-2, Bcl-xl, and PD-L1 and other relevant immune markers in tumor tissue, real-time polymerase chain reaction (RT-qPCR) was performed on RNA extracted from homogenized tumor samples of C57BL/6 mice. Tumors were first homogenized in TRIzol reagent to isolate total RNA, followed by chloroform extraction and isopropanol precipitation. The RNA was then purified using a commercial RNA purification kit, and its concentration and quality were assessed using a spectrophotometer. cDNA synthesis was conducted using a reverse transcription kit, following the manufacturer’s protocol. Specific primers for PD-L1 (Table 1) were designed and validated for RT-qPCR. The qPCR reaction was set up using SYBR Green Master Mix, and 1 µg of cDNA was added to each reaction well. The cycling conditions included an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. Melting curve analysis was performed to confirm the specificity of the amplified products. The relative expression levels of target genes were normalized to the housekeeping gene GAPDH, and the results were analyzed using the 2-ΔΔCT method to quantify the expression of PD-L1 and other immune markers in the tumor microenvironment.

Statistical Analysis
Statistical analysis was carried out using SPSS software. The Shapiro–Wilk test was utilized to assess the data’s normality. A one-way analysis of variance (ANOVA) was performed to compare multiple groups. Following this, Tukey’s test was applied to pinpoint specific group differences. A p-value lower than 0.05 was considered statistically significant. All data are expressed as means ± standard deviations (SD).

Results

In vitro Cytotoxicity
The MTT assay evaluated the effects of different concentrations of BMS-202 on the viability of CMT167 cancer cells (Figure 1A) and HDF as normal control (Figure 1B). For the CMT167 cells, as the concentration of BMS-202 increases, the cell viability decreases concentration-dependent. At 0 μM, the viability is around 98.1 ± 1%, but this drops to 75.5 ± 2.7%, 62.7 ± 3.1%, 49.1 ± 1.6%, and 30.1 ± 2.4% at 5 μM, 10 μM, 20 μM, and 40 μM concentrations, respectively (P<0.001). This indicates a significant cytotoxic effect of higher BMS-202 concentrations on this cancer cell line. In contrast, the HDF control cells show much less sensitivity to BMS-202. The viability remains high even at the higher BMS-202 concentrations. This suggests that the cytotoxic effects of BMS-202 are more selective towards the CMT167 cancer cells than the normal HDF. Overall, the data demonstrates a concentration-dependent inhibitory effect of BMS-202 on the viability of CMT167 cells, while the normal HDF cells are relatively resistant to the treatment at the concentrations tested.

In vitro Apoptosis Assay
AO/PI staining of CMT167 cells exposed to BMS-202 reveals distinct fluorescence patterns influenced by dosage. As shown in Figure 2, the untreated cells (control) showed consistent green fluorescence, signifying healthy cells. At a concentration of 10 μM, some cells display green and orange/red fluorescence, indicating viable and early apoptotic cells. The 20 μM concentration exhibits a rise in orange/red fluorescence, suggesting a more significant number of late-apoptotic or necrotic cells. At 40 μM, numerous cells show orange/red fluorescence, reflecting elevated levels of apoptosis and cell death.

Tumor Weight
In this study, the impact of BMS-202 on lung carcinogenesis was evaluated using a subcutaneous tumor model in C57BL/6 mice. The mean tumor weight for the untreated control group was 884.75 ± 53.3 mg, while the BMS-202 treatment at 30 mg/kg significantly reduced it to 609 ± 41.5 mg. Furthermore, the higher dosage of BMS-202, 60 mg/kg, yielded the lowest mean tumor weight, 371.88 ± 47.5 mg (P<0.001). Accordingly, the tumor growth inhibition rates for groups treated with BMS202 at 30 mg/kg or 60 mg/kg were 31.6% and 57.97%, respectively. These results indicate a dose-dependent effect of BMS-202 on tumor growth, as shown in Figure 3.

Flow Cytometry Analysis of Lymphocytes
The impact of BMS-202 treatment on the tumor immune microenvironment was assessed by flow cytometric analysis of tumor-infiltrating lymphocytes. In the CMT167 subcutaneous tumor model, the percentage of CD3+CD8+ cytotoxic T cells within the tumor tissue was significantly increased from 6.8 ± 1.2% in the untreated group to 26.2 ± 3.1% in the BMS-202 (60 mg/kg) treatment group (Figure 4A; P<0.001). Additionally, the proportion of PD-1 expressing CD8+ T cells was reduced from 77.4 ± 5.7% in the untreated tumors to 39.6 ± 3.2% in the BMS-202 treated group (Figure 4B; P<0.001). These results indicate that BMS-202 treatment enhanced the infiltration and activation of cytotoxic T cells within the CMT167 tumor microenvironment.

In vivo Apoptosis Assay
The assessment of apoptosis induced by BMS-202 in the CMT167-induced subcutaneous tumors in mice was conducted using the Annexin V-FITC/PI assay (Figure 5A–5D). The results demonstrated a significant increase in apoptotic cell populations with BMS-202 treatment. Specifically, the untreated group exhibited a low level of apoptosis at 2.1 ± 0.17% (Figure 5A). In contrast, treatment with BMS-202 at 30 mg/kg significantly increased apoptosis, with a value of 35 ± 2.1% (P < 0.001) (Figure 5B). Furthermore, the higher dosage of BMS-202 at 60 mg/kg led to an even more pronounced effect, with apoptosis reaching 57.6 ± 3.4% (P < 0.001) (Figure 5C). A comparative quantification of apoptotic percentages across all groups is presented in (Figure 5D). These findings indicate that BMS-202 effectively promotes apoptosis in CMT167-induced tumors dose-dependently.

Caspase 3 Activity Assay
The activity of active caspase-3 in tumor tissue was evaluated to assess the apoptotic response to BMS-202 treatment in a subcutaneous tumor model using C57BL/6 mice, with results presented relative to untreated cells as the control (normalized OD). As shown in Figure 6, treatment with BMS-202 at 30 mg/kg significantly increased caspase-3 activity, with a fold change of 2.8 ± 0.24 (P<0.001). Furthermore, the higher dosage of BMS-202 at 60 mg/kg led to an even more significant fold change of 3.82 ± 0.2 (P<0.001). These findings indicate that BMS-202 effectively enhances caspase-3 activity in tumor tissues compared to normal HDF cells.

Real-Time PCR
The expression levels of apoptosis-related genes were assessed in tumor tissue following BMS-202 (60 mg/kg) treatment using real-time PCR (Figure 7). The results indicated significant changes in the expression of key apoptotic regulators. Specifically, the pro-apoptotic genes Bad, Bax, and Apaf1 exhibited increased expression levels of 2.78 ± 0.16, 3.48 ± 0.2, and 3.1 ± 0.34, respectively, indicating enhanced apoptotic signaling in response to BMS-202 treatment (P<0.001), the primers of these genes were shown in the Table 1. Conversely, the anti-apoptotic genes Bcl2 and Bcl-xl showed decreased expression levels of 0.38 ± 0.07 and 0.52 ± 0.09, respectively, further supporting the induction of apoptosis (P<0.001). These findings collectively highlight the impact of BMS-202 on the regulation of apoptosis-related genes in lung carcinogenesis, underscoring its therapeutic potential.

Inflammatory Cytokines Assay
As illustrated in Figure 8, the levels of inflammatory cytokines in the blood were measured following BMS-202 treatment using the ELISA assay. The results revealed a notable increase in the levels of key pro-inflammatory factors in treated cells compared to untreated controls. Specifically, untreated cells exhibited IFN-γ, TNF-α, and IL-6 levels of 235.6 ± 26 pg/mL, 102.8 ± 11 pg/mL, and 172.4 ± 15 pg/mL, respectively. In contrast, BMS-202-treated cells showed significantly elevated levels of IFN-γ at 521.6 ± 32 pg/mL (P<0.001), indicating a robust immune response. Additionally, TNF-α levels increased to 128 ± 11.9 pg/mL (P<0.05), while IL-6 levels rose to 191.4 ± 10.2 pg/mL (P<0.01). These findings suggest that BMS-202 treatment enhances the production of inflammatory cytokines, which may play a crucial role in modulating the immune response during lung carcinogenesis.

Discussion
The increasing rate of cancer-related deaths underscores an urgent and critical necessity to enhance cancer therapies through ongoing and dedicated research efforts.29,30 Cancer remains one of the leading causes of mortality worldwide, with new cases and deaths continuing to rise each year.31 Current cancer medications, while effective in many cases, face significant challenges. These include difficulties in specifically targeting tumors without affecting surrounding healthy tissue, managing adverse side effects, and overcoming the potential for resistance that can develop over time. Such issues highlight the need for innovative approaches and novel compounds to improve treatment outcomes.32,33
In this context, the present study evaluated the effects of BMS-202 on CMT167 cancer cells through various assays. The MTT assay revealed a concentration-dependent decrease in CMT167 cell viability, with significant reductions at higher concentrations of BMS-202 (P<0.001), while HDF cells remained relatively resistant. Apoptosis assays indicated that BMS-202 treatment increased fluorescence, indicative of late-apoptotic and necrotic cells at higher doses. In vivo, BMS-202 significantly reduced tumor weight in a mouse model, showing a dose-dependent effect (P<0.001). Flow cytometry analysis demonstrated enhanced infiltration of CD3+CD8+ T cells and reduced PD-1 expression in treated tumors (P<0.001). The Annexin V-FITC/PI assay confirmed increased apoptosis rates with BMS-202 treatment, reaching 57.6 ± 3.4% at 60 mg/kg (P<0.001). Caspase-3 activity and real-time PCR results indicated elevated pro-apoptotic gene expression and reduced anti-apoptotic gene expression following treatment (P<0.001).
Furthermore, BMS-202 treatment increased inflammatory cytokine levels, suggesting a robust immune response. The results of this study underscore the potential of BMS-202 as a therapeutic agent in lung cancer treatment. The significant cytotoxic effects observed in CMT167 cells and the selective sparing of normal HDF cells highlight the drug’s efficacy and specificity. The concentration-dependent decrease in cell viability and the corresponding increase in apoptosis suggest that BMS-202 initiates a robust apoptotic pathway, as evidenced by elevated caspase-3 activity and alterations in apoptosis-related gene expression. The enhancement of cytotoxic T-cell infiltration and reduction in PD-1 expression indicate that BMS-202 not only affects tumor cells directly but may also modulate the immune microenvironment favorably. The increased levels of pro-inflammatory cytokines further support the notion that BMS-202 could enhance anti-tumor immunity. Collectively, these findings provide compelling evidence for the therapeutic potential of BMS-202 in lung carcinogenesis, warranting further investigation into its mechanisms and clinical application.
The immune checkpoint pathway, PD-1/PD-L1, plays a crucial role in the immune evasion mechanisms of cancer. Tumor cells exploit this pathway to escape immune surveillance, leading to disease progression.34 The PD-1 receptor on T cells binds to its ligands, PD-L1 and PD-L2, which are often upregulated in cancers such as NSCLC and melanoma.35 This interaction inhibits T cell activation, promotes apoptosis, and leads to T cell exhaustion, facilitating tumor immune escape.36 Pro-inflammatory cytokines, particularly IFN-γ, induce PD-L1 expression, exemplifying the adaptive immune resistance mechanism where tumors enhance PD-L1 levels in response to immune pressure.37 Oncogenic mutations, such as those in the PTEN gene, further contribute to this immune suppression.
BMS-202, has significantly affected tumor-infiltrating lymphocytes (TILs) and overall immune response.23 BMS-202 effectively inhibits PD-L1, contributing to immune evasion by binding to PD-1 on T lymphocytes.23 T-cells are categorized into CD4+ and CD8+ subgroups, with CD8+ T lymphocytes playing a crucial role in combating tumors.38 Their effectiveness can be hindered by immunosuppressive factors like PD-1, which is present in immune cells. Elevated PD-1 levels in tumors can weaken the cytotoxic function of CD8+ T lymphocytes.39 Inhibiting PD-1 expression may boost the immune response against tumor cells. BMS-202 enhances T cell activation and proliferation by blocking this interaction, leading to a more robust anti-tumor immune response.24 Studies have demonstrated that BMS-202 increases the cytotoxic activity of CD8+ T cells. In preclinical models, treatment with BMS-202 resulted in elevated IFN-γ production levels by T cells, which is crucial for effective anti-tumor immunity. This indicates that BMS-202 promotes T-cell activation and improves their functional capabilities against tumor cells.40 BMS-202 has decreased the frequency of Tregs in the tumor microenvironment. This reduction is significant because Tregs typically suppress anti-tumor immune responses. By lowering Treg populations, BMS-202 enhances the overall immune response against tumors.19 The compound has been observed to rescue PD-L1-mediated inhibition of cytokine production in human CD3+ T cells. This suggests that BMS-202 can restore the ability of T cells to produce key cytokines essential for orchestrating an effective immune response against tumors.18
In vivo studies using mouse models have shown that BMS-202 significantly inhibits tumor growth.41 The anti-tumor effects are attributed to enhanced lymphocyte activity and direct cytotoxic effects on tumor cells, indicating a dual mechanism of action. In this regard, BMS-202 can induce apoptosis in cancer cells by activating the mitochondrial apoptosis pathway. BMS-202 influences the mitochondrial membrane potential, crucial for maintaining mitochondrial function and cellular energy production.42,43 By disrupting this potential, BMS-202 promotes mitochondrial outer membrane permeabilization (MOMP), releasing pro-apoptotic factors such as cytochrome c into the cytosol.42 The compound enhances the activity of pro-apoptotic members of the Bcl-2 family, such as Bax and Bak. These proteins translocate to the mitochondria upon activation, where they oligomerize and form pores in the outer mitochondrial membrane.42,44,45 This process is critical for initiating apoptosis as it allows for the release of cytochrome c, which subsequently activates caspases, the executioners of apoptosis.46 BMS-202 also appears to inhibit anti-apoptotic proteins like Bcl-2 and Bcl-xL. By reducing their levels of activity, BMS-202 shifts the balance towards apoptosis. The inhibition of these proteins facilitates the oligomerization of Bax and Bak, further promoting MOMP and apoptosis.47 Following the release of cytochrome c into the cytosol, it binds to Apaf-1, forming an apoptosome that activates caspase-9. This activation cascades into downstream caspases (such as caspase-3), leading to cellular dismantling and death.
Regarding translational status, no human clinical trials of BMS-202 are currently registered in major trial registries (ClinicalTrials.gov; EU CTR/CTIS; accessed November 12, 2025). Available evidence for BMS-202 is pre-clinical, including structural/mechanistic studies and antitumor activity in murine and xenograft models, supporting further translational investigation. To further elucidate the clinical relevance of BMS-202, future studies should focus on its long-term effects and potential combination therapies with existing treatments. Investigating the synergistic impact of BMS-202 with chemotherapy or other immunotherapies could provide insights into optimizing treatment regimens for lung cancer patients. Additionally, exploring biomarkers that predict response to BMS-202 would enhance patient selection and improve treatment outcomes. Understanding the pharmacokinetics and pharmacodynamics of BMS-202 in diverse populations is crucial for assessing its translational potential in clinical practice. Furthermore, evaluating the safety profile and potential adverse effects of BMS-202 in human trials will be essential to ensure its viability as a therapeutic option. Recent advances in targeted protein degradation provide an important conceptual framework for interpreting our findings with BMS-202 in the C57BL/6 subcutaneous lung carcinogenesis model. Yan et al describe how PROTAC technology harnesses the ubiquitin–proteasome system to selectively degrade oncogenic and immune-regulatory proteins within the tumor microenvironment, thereby overcoming several limitations of traditional occupancy-based inhibitors and enabling deeper, more durable pathway suppression.48 Qin and Xiao further emphasize that, over the next five years, PROTAC platforms are expected to transform cancer therapy by expanding druggable targets, incorporating nanotechnology for improved tumor delivery, and reshaping local immune and stromal signaling.49
Additionally, this research has several limitations that should be acknowledged. Firstly, the sample size may not be representative of the broader population, which could affect the generalizability of the findings. Additionally, the reliance on self-reported data may introduce bias, as participants might not accurately reflect their actual behaviors or opinions. Furthermore, the study’s cross-sectional design limits the ability to establish causal relationships between variables. Lastly, external factors such as environmental influences and temporal changes during the study period may have impacted the results, thereby limiting the conclusions that can be drawn.

Conclusion
In summary, BMS-202’s multifaceted action, inducing apoptosis, enhancing T-cell infiltration, and modulating inflammatory cytokine levels highlights its potential as a therapeutic agent in lung cancer. These findings warrant further investigation into the underlying molecular mechanisms and the potential for BMS-202 to be integrated into existing treatment regimens, particularly in combination with other immunotherapeutic strategies. Future studies should also explore the long-term effects of BMS-202 on tumor recurrence and metastasis and its impact on the overall survival of patients with lung cancer.