4‐FPAC‐Modulated Tumor‐Associated Macrophage Conditioned Media Suppresses Malignant Phenotypes and Promotes Cell‐Cycle Arrest and Apoptosis in NSCLC

ABSTRACT

Non‐small cell lung cancer (NSCLC) progression is strongly influenced by tumor‐associated macrophages (TAMs), which frequently acquire an M2‐like phenotype that promotes proliferation, epithelial‐mesenchymal transition (EMT), angiogenesis, stemness, and therapy resistance. Previous studies from our group established the direct cytostatic and macrophage‐polarizing effects of the synthetic coumarin derivative 4‐fluorophenylacetamide‐acetyl coumarin (4‐FPAC), including ROS‐mediated apoptosis, G0/G1 cell‐cycle arrest, suppression of EMT‐associated signaling in A549 cells, and promotion of M1‐like macrophage polarization. In the present study, we investigated whether 4‐FPAC‐modulated TAM‐derived conditioned media could suppress tumor‐promoting behavior in A549 NSCLC cells. THP‐1 monocytes were differentiated into macrophages and polarized using A549‐conditioned medium to generate TAM‐like macrophages. These TAMs were subsequently treated with 4‐FPAC, and conditioned media from control or treated TAMs were applied to A549 cells to evaluate macrophage‐associated anti‐tumor effects. MTT analysis further showed that direct exposure of A549 cells to 4‐FPAC under TAM‐CM‐supported conditions reduced cell viability in a dose‐dependent manner. Functional assays demonstrated that conditioned media derived from 4‐FPAC‐treated TAMs significantly reduced A549 proliferation, inhibited migration and invasion, and impaired angiogenic responses in a chick chorioallantoic membrane model. Flow cytometry revealed G0/G1 cell‐cycle arrest and reduced NANOG‐positive stem‐like cells, while DNA fragmentation and TUNEL assays confirmed enhanced apoptosis. Mechanistically, qRT‐PCR and immunoblotting demonstrated EMT reversal, suppression of AKT signaling, VEGFα, IL8, OCT3/4, and NANOG, and upregulation of p21, p53, and caspase‐3/cleaved caspase‐3. Collectively, these findings demonstrate that 4‐FPAC directly suppresses A549 viability under TAM‐CM‐supported conditions and modulates TAM‐derived conditioned‐media activity, highlighting its potential to target both tumor cells and tumor‐macrophage interactions within the NSCLC microenvironment.

4‐FPAC‐modulated TAM conditioned media attenuated proliferation, EMT, angiogenesis, and stemness while inducing cell‐cycle arrest and apoptosis in NSCLC cells. These anti‐tumor effects were associated with disruption of the IL8–CD163 axis, supporting the potential of 4‐FPAC as a tumor microenvironment‐targeted therapeutic agent.

1. Introduction

Lung cancer is one of the most prevalent malignancies worldwide and remains the leading cause of cancer‐related mortality, accounting for nearly 18% of global cancer deaths [1, 2]. Non‐small cell lung cancer (NSCLC), which constitutes approximately 85% of all lung cancer cases, is frequently diagnosed at advanced stages where currently available therapeutic strategies provide only limited clinical benefit [3, 4]. Despite substantial advances in targeted therapies and immunotherapy, NSCLC continues to exhibit poor prognosis due to intrinsic and acquired therapeutic resistance, metastatic progression, and high recurrence rates [5, 6]. These limitations highlight the urgent need for therapeutic approaches that not only directly target tumor cells but also modulate the tumor microenvironment (TME), which plays a central role in tumor progression and therapy resistance [7, 8].

The TME comprises tumor cells, stromal components, extracellular matrix, and diverse immune cell populations that collectively regulate cancer growth, invasion, and dissemination. Among these, tumor‐associated macrophages (TAMs) represent one of the most abundant and functionally influential immune cell populations within NSCLC [9, 10]. Due to their remarkable plasticity, macrophages can polarize toward either a pro‐inflammatory M1 phenotype or an immunosuppressive and pro‐tumorigenic M2 phenotype in response to microenvironmental cues. M2‐polarized TAMs promote tumor proliferation, epithelial–mesenchymal transition (EMT), invasion, angiogenesis, stemness, immune evasion, and therapeutic resistance [11, 12]. Clinically, elevated M2‐TAM infiltration has been strongly associated with increased metastatic burden, poor therapeutic response, and unfavorable prognosis in NSCLC patients [13, 14]. Consequently, pharmacological reprogramming of M2‐like TAMs toward an M1‐like phenotype has emerged as a promising strategy for disrupting tumor‐supportive signaling within the TME [15].

Coumarins constitute a versatile class of naturally occurring heterocyclic compounds with well‐documented anti‐inflammatory, antioxidant, and anticancer properties [16]. Structural modification of the coumarin scaffold has resulted in derivatives with enhanced biological and immunomodulatory activities. In this context, 4‐fluorophenylacetamide‐acetyl coumarin (4‐FPAC), a newly synthesized coumarin derivative, has attracted considerable interest due to its potent anticancer and immunomodulatory potential. In our previous study, direct exposure of A549 NSCLC cells to 4‐FPAC demonstrated pronounced cytostatic and anti‐metastatic effects at nanomolar concentrations, including G0/G1 cell‐cycle arrest, ROS‐mediated apoptosis, suppression of epithelial–mesenchymal transition (EMT), and inhibition of migratory and invasive properties through modulation of p53 and PI3K/AKT signaling pathways [17]. Furthermore, comparative analyses performed using additional NSCLC cell lines, including NCI‐H23 and NCI‐H522, revealed that A549 cells were markedly more sensitive to 4‐FPAC treatment, thereby establishing A549 as a highly responsive and biologically relevant in vitro model for investigating the anticancer effects of this compound [17].

More recently, we demonstrated that 4‐FPAC also exerts significant immunomodulatory effects on THP‐1‐derived macrophages by promoting polarization toward a pro‐inflammatory M1 phenotype while suppressing M2‐associated characteristics [17]. This macrophage reprogramming was validated through enhanced intracellular reactive oxygen species (ROS) and nitric oxide (NO) production, elevated IL‐12 levels, reduced IL‐10 expression, upregulation of M1‐associated markers such as CD80, STAT1, and AKT2, and suppression of M2‐associated markers including CD163, STAT3, and AKT1 [17]. In addition, molecular and in silico analyses demonstrated that 4‐FPAC modulates key signaling pathways associated with macrophage polarization, including NF‐κB, p38 MAPK, and PI3K/AKT signaling [17]. Collectively, these findings established that 4‐FPAC possesses both direct anticancer activity against NSCLC cells and the capacity to functionally reprogram macrophages toward an anti‐tumorigenic phenotype.

Given the pivotal role of M2‐TAMs in promoting NSCLC progression and metastasis, it is important to determine whether 4‐FPAC can counteract the tumor‐supportive influence of TAM‐derived soluble mediators. M2‐TAM‐secreted factors are known to enhance EMT, proliferation, migration, angiogenesis, stemness, and survival signaling, thereby creating a self‐reinforcing network that drives metastatic progression and therapy resistance [18]. While our previous studies independently established the direct cytotoxic effects of 4‐FPAC on NSCLC cells and its macrophage‐polarizing activity, the functional consequences of 4‐FPAC‐mediated TAM reprogramming on tumor cell behavior have not yet been explored. Therefore, the present study was specifically designed to investigate the indirect, macrophage‐mediated anti‐tumor effects of 4‐FPAC rather than re‐evaluating its already established direct cytotoxic effects on A549 cells.

To address this objective, we employed an experimental workflow in which A549‐derived conditioned medium (A549‐CM) was first used to induce TAM‐like polarization in THP‐1‐derived macrophages, followed by treatment of these TAMs with 4‐FPAC. Conditioned media collected from control and treated TAMs were subsequently applied to A549 cells to evaluate whether 4‐FPAC‐mediated macrophage reprogramming could disrupt tumor‐supportive signaling within the NSCLC microenvironment. This biologically relevant in vitro model enabled us to specifically examine how alterations in the macrophage secretome influence tumor cell behavior.

Based on this rationale, we hypothesized that treatment of TAMs with 4‐FPAC would reprogram their secretory profile toward an anti‐tumorigenic state, and that conditioned media derived from 4‐FPAC‐treated TAMs would suppress TAM‐driven proliferation, migration, EMT, angiogenesis, stemness, and survival in A549 NSCLC cells. To test this hypothesis, we investigated the effects of 4‐FPAC‐modulated TAM‐conditioned media on A549 cellular behavior using a comprehensive series of functional and molecular assays. Specifically, we examined changes in proliferation, migration, invasion, EMT‐associated markers, angiogenic potential, cell‐cycle progression, apoptotic activation, stemness characteristics, and key gene and protein expression profiles. Collectively, this study extends our previous findings by elucidating whether macrophage‐centered immunomodulation by 4‐FPAC can indirectly suppress NSCLC progression through remodeling of TAM‐derived signaling networks.

2. Materials and Methods

2.1. Materials

Roswell Park Memorial Institute (RPMI‐1640) medium, phorbol‐12‐myristate‐13‐acetate (PMA), and 3‐(4,5‐dimethylthiazol‐2‐yl)−2,5‐diphenyltetrazolium bromide (MTT) were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Gibco®, Thermo Fisher Scientific (Waltham, MA, USA) supplied fetal bovine serum (FBS) and penicillin–streptomycin (100 U/mL). Additional analytical‐grade reagents were procured from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). The coumarin derivative, 4‐FPAC was synthesized and structurally validated by the research group of Dr. S. S. Soman, Department of Chemistry, The M.S. University of Baroda, and provided as a gift sample. A primary stock of 4‐FPAC was dissolved in dimethylformamide (DMF), followed by dilution in phosphate‐buffered saline (PBS) to prepare working concentrations. Care was taken to ensure that the final DMF content in all experimental setups did not exceed 0.5%.

2.2. Maintenance of Cell Lines

THP‐1 and A549 cell lines were procured from the National Centre for Cell Science (NCCS), Pune, India. THP‐1 cells were cultured in RPMI‐1640 medium, while A549 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), both supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‐streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). All cultures were grown at 37°C in a humidified atmosphere containing 5% CO₂ (Thermo Fisher Scientific, Waltham, MA, USA). For macrophage differentiation, THP1 monocytes were treated with 50 nM PMA for 24 h, followed by a 24‐h resting period in PMA‐free medium to allow complete differentiation.

2.3. Activation of Tumor‐Associated Macrophages and Collection of Conditioned Media

For the preparation of conditioned medium (CM), A549 cells were cultured in serum‐free medium for 24 h, and the supernatant was collected. THP‐1‐derived macrophages were subsequently treated with this A549‐derived CM for 24 h to promote their polarization into tumor‐associated macrophages (TAMs). After activation, the medium was replaced with serum‐free medium and incubated for an additional 24 h to obtain TAM‐derived CM.

For drug‐treated conditioned media, TAMs were exposed to 4‐FPAC for 48 h, followed by incubation in serum‐free medium for another 24 h. The resulting supernatant was collected as 4‐FPAC–treated TAM‐derived CM. All collected media were centrifuged to remove cellular debris, and the clarified supernatants were used for subsequent A549 cell experiments. The complete workflow used for the preparation of all conditioned media types is illustrated in Figure 1.

Figure 1.

Workflow illustrating sequential preparation of A549‐CM, TAM‐CM, and 4‐FPAC–treated TAM‐CM for subsequent treatment of A549 cells.

2.4. Cell Viability (MTT Assay)

A total of 10,000 A549 cells per well were seeded in triplicate into 96‐well plates in the presence of 200 μL of TAM‐derived CM and incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO₂. After incubation, the cells were then exposed to increasing concentrations of 4‐fluorophenylacetamide‐acetyl coumarin (0, 0.5, 1, 10, 25, 50, 75, and 100 μM) for 48 h. As controls, vehicle‐treated wells received 0.1% (v/v) DMF, while positive control wells were treated with 100 μM hydrogen peroxide (H₂O₂), a standard cytotoxic agent, to validate assay performance. Following the 48 h treatment, 20 μL of MTT solution (5 mg/mL) was added to each well and incubated for an additional 4 h at 37°C. The medium was then carefully removed, and 100 μL of acidified isopropanol was added to dissolve the formazan crystals generated by viable cells. Absorbance was recorded at 570 nm using a iMark Microplate absorbance plate reader (Biorad). Cell viability was determined using the formula:

$$\text{Cell viability}(\%)=\left(\frac{\text{Absorbance of treated cells}}{\text{Absorbance of control group}}\right)\mathrm{x}100$$

2.5. BrdU Incorporation Assay for Proliferation Analysis

The proliferative capacity of A549 cells in response to soluble factors secreted by tumor‐associated macrophages was assessed using a BrdU (5‐bromo‐2′‐deoxyuridine) incorporation assay. Briefly, A549 cells were seeded into 6‐well plates at a density of 1 × 10⁴ cells per well and allowed to adhere overnight in complete DMEM. Cells were then treated with CM obtained from control or 4‐FPAC‐treated TAMs, prepared in serum‐free media, and incubated for 48 h. Following treatment, 25 μM BrdU was added to each well, and cells were incubated for an additional 24 h. Thereafter, cells were washed and fixed with 4% paraformaldehyde for 20 min at 4°C. DNA denaturation was carried out by incubation with 1 N HCl at 37°C for 15 min. Fixed cells were then incubated with an anti‐BrdU primary antibody prepared in PBS containing 0.1% Triton X‐100 and 2.5% BSA for 24 h (overnight) at 4°C. After three washes with PBS, cells were incubated with an Alexa Fluor 488‐conjugated secondary antibody for 1 h at 37°C. Finally, BrdU‐positive nuclei were visualized using a fluorescence microscope.

2.6. Wound Healing Assay

The migratory potential of A549 cells was evaluated using a wound‐healing assay. A549 cells were seeded into six‐well plates at a density of 2 × 10⁵ cells per well in complete DMEM supplemented with 10% FBS and allowed to adhere overnight to form a confluent monolayer. Upon reaching approximately 90% confluency, a uniform scratch was created across the cell monolayer using a sterile 20 μL pipette tip. The wells were gently rinsed with PBS to remove detached cells, and the medium was replaced with serum‐reduced (1% FBS) DMEM containing conditioned media derived from control TAMs or 4‐FPAC–treated TAMs. Images of the wound area were captured immediately after scratching (0 h) and subsequently at 24 h and 48 h of incubation using a Lawrence & Mayo inverted microscope. The extent of wound closure was analyzed to determine the migratory response of A549 cells using wound healing tool in ImageJ software [19].

2.7. Transwell Invasion and Migration Assay

The migratory and invasive behavior of A549 cells in response to soluble factors secreted by TAMs was evaluated using Transwell chambers (8 µm pore size; Corning, USA). A549 cells were cultured in conditioned media obtained from control TAMs or 4‐FPAC–treated TAMs for 48 h. After incubation, the cells were trypsinized, centrifuged at 600 × g for 5 min at room temperature, and resuspended in serum‐free DMEM. The cell suspension was adjusted to a density of 1 × 10⁵ cells/mL, and 200 µL was added to the upper chamber. For invasion assays, the membranes were pre‐coated with Matrigel (50 µg/mL) and incubated at 37°C for 30 min, whereas migration assays were performed without Matrigel coating. The lower chambers were filled with 600 µL of DMEM containing 10% FBS as a chemoattractant. Following 24 h incubation, non‐migrated cells on the upper surface were gently removed using a sterile cotton swab. The membranes were fixed with 100% methanol for 30 min at room temperature and stained with 0.1% crystal violet for 20 min. Stained cells on the lower surface were visualized and counted under a light microscope (Lawrence and Mayo, magnification × 40). The migration and invasion assays were performed under identical conditions, except for the presence of Matrigel in the invasion setup. Images were quantified using ImageJ software.

2.8. Chorioallantoic Membrane (CAM) Assay

To assess the angiogenic potential of tumor cells influenced by TAM‐derived soluble factors, a CAM assay was performed using fertilized chick embryos. A549 cells were pre‐incubated for 48 h with conditioned media obtained from control TAMs or 4‐FPAC–treated TAMs. After incubation, the cells were harvested, resuspended in sterile PBS, and approximately 1 × 10⁶ cells were injected into the air sac of day 0 fertilized chick embryos under sterile conditions. The embryos were further incubated at 37°C with 60%–70% humidity for 3 days. On embryonic days 3 post‐injection, the chorioallantoic membranes were excised and photographed under a stereomicroscope. The vascular architecture was analyzed using AngioTool 0.6 software (RRID:SCR_016393) to quantify key angiogenic parameters, including vessel density, number of junctions, total vessel length, and lacunarity. All experiments involving chick embryos were performed in accordance with the guidelines of the Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India. The study protocol was approved by the Institutional Animal Ethics Committee (IAEC), The Maharaja Sayajirao University of Baroda (Approval No. MSU‐Z/IAEC‐04/10‐2020). Statistical analysis was performed using Student's t‐test, with a significance threshold set at p ≤ 0.05.

2.9. Flow Cytometric Analysis of Cell Cycle Progression and Nanog Expression

The effect of conditioned media derived from control and 4‐FPAC–treated TAMs on stemness and cell cycle dynamics of A549 cells was examined using flow cytometry. A549 cells were seeded in T25 flasks at a density of 6 × 10⁶ cells and synchronized in serum‐free medium for 24 h. Subsequently, the cells were treated with CM from control or 4‐FPAC–treated TAMs for 24 h under standard culture conditions.

For cell cycle analysis, treated cells were harvested, washed with PBS, and fixed overnight in ice‐cold 70% ethanol at −20°C. The following day, cells were centrifuged, washed twice with PBS, and incubated with RNase A (100 µg/mL) and propidium iodide (PI, 50 µg/mL) for 30 min at 37°C in the dark.

For stemness evaluation, cells were fixed with 4% paraformaldehyde for 15 min at 4°C, permeabilized with 0.1% Triton X‐100, and blocked with 1% BSA in PBS to reduce non‐specific binding. The cells were then incubated with an anti‐Nanog rabbit primary antibody (Cloud‐Clone Corp., USA; 1:300) for 1 h at room temperature, followed by incubation with an FITC‐conjugated anti‐rabbit secondary antibody (Sigma‐Aldrich, USA; 1:500) for 45 min in the dark.

Fluorescence acquisition was performed using a BD FACSAria cell sorter (BD Biosciences, San Jose, CA, USA), and data were processed using the Manufacturer's software. A minimum of 10,000 events per sample were recorded to quantify the distribution of cells across G₀/G₁, S, and G₂/M phases and determine Nanog expression.

2.10. DNA Fragmentation Assay

To evaluate the apoptotic potential of soluble factors secreted by tumor‐associated macrophages and their modulation by 4‐FPAC, a DNA fragmentation assay was performed. A549 cells were cultured in CM derived from control or 4‐FPAC–treated TAMs for 24 h under standard culture conditions. Following treatment, the cells were harvested, washed twice with cold PBS, and lysed using a DNA lysis buffer containing 10 mM Tris‐HCl (pH 8.0), 10 mM EDTA, and 0.5% Triton X‐100. The lysates were incubated on ice for 30 min and subsequently centrifuged at 12,000 × g for 15 min at 4°C to separate the fragmented DNA in the supernatant from intact chromatin in the pellet. The supernatant was treated with RNase A (100 µg/mL) at 37°C for 1 h, followed by proteinase K digestion (200 µg/mL) at 50°C for 1 h. DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1, v/v) and precipitated overnight at −20°C with two volumes of cold ethanol and 0.3 M sodium acetate. The DNA pellet was collected by centrifugation, washed with 70% ethanol, air‐dried, and resuspended in TE buffer (10 mM Tris‐HCl, 1 mM EDTA, pH 8.0). Extracted DNA samples were electrophoresed on a 1.8% agarose gel containing ethidium bromide (0.5 µg/mL) at 80 V for 90 min in 1× TBE buffer. DNA fragmentation was visualized under UV illumination using a gel documentation system (Bio‐Rad, USA). The appearance of a characteristic DNA “ladder” pattern was considered indicative of apoptosis induction.

2.11. In Situ Apoptosis Detection Using TUNEL Assay

Apoptotic DNA fragmentation in A549 cells treated with conditioned media derived from control and 4‐FPAC–treated TAMs was evaluated using the Click‐iT Plus TUNEL Assay Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). A549 cells were seeded on sterile glass coverslips placed in 6‐well plates and incubated with TAM‐CM or 4‐FPAC–treated TAM‐CM for 48 h. After treatment, cells were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, followed by permeabilization with 0.25% Triton X‐100 in PBS for 10 min at 4°C.

The TUNEL reaction was then performed according to the manufacturer's protocol. Briefly, permeabilized cells were incubated with a reaction mixture containing terminal deoxynucleotidyl transferase (TdT) enzyme and fluorescein‐labeled dUTP at 37°C for 1 h in a dark, humidified chamber. After labeling, the cells were rinsed three times with PBS and counterstained with DAPI (1 µg/mL) to visualize nuclei. The coverslips were mounted with antifade mounting medium, and fluorescence images were acquired using a fluorescence microscope (Lawrence and Mayo, India) equipped with FITC and DAPI filter sets.

2.12. Quantitative Real‐Time PCR (qRT‐PCR) Analysis

Total RNA was isolated from A549 cells treated with CM derived from control and 4‐FPAC–treated TAMs using the TRIzol reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. The purity and concentration of RNA were determined spectrophotometrically, and 1 µg of total RNA was reverse transcribed into complementary DNA (cDNA) using a one‐step cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA).

Gene‐specific primers targeting epithelial–mesenchymal transition, angiogenesis, proliferation, and apoptosis‐related markers were designed based on sequences obtained from the NCBI database (Supporting Information S1: Table 1). Quantitative PCR amplification was carried out using the LightCycler® 96 Real‐Time PCR System (Roche Diagnostics, Basel, Switzerland). The reaction conditions included an initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 10 s, annealing at 61°C for 20 s, and extension at 72°C for 20 s. A final melt curve analysis was performed to confirm the specificity of the amplified products.

The mean cycle quantification (Cq) values were normalized against GAPDH, which served as the internal reference gene. Relative expression levels of target genes were calculated using the 2⁻ΔΔCq method, as described by Livak and Schmittgen [20].

2.13. Western Blot Analysis

To evaluate the effect of CM derived from control and 4‐FPAC–treated TAMs on signaling pathway modulation in A549 cells, total protein was extracted using RIPA lysis buffer supplemented with a protease inhibitor cocktail (P8340, Sigma‐Aldrich, St. Louis, MO, USA). The lysates were centrifuged at 12,000 × g for 15 min at 4°C, and the supernatants were collected. Protein concentration was quantified using the Bradford assay [21].

Equal amounts of protein (40 μg per sample) were mixed with loading dye, denatured, and subjected to SDS‐PAGE on a 12% resolving gel with a 4% stacking gel. Separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes via semi‐dry transfer at 100 mA for 20 min. Membranes were then blocked for 1 h in Tris‐buffered saline (TBS) containing 5% skim milk to prevent non‐specific binding.

After blocking, membranes were incubated overnight at 4°C with primary antibodies against p‐E‐cadherin IgG Mouse, N‐cadherin IgG Mouse, phospho‐AKT (p‐AKT) IgG Rabbit, VEGF‐α IgG Mouse, Nanog IgG Rabbit, p53 IgG Rabbit, and cleaved Caspase‐3 IgG Rabbit (Cell Signaling Technology, Danvers, MA, USA). GAPDH IgG Mouse (Sigma‐Aldrich, St. Louis, MO, USA) was used as a loading control. Following primary incubation, membranes were washed three times (15 min each) with TBST buffer (50 mM Tris‐HCl, pH 7.6; 150 mM NaCl; 0.1% Tween 20) and then incubated with the appropriate biotinylated secondary antibodies (Sigma‐Aldrich, St. Louis, MO, USA) for 45 min at room temperature.

Subsequently, membranes were incubated with alkaline phosphatase–conjugated streptavidin for 45 min and visualized using the BCIP/NBT substrate (Sigma‐Aldrich, St. Louis, MO, USA). Band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and relative protein expression levels were normalized to GAPDH.

2.14. In Silico Analysis of Copy Number Alterations, Macrophage Infiltration, and IL8 Prognostic Significance in LUAD

Gene‐level copy number alteration (CNA), RNA‐seq expression, immune infiltration, and clinical survival data for lung adenocarcinoma (LUAD) were obtained from the TCGA Pan‐Cancer Atlas using the TIMER. CNA states were classified as deep deletion, arm‐level deletion, diploid/normal, arm‐level gain, and high amplification. Macrophage infiltration levels were compared across CNA groups using diploid/normal tumors as the reference and visualized using box‐and‐whisker plots with individual data points representing tumor samples. Correlation analysis between IL8 expression and the M2 macrophage marker CD163 was performed using Spearman's correlation on normalized expression values (log2 TPM). The prognostic significance of IL8 was evaluated using Kaplan–Meier overall survival analysis, with patients stratified into high‐ and low‐expression groups based on median IL8 expression and survival differences assessed using the log‐rank test. All statistical analyses were conducted using default platform parameters, and statistical significance was defined as p ≤ 0.05.

2.15. Statistical Analysis

All experimental data were expressed as the mean ± standard error of the mean (SEM), with each experiment performed independently at least three times to ensure reproducibility and reliability. Statistical analyses were conducted using GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA). Data distribution was assessed for normality prior to comparison, and appropriate parametric tests were applied.

Comparisons between two experimental groups (control and treated) were evaluated using the student's t‐test, whereas comparisons among multiple groups, where applicable, were analyzed using one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparison post hoc test to determine intergroup significance. The degree of statistical significance was represented using p‐values, with p ≤ 0.05 considered statistically significant.

All results were graphically represented using mean values with error bars indicating SEM, and statistically significant differences were denoted using standard notation (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Statistical evaluation was performed to confirm the consistency of observed trends across biological replicates, ensuring the robustness and validity of the experimental conclusions.

3. Results

3.1. Tumor‐Derived Conditioned Media Promotes Polarization of THP‐1–Derived Macrophages Toward a CD163⁺ Tumor‐Associated Phenotype

Morphological and flow cytometric analyses were performed to evaluate the differentiation and polarization status of THP‐1–derived macrophages in response to A549‐conditioned media. Phase‐contrast imaging demonstrated that THP‐1 monocytes exhibited a small, round, non‐adherent morphology, whereas PMA‐differentiated macrophages (M0) displayed an adherent phenotype with increased cell size and cytoplasmic complexity. Upon exposure to A549‐conditioned media, M0 macrophages showed pronounced morphological changes, including enhanced cell spreading and elongated projections, indicative of macrophage activation and tumor‐associated polarization.

Flow cytometric analysis further confirmed these phenotypic changes. Sequential gating of the main cell population and singlets revealed a progressive increase in APC‐conjugated CD163 expression across the three conditions. As summarized in Supporting Information S1: Table 2, THP‐1 monocytes exhibited a basal level of CD163 positivity, which was significantly elevated in differentiated M0 macrophages. Notably, exposure of M0 macrophages to A549‐conditioned media resulted in a further significant increase in CD163‐positive cells, reflecting strong induction of a tumor‐associated macrophage phenotype.

Collectively, the data presented in Figure 2 and Supporting Information S1: Table 2 demonstrate that soluble factors released by A549 lung cancer cells effectively drive polarization of THP‐1–derived macrophages toward a CD163⁺ tumor‐associated phenotype. This tumor‐mediated macrophage reprogramming provides a mechanistic basis for the pro‐tumorigenic interactions observed between macrophages and lung cancer cells in subsequent functional assays.

Figure 2.

Morphological and flow cytometric characterization of THP‐1 differentiation and macrophage polarization under A549‐conditioned media. Top panel shows phase‐contrast images of (A) THP‐1 monocytes, (B) PMA‐differentiated macrophages (M0), and (C) M0 macrophages exposed to A549‐conditioned media (M0‐A549 CM), demonstrating morphological changes associated with differentiation and activation. (Scale bar: 50 µm). Bottom panels depict flow cytometric analysis showing sequential gating of the main cell population (FSC‐A vs. SSC‐A), singlet discrimination (FSC‐A vs. FSC‐H), and APC fluorescence histograms representing marker expression associated with macrophage polarization. Quantitative bar graphs (right) summarize the relative increase in CD163‐APC‐positive cells, indicating enhanced activation/polarization of macrophages upon exposure to A549‐conditioned media. Data are representative of three independent experiments.

3.2. Direct 4‐FPAC Exposure Under TAM‐CM‐Supported Conditions Reduces A549 Cell Viability in a Dose‐Dependent Manner

MTT assays performed in the presence of TAM‐CM showed that direct exposure of A549 cells to increasing concentrations of 4‐FPAC under TAM‐CM‐supported conditions markedly diminished cell viability in a dose‐dependent manner (Figure 3; Table 1). Even at low micromolar concentrations, 4‐FPAC attenuated the TAM‐CM‐supported survival advantage, with progressively greater suppression observed at higher doses. Dose–response analysis yielded an IC50 of 22.63 μM, while an IC20 value of 9 μM was selected for downstream experiments to allow functional assessment under minimally cytotoxic conditions. These findings indicate that 4‐FPAC directly reduces A549 viability even in the presence of tumor‐promoting TAM‐derived soluble factors. Therefore, the MTT assay should be interpreted as evidence of direct 4‐FPAC cytotoxicity under TAM‐CM‐supported conditions, whereas the subsequent conditioned‐media experiments were designed to evaluate the indirect effects of soluble mediators derived from 4‐FPAC‐treated TAMs.

Figure 3.

MTT assay showing the dose‐dependent cytotoxic effect of 4‐FPAC on A549 cells in the presence of TAM‐conditioned media. A549 cells cultured with TAM‐derived conditioned media were treated with increasing concentrations of 4‐FPAC (0.5–100 µM) for 48 h, and cell viability was quantified using the MTT assay. A marked and progressive decline in cell viability was observed with rising drug concentrations compared to the control. Data are expressed as mean ± SEM (n = 3). Statistical significance: ***p ≤ 0.001.

Table 1.

Concentration‐dependent effect of 4‐FPAC on cell viability of A549 cells in presence of TAM‐conditioned media.

Concentrations (µM)	Percentage Cell viability
0	100%
0.5	82.9%
1	71.4%
10	64.7%
25	35.4%
50	16.0%
75	9.8%
100	5.2%

Note: The IC₅₀ value was determined to be 22.63 µM, and an IC₂₀ value of 9 µM was selected for subsequent experiments. All experiments were performed in triplicate (n = 3).

3.3. 4‐FPAC–Reprogrammed TAM‐Conditioned Media Diminishes A549 Cell Proliferation as Shown by BrdU Incorporation

BrdU incorporation analysis revealed that 4‐FPAC‐mediated reprogramming of tumor‐associated macrophages markedly attenuates the proliferative activity of A549 cells. When cultured in control TAM‐CM, A549 cells displayed abundant BrdU‐positive nuclei, reflecting active DNA synthesis and strong S‐phase entry consistent with TAM‐supported proliferative signaling. Exposure to conditioned media derived from 4‐FPAC–treated TAMs, however, resulted in a pronounced decline in BrdU incorporation phase (Figure 4), indicating reduced DNA replication and impaired progression through S‐ This decrease in proliferative output suggests that 4‐FPAC modifies the TAM secretome in a manner that weakens macrophage‐derived pro‐growth cues, thereby suppressing tumor cell proliferation despite an otherwise supportive microenvironment.

Figure 4.

BrdU incorporation assay showing the effect of 4‐FPAC‐treated TAM‐conditioned media on A549 cell proliferation. Representative fluorescence micrographs illustrate A549 cells cultured with conditioned media (CM) from control TAMs (top panel) and 4‐FPAC‐treated TAMs (bottom panel) for 48 h, followed by BrdU labeling for 24 h. (a) DAPI staining (blue) marks cell nuclei, (b) BrdU staining (red) indicates proliferating cells incorporating BrdU during DNA synthesis, and (c) merged images show colocalization of DAPI and BrdU signals. A notable reduction in BrdU‐positive nuclei was observed in A549 cells treated with CM from 4‐FPAC‐modulated TAMs, suggesting decreased proliferative activity. (Scale bar: 50 μm).

3.4. 4‐FPAC–Reprogrammed TAM‐Conditioned Media Suppresses the Migratory Capacity of A549 Cells in Wound‐Healing Assays

Assessment of A549 cell migration using the wound‐healing assay showed that 4‐FPAC‐modulated TAM‐conditioned media markedly diminishes TAM‐driven pro‐migratory activity. As illustrated in Figure 5 and summarized in Supporting Information S1: Table 3, cells cultured with control TAM‐CM exhibited rapid wound closure over 24–48 h, consistent with the strong migratory stimulus typically provided by M2‐like macrophage secretions. In contrast, exposure to conditioned media from 4‐FPAC‐treated TAMs resulted in substantially slower wound closure, with reductions that were statistically significant at both time points (***p ≤ 0.001). This impaired migratory response indicates that 4‐FPAC alters the macrophage‐derived soluble factors that otherwise promote motility in A549 cells, thereby attenuating one of the key functional hallmarks of TAM‐driven tumor progression.

Figure 5.

Effect of 4‐FPAC‐treated TAM‐conditioned media on the migratory potential of A549 cells as assessed by wound healing assay. Representative phase‐contrast images show wound closure in A549 cells cultured with TAM‐conditioned media (control) and 4‐FPAC–treated TAM‐conditioned media at 0, 24, and 48 h. A549 cells exposed to 4‐FPAC–treated TAM‐CM exhibited a markedly reduced rate of wound closure compared to control. Quantitative analysis of percent wound closure is presented on the right. Data are expressed as mean ± SEM (n = 3). Statistical significance: ***p ≤ 0.001. (Scale bar: 20 µm).

3.5. 4‐FPAC‐Reprogrammed TAM‐Conditioned Media Significantly Reduces Migration and Invasion of A549 Cells

Transwell assays revealed a marked reduction in the migratory and invasive capacity of A549 cells when exposed to conditioned media from 4‐FPAC‐treated TAMs. As shown in Figure 6 and detailed in Supporting Information S1: Table 4, control TAM‐CM strongly promoted invasion, reflected by a high number of cells traversing the Matrigel‐coated membrane, whereas 4‐FPAC‐TAM‐CM reduced invasive cell numbers by more than half (***p ≤ 0.001). A similar inhibitory pattern was observed in the migration assay, where the robust migratory response induced by control TAM‐CM was significantly diminished in the presence of 4‐FPAC‐modulated macrophage secretions (***p ≤ 0.001). These findings demonstrate that 4‐FPAC alters the TAM‐derived soluble milieu in a manner that weakens the pro‐metastatic signals typically conveyed to tumor cells, thereby suppressing both migration and invasion, key functional determinants of metastatic potential in NSCLC.

Figure 6.

Effect of 4‐FPAC‐treated TAM‐conditioned media on A549 cell invasion and migration as assessed by Transwell assays. (A) Schematic of Transwell assay setup showing invasion (with Matrigel) and migration (without Matrigel) assays. (B–C) Representative images show reduced invasion and migration of A549 cells treated with 4‐FPAC TAM‐CM compared to control TAM‐CM, indicating suppression of metastatic behavior. Quantitative analysis of invasion and migration is depicted on the right. Data are presented as mean ± SEM (n = 3). Statistical significance: ***p ≤ 0.001. (Scale bar: 10 μm).

3.6. 4‐FPAC–Reprogrammed TAM‐Conditioned Media Suppresses Angiogenic Responses in the CAM Model

Evaluation of angiogenic activity using the chick chorioallantoic membrane (CAM) assay revealed that conditioned media from 4‐FPAC‐treated TAMs markedly attenuates the pro‐angiogenic influence of TAM‐derived signals. CAMs exposed to control TAM‐CM developed dense, highly branched vascular networks characteristic of strong angiogenic stimulation, whereas those treated with 4‐FPAC‐TAM‐CM displayed visibly reduced vessel formation, with fewer branches, lower vessel density, and a more simplified vascular architecture (Figure 7A–C). Quantitative metrics supported these observations: both vessel area and junction number were substantially higher in control CAMs, while 4‐FPAC‐TAM‐CM produced significant reductions across these parameters (Table 2; Figure 7D–G). Total vessel length was similarly diminished in the treated group, and increased lacunarity indicated a more fragmented and less organized vascular pattern. Together, these findings demonstrate that 4‐FPAC alters TAM‐derived soluble mediators in a way that disrupts endothelial network formation, consistent with a shift toward an anti‐angiogenic macrophage phenotype and reduced capacity to support tumor‐associated neovascularization.

Figure 7.

Effect of 4‐FPAC‐treated TAM‐conditioned media on angiogenesis assessed by the chick chorioallantoic membrane (CAM) assay of day 3 chick embryo. (A–C) Representative CAM, skeletal, and architectural images showing reduced vessel branching and density in CAMs treated with 4‐FPAC‐treated TAM‐CM compared to control CM. (D–G) Quantitative analysis of vessel area, junctions, vessel length, and lacunarity confirmed significant inhibition of angiogenesis by 4‐FPAC‐treated CM. Data are expressed as mean ± SEM (n = 3). Statistical significance: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Table 2.

Quantitative analysis of angiogenic parameters in the CAM assay following exposure to normal media, control TAM‐conditioned media, and 4‐FPAC–treated TAM‐conditioned media.

CAM Parameters	Normal	Control	Treated
Vessel area (mm2)	1.5 × 105 ± 0.038	2.6 × 105 ± 0.032***	3.5 × 104 ± 0.067***
Total no. of junctions	121.3 ± 1.85	308 ± 2.64***	163.3 ± 2.60***
Total Vessel length (mm)	139.5 ± 0.59	252.2 ± 3.43***	41.4 ± 4.23***
Mean lacunarity	0.25 ± 0.01	0.13 ± 0.005***	0.19 ± 0.006*

Note: Vessel area, total number of junctions, total vessel length, and mean lacunarity were measured to assess angiogenic responses under different treatment conditions. Data presented as mean ± SEM; n = 3; *p ≤ 0.05, ***p ≤ 0.001 versus control.

3.7. 4‐FPAC–Reprogrammed TAM‐Conditioned Media Induces G0/G1 Cell Cycle Arrest in A549 Cells

Flow cytometric profiling (Figure 8) demonstrated that conditioned media derived from 4‐FPAC‐reprogrammed TAMs profoundly disrupts cell‐cycle progression in A549 cells, driving them toward a growth‐arrested phenotype. Whereas control TAM‐CM maintained a typical proliferative distribution across G0/G1, S, and G2/M phases, exposure to 4‐FPAC‐TAM‐CM led to a pronounced accumulation of cells in G0/G1, accompanied by marked reductions in S‐phase and G2/M fractions. Quantitative analysis (Supporting Information S1: Table 5) confirmed this shift, reflecting a strong suppression of DNA synthesis and cell‐cycle transit. These findings indicate that soluble mediators released from 4‐FPAC‐treated TAMs curb A549 proliferative capacity by imposing a PI‐detectable G0/G1 arrest, consistent with the downregulation of cell‐cycle regulators observed at the transcriptional level (Figure 12; Supporting Information S1: Table 7).

Figure 8.

Flow cytometric analysis of cell cycle distribution in A549 cells treated with control TAM‐CM and 4‐FPAC–treated TAM‐CM. FSC‐A versus SSC‐A plots (top panels) show gating of the main cell population (P1). Singlet discrimination using FSC‐A versus FSC‐H (middle panels) isolates single cells (P2). PE‐A fluorescence histograms (bottom panels) depict DNA content profiles, with gates representing G0/G1 (P3), S‐phase (P5), and G2/M (P4) populations. Treatment with 4‐FPAC–modulated TAM‐CM results in altered cell cycle distribution compared to control TAM‐CM, indicating cell cycle arrest induced by 4‐FPAC–treated macrophage‐secreted factors. Data are presented as mean ± SEM (n = 3). Statistical significance: **p ≤ 0.01 and ***p ≤ 0.001.

Figure 12.

Effect of 4‐FPAC‐treated TAM‐conditioned media on gene expression in A549 cells. qRT‐PCR analysis showing differential expression of (A) proliferation and cell survival markers, (B) cell cycle markers, (C) angiogenesis markers, (D) stem cell markers, and (E) tumor suppressor markers in A549 cells treated with control or 4‐FPAC‐treated TAM‐CM. 4‐FPAC‐treated CM reduced mesenchymal, proliferative, angiogenic, and stemness markers while upregulating epithelial and tumor suppressor genes. Statistical significance is presented as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ns = non‐significant versus control. Data represents pooled biological samples (N = 3), each analyzed in duplicate technical replicates (n = 6).

3.8. 4‐FPAC–Reprogrammed TAM‐Conditioned Media Diminishes NANOG‐Positive Stem‐Like A549 Cell Populations

Flow cytometric analysis (Figure 9) demonstrated that conditioned media derived from 4‐FPAC‐reprogrammed TAMs significantly diminishes the stem‐like subpopulation of A549 cells, as reflected by reduced NANOG expression. Control TAM‐CM maintained a clear NANOG‐positive subset, consistent with the capacity of TAM‐derived soluble factors to reinforce stemness programs. In contrast, cells exposed to 4‐FPAC‐TAM‐CM exhibited a substantial reduction in NANOG positivity, confirmed by quantitative measurements in Supporting Information S1: Table 6, where the NANOG⁺ population declined by more than half (**p ≤ 0.01). These findings indicate that soluble mediators released from 4‐FPAC‐treated TAMs impair pathways sustaining cancer stem cell‐like characteristics, thereby attenuating features linked to tumor aggressiveness, therapeutic resistance, and recurrence.

Figure 9.

Flow cytometric analysis of NANOG‐positive stem‐like cells in A549 cells treated with control TAM‐CM and 4‐FPAC‐treated TAM‐CM. Cells were gated by FSC‐A versus SSC‐A (left) and singlets were selected using FSC‐A versus FSC‐H (middle). FITC fluorescence histograms (right) represent NANOG‐positive cells. A significant reduction in NANOG⁺ stem‐like cells was observed in the 4‐FPAC‐treated TAM‐CM group compared to the control TAM‐CM group, indicating suppression of stemness. Data are expressed as mean ± SEM (n = 3). Statistical significance: **p ≤ 0.01.

3.9. 4‐FPAC–Reprogrammed TAM‐Conditioned Media Promotes Apoptotic DNA Fragmentation in A549 Cells

Assessment of apoptotic DNA fragmentation revealed a clear pro‐apoptotic shift in A549 cells exposed to conditioned media from 4‐FPAC‐treated TAMs. As shown in Figure 10, cells cultured with control TAM‐CM exhibited intact, high‐molecular‐weight genomic DNA, indicative of minimal apoptosis. In contrast, cells treated with 4‐FPAC–TAM‐CM displayed a pronounced DNA laddering pattern, reflecting internucleosomal cleavage characteristic of apoptotic cell death. This distinct fragmentation profile demonstrates that soluble mediators released by 4‐FPAC‐reprogrammed TAMs activate apoptotic pathways in A549 cells, supporting the transition of TAM‐derived signals from a tumor‐supportive to an anti‐tumorigenic, pro‐apoptotic phenotype.

Figure 10.

DNA fragmentation assay showing apoptotic DNA laddering in A549 cells treated with 4‐FPAC‐treated TAM‐conditioned media. Lane C: control TAM‐CM‐treated cells showing intact genomic DNA; Lane T: 4‐FPAC‐treated TAM‐CM–treated cells displaying characteristic DNA ladder pattern indicative of apoptosis.

3.10. 4‐FPAC–Reprogrammed TAM‐Conditioned Media Enhances Apoptosis in A549 Cells as Demonstrated by TUNEL Assay

TUNEL analysis further confirmed that soluble mediators released from 4‐FPAC‐reprogrammed TAMs promote apoptosis in A549 cells. As shown in Figure 11, cells cultured with control TAM‐CM displayed minimal TUNEL labeling, consistent with low levels of DNA fragmentation. In contrast, A549 cells exposed to 4‐FPAC‐TAM‐CM exhibited a pronounced increase in TUNEL‐positive nuclei, indicating robust induction of apoptosis. DAPI staining demonstrated preserved nuclear structure across groups, while Alexa Fluor 488 selectively highlighted apoptotic nuclei undergoing DNA cleavage. The merged images revealed clear colocalization of apoptotic signals with nuclear staining in the treated group, reinforcing that 4‐FPAC alters TAM‐derived secretions in a manner that drives apoptotic activation. These observations further support the shift of TAM signaling from a tumor‐supportive to an anti‐tumorigenic, pro‐apoptotic profile following 4‐FPAC treatment.

Figure 11.

TUNEL assay demonstrating apoptosis in A549 cells treated with 4‐FPAC‐modulated TAM‐conditioned media. (a) DAPI staining (blue) identifies nuclear morphology of A549 cells. (b) Alexa Fluor 488 fluorescence (green) marks TUNEL‐positive nuclei, indicating DNA fragmentation characteristic of apoptosis. (c) Merged images display colocalization of nuclear (DAPI) and apoptotic (Alexa Fluor 488) signals. A substantial increase in TUNEL‐positive cells was observed in the 4‐FPAC‐treated TAM‐CM group (bottom panel) compared to control TAM‐CM (top panel), confirming enhanced apoptotic activity induced by 4‐FPAC‐mediated modulation of TAM‐secreted factors. (Scale bar: 20 μm).

3.11. 4‐FPAC–Reprogrammed TAM‐Conditioned Media Drives Broad Transcriptional Reprogramming in A549 Cells

qRT‐PCR analysis (Figure 12; Supporting Information S1: Table 7) revealed that conditioned media from 4‐FPAC‐reprogrammed TAMs induces extensive transcriptional reprogramming in A549 cells, shifting them toward an epithelial, anti‐proliferative, and pro‐apoptotic phenotype. Mesenchymal and survival‐associated genes, including N‐cadherin, Vimentin, and AKT1, were markedly downregulated, while epithelial markers such as E‐cadherin and tight‐junction components Claudin‐3 and Claudin‐7 were strongly upregulated, consistent with EMT reversal. Genes linked to angiogenesis (IL8, VEGFα) were substantially reduced, aligning with the impaired vascular responses observed in the CAM assay. This transcriptional pattern paralleled cell‐cycle suppression, as key regulators (Cyclin D1, Cyclin E1, CDK4) were downregulated in accordance with the G0/G1 arrest documented in Figure 8, Supporting Information S1: Table 5. Stemness markers (OCT3/4, NANOG) were also diminished, corroborating the reduced NANOG⁺ subpopulation quantified by flow cytometry (Figure 9, Supporting Information S1: Table 6). Conversely, tumor‐suppressive and pro‐apoptotic genes, most notably p21, p53, and Caspase‐3, were robustly upregulated, reflecting enhanced apoptotic signaling consistent with DNA fragmentation (Figure 10) and TUNEL positivity (Figure 11). Collectively, the transcriptomic data (Figure 12; Supporting Information S1: Table 7 demonstrate that soluble mediators released by 4‐FPAC‐treated TAMs coordinately suppress pathways associated with proliferation, migration, angiogenesis, and stemness, while activating tumor‐suppressive and apoptotic responses in A549 cells.

3.12. 4‐FPAC–Reprogrammed TAM‐Conditioned Media Modulates EMT, Survival, Angiogenesis, Stemness, and Apoptotic Protein Expression in A549 Cells

Western blot analysis (Figure 13) demonstrated that exposure to conditioned media from 4‐FPAC‐treated TAMs induces prominent changes in key regulatory proteins governing epithelial–mesenchymal transition (EMT), proliferation, angiogenesis, stemness, and apoptosis in A549 cells. Quantitative densitometry (Supporting Information S1: Table 8) showed increased E‐cadherin and reduced N‐cadherin, consistent with reinforcement of epithelial characteristics and suppression of EMT. The pro‐survival kinase p‐AKT was markedly downregulated in the treated group, indicating inhibition of AKT‐dependent survival signaling, while the stemness‐associated factor Nanog was also decreased, aligning with the reduced NANOG⁺ population observed by flow cytometry (Figure 9; Supporting Information S1: Table 6). A modest but clear reduction in VEGF‐α protein levels paralleled the transcriptional and functional evidence of impaired angiogenesis (Figure 6; Table 2). In contrast, tumor‐suppressive and pro‐apoptotic proteins were elevated following treatment with 4‐FPAC–TAM‐CM, as reflected by increased p53 and enhanced levels of cleaved Caspase‐3, a key executor of apoptosis. Together, these protein‐level alterations (Figure 13; Supporting Information S1: Table 8) corroborate the transcriptional changes (Figure 12; Supporting Information S1: Table 7) and demonstrate that soluble mediators derived from 4‐FPAC‐reprogrammed TAMs attenuate pro‐tumorigenic signaling while augmenting tumor‐suppressive and apoptotic pathways in A549 cells, supporting the anti‐cancer potential of 4‐FPAC through modulation of the macrophage‐tumor cell axis.

Figure 13.

Western blot analysis of key regulatory proteins in A549 cells treated with control TAM‐CM and 4‐FPAC–treated TAM‐CM. Immunoblot results show differential expression of EMT, proliferation, angiogenesis and apoptosis‐related proteins between control and treated groups. Quantitative densitometry (right panel) represents normalized band intensities relative to GAPDH. Treatment with 4‐FPAC–modulated TAM‐CM resulted in decreased expression of pro‐tumorigenic markers and increased levels of tumor‐suppressive/apoptotic proteins. Data are presented as mean ± SEM; n = 3; *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

3.13. Copy Number Alterations Modulate Macrophage Infiltration and Identify IL8 as a Key Immunoregulatory Gene in LUAD

Analysis of TCGA‐LUAD data revealed that copy number gains and amplifications of multiple genes are associated with significantly increased macrophage infiltration, whereas deletion states generally exhibited lower infiltration levels (Figure 14A). Gene‐wise statistical comparisons identifying the most significant associations are summarized in Table 3. Among the analyzed genes, CDH1 and IL8 showed the strongest statistical association with macrophage infiltration, particularly under high amplification conditions. Notably, IL8 expression demonstrated a significant positive correlation with the M2 macrophage marker CD163, indicating enrichment of immunosuppressive tumor‐associated macrophages in IL8‐high tumors (Figure 14B). Furthermore, Kaplan–Meier survival analysis revealed that elevated IL8 expression is significantly associated with reduced overall survival, underscoring its adverse prognostic significance in lung adenocarcinoma.

Figure 14.

Copy number–associated macrophage infiltration and IL8‐driven immunosuppressive signaling in LUAD. (A) Box‐and‐whisker plots depicting macrophage infiltration levels across different copy number alteration (CNA) states (deep deletion, arm‐level deletion, diploid/normal, arm‐level gain, and high amplification) of the indicated genes in lung adenocarcinoma (LUAD). Individual points represent tumor samples, boxes indicate median and interquartile range, and asterisks denote statistically significant differences relative to diploid/normal tumors (*p < 0.05; **p < 0.01; ***p < 0.001). (B) Scatter plot showing a significant positive correlation between IL8 expression and the M2 macrophage marker CD163 in LUAD, alongside Kaplan–Meier overall survival analysis demonstrating that high IL8 expression is associated with reduced overall survival, highlighting the prognostic and immunological relevance of IL8 in lung adenocarcinoma.

Table 3.

Genes significantly associated with macrophage infiltration under copy number amplification in LUAD.

Gene	p‐value (Macrophage, High Amplification)	Significance
CDH1	3.04 × 10−19	Highly significant
IL8	2.36 × 10−11	Highly significant
VEGFα	5.85 × 10−4	Significant
VIM	1.16 × 10−3	Significant
P21	1.79 × 10−2	Moderately significant

Note: Genes are ranked by p‐value reflecting the strength of association between high copy number amplification and macrophage infiltration in lung adenocarcinoma (LUAD). Statistical significance was defined as p ≤ 0.05.

4. Discussion

Tumor‐associated macrophages (TAMs) represent one of the most abundant and influential immune cell populations within the tumor microenvironment of NSCLC and are widely recognized as key regulators of tumor progression, metastasis, angiogenesis, immune evasion, and resistance to therapy [22, 23]. Their inherent plasticity allows them to shift between an M1‐like anti‐tumor phenotype and an M2‐like pro‐tumor phenotype in response to microenvironmental cues [24, 25]. Numerous clinical studies demonstrate that an enrichment of M2‐polarized TAMs correlates with increased tumor aggressiveness, enhanced EMT, poor response to therapy, and shortened survival [26, 27]. Consequently, therapeutic strategies aimed at reprogramming M2‐like TAMs toward an M1‐like state have gained substantial attention as a means to counteract tumor‐supportive signaling within the TME [28].

Consistent with this rationale, the observed morphological and phenotypic changes confirm efficient differentiation of THP‐1 monocytes into macrophages and demonstrate that tumor‐derived soluble factors profoundly influence macrophage activation states. The enhanced marker expression in macrophages exposed to A549‐conditioned media suggests tumor‐driven macrophage reprogramming, consistent with a pro‐tumorigenic microenvironment. These findings highlight the dynamic crosstalk between lung cancer cells and macrophages and provide a cellular basis for subsequent functional and molecular analyses.

Importantly, the present study was specifically designed to investigate the functional consequences of 4‐FPAC‐modulated TAM‐derived conditioned media on tumor cell behavior rather than to reassess the already established direct cytotoxic effects of 4‐FPAC on A549 cells. In our previous study, direct exposure of A549 cells to 4‐FPAC induced ROS‐mediated apoptosis, G0/G1 cell‐cycle arrest, suppression of EMT‐associated signaling, and inhibition of migratory and invasive behavior through modulation of p53 and PI3K/AKT pathways [17]. That study also demonstrated that A549 cells were considerably more sensitive to 4‐FPAC than other NSCLC cell lines, including NCI‐H23 and NCI‐H522, thereby justifying their selection as the primary experimental model in the current investigation [17]. Building upon these findings, our subsequent study demonstrated that 4‐FPAC promotes M1 macrophage polarization while suppressing M2‐associated phenotypes through modulation of NF‐κB, p38 MAPK, and PI3K/AKT signaling pathways [17]. Macrophage reprogramming was further validated through increased ROS and nitric oxide production, elevated IL‐12 levels, and suppression of IL‐10 and other M2‐associated markers [17]. Collectively, these previously published findings provide the mechanistic basis for the present study, which was undertaken to determine whether 4‐FPAC treatment could alter TAM‐derived conditioned‐media activity and thereby suppress NSCLC progression through macrophage‐associated mechanisms.

Our findings demonstrate that 4‐FPAC influences A549 tumor cell behavior through both direct and macrophage‐associated mechanisms, depending on the assay design. In the MTT assay, A549 cells were directly exposed to increasing concentrations of 4‐FPAC in the presence of TAM‐CM, and therefore the observed dose‐dependent reduction in viability reflects direct 4‐FPAC cytotoxicity under TAM‐CM‐supported conditions rather than a purely indirect conditioned‐media effect. This result indicates that 4‐FPAC can reduce A549 viability even when tumor‐promoting soluble factors from TAMs are present. This interpretation is consistent with our previous report showing that direct exposure of A549 cells to 4‐FPAC induces cytostatic and pro‐apoptotic effects through modulation of ROS‐mediated p53 and AKT signaling pathways [17]. In contrast, the BrdU proliferation assay and subsequent migration, invasion, angiogenesis, cell‐cycle, apoptosis, stemness, qRT‐PCR, and immunoblotting experiments evaluated A549 responses to conditioned media derived from control or 4‐FPAC‐treated TAMs. These later assays therefore provide evidence that 4‐FPAC treatment alters TAM‐derived soluble signaling in a manner that weakens macrophage‐associated pro‐tumorigenic support, consistent with the established role of TAM‐derived cytokines and growth factors in activating pro‐survival pathways such as STAT3, PI3K/AKT, and NF‐κB [29, 30]. Taken together, these results support a dual interpretation: 4‐FPAC directly suppresses A549 viability under TAM‐CM‐supported conditions and also modulates TAM‐derived conditioned media to reduce proliferation, motility, angiogenesis, stemness, and survival signaling in A549 cells.

Functional migration and invasion assays further demonstrated that 4‐FPAC‐modulated TAM‐conditioned media (TAM‐CM) markedly suppresses A549 cell motility and invasive potential. A549 cells exposed to control TAM‐CM exhibited pronounced wound closure together with elevated Transwell migration and invasion, consistent with the well‐established role of M2‐TAM‐derived mediators, including TGF‐β, matrix metalloproteinases (MMPs), and chemokines, in promoting epithelial–mesenchymal transition (EMT) and metastatic progression [31, 32]. In contrast, conditioned media derived from 4‐FPAC‐treated TAMs significantly attenuated these pro‐metastatic behaviors. At the molecular level, A549 cells treated with 4‐FPAC‐TAM‐CM displayed marked downregulation of mesenchymal markers such as N‐cadherin, Vimentin, and AKT1, accompanied by upregulation of epithelial markers including E‐cadherin, Claudin‐3, and Claudin‐7, collectively indicating reversal of EMT. Since EMT is characterized by the loss of epithelial features and acquisition of mesenchymal traits that facilitate migration and invasion [33], restoration of epithelial marker expression strongly suggests that conditioned media from 4‐FPAC‐treated TAMs disrupts EMT‐promoting signaling within the tumor microenvironment. These findings are in agreement with earlier reports demonstrating that M2‐TAMs enhance metastatic behavior through proteolytic remodeling pathways, including MMP‐9 activation via PI3K/AKT/Snail signaling [34], as well as TGF‐β‐mediated SOX9 induction through c‐Jun/SMAD3 pathways [35]. Notably, our previous direct‐treatment study similarly demonstrated suppression of EMT‐associated signaling and metastatic behavior in A549 cells following direct exposure to 4‐FPAC [17]. The present findings therefore extend those observations by demonstrating that 4‐FPAC can additionally suppress EMT through modulation of TAM‐derived conditioned‐media effects, thereby highlighting the dual anti‐metastatic potential of the compound. Collectively, these observations suggest that 4‐FPAC attenuates TAM‐derived EMT‐inducing signals, thereby limiting the migratory and invasive capabilities of NSCLC cells.

Angiogenesis, another hallmark driven heavily by TAMs, was also significantly impaired by 4‐FPAC‐treated TAM‐CM. CAM assays revealed reduced vessel branching, lower vessel area, fewer junctions, and increased lacunarity, collectively indicating disrupted neovascular architecture. These structural alterations were accompanied by reduced expression of key angiogenic mediators, including IL8 and VEGFα. This is noteworthy, as TAMs are central drivers of angiogenesis, secreting VEGF, IL8, and additional cytokines that promote endothelial proliferation, neovessel formation, and vascular permeability [36, 37, 38]. VEGF also recruits more TAMs into hypoxic tumor regions, reinforcing a proangiogenic feedback loop [39]. By altering TAM‐derived soluble mediators, 4‐FPAC appears to interrupt this circuitry, thereby limiting the vascular support needed for tumor expansion and dissemination.

In addition to its anti‐proliferative and anti‐migratory effects, 4‐FPAC‐treated TAM‐conditioned media (TAM‐CM) exerted a pronounced influence on cell‐cycle regulation and apoptotic signaling in A549 cells. The observed downregulation of key cell‐cycle regulators, including Cyclin D1, Cyclin E1, and CDK4, together with marked upregulation of the tumor suppressor genes p21 and p53 and the apoptotic effector Caspase‐3, indicates a coordinated shift toward growth arrest and programmed cell death. Flow cytometric analysis further confirmed significant G0/G1 phase accumulation accompanied by reduced S and G2/M phase populations, supporting the induction of cell‐cycle arrest. These transcriptional alterations were corroborated at the protein level, where elevated p53 and cleaved Caspase‐3 expression reflected enhanced apoptotic activity. Consistently, DNA fragmentation and TUNEL assays further substantiated apoptosis induction in A549 cells exposed to 4‐FPAC‐treated TAM‐CM. These findings are particularly significant because M2‐like TAMs are well known to secrete growth‐promoting and anti‐apoptotic factors that enhance tumor survival and therapy resistance [40]. In addition, cisplatin‐resistant lung cancer cells can intensify this tumor‐supportive loop through the release of macrophage inhibitory factor (MIF), which further promotes M2 polarization and enhances stemness and metastatic potential [41]. Importantly, our previously published study demonstrated that direct treatment of A549 cells with 4‐FPAC similarly activated ROS‐mediated apoptotic pathways and induced G0/G1 arrest through modulation of p53, p21, CDK2, and CDK4 [17]. The present findings therefore extend those observations by demonstrating that 4‐FPAC not only directly targets tumor cells but can also modify TAM‐derived conditioned‐media activity to reinforce anti‐proliferative and pro‐apoptotic signaling. By counteracting these M2‐driven survival mechanisms, 4‐FPAC disrupts the reciprocal tumor–macrophage signaling network that contributes to therapy resistance and sustained tumor progression.

Another key finding of this study is the impact of conditioned media from 4‐FPAC‐treated TAMs on cancer stem cell (CSC)‐associated features. TAMs are known to support CSC maintenance through cytokine secretion and paracrine interactions that enhance self‐renewal markers such as SOX2, NANOG, and OCT4 [42, 43, 44]. In our study, 4‐FPAC‐treated TAM‐CM significantly reduced the expression of NANOG and OCT3/4 and decreased the proportion of NANOG⁺ A549 cells, indicating loss of stem‐like characteristics. Given that CSCs contribute to metastasis, tumor relapse, and resistance, the suppression of TAM‐driven stemness suggests a shift toward a less aggressive tumor phenotype.

Protein‐level analyses corroborated transcriptional trends, showing increased E‐cadherin and decreased N‐cadherin, p‐AKT, Nanog, and VEGF‐α in A549 cells treated with 4‐FPAC‐TAM‐CM. These changes validate that 4‐FPAC‐mediated modulation of TAM‐derived conditioned media translates into functional attenuation of EMT, survival, angiogenesis, and stemness pathways. The elevation of p53 and cleaved Caspase‐3 further confirms the activation of apoptotic cascades.

Collectively, these results demonstrate that 4‐FPAC reshapes TAM‐derived signaling in a manner that shifts A549 cells away from a mesenchymal, proliferative, angiogenic, and stem‐like phenotype toward an epithelial, growth‐arrested, and apoptosis‐prone state. These effects align strongly with our hypothesis that 4‐FPAC acts as a dual‐function molecule exerting direct cytotoxic effects under TAM‐CM‐supported conditions and macrophage‐associated anticancer effects through modulation of TAM‐derived conditioned media. Importantly, the present findings should be interpreted in conjunction with our previous reports demonstrating both direct anticancer effects on NSCLC cells and macrophage‐polarizing activity of 4‐FPAC [17]. Together, these studies establish a broader mechanistic framework in which 4‐FPAC simultaneously targets tumor cells and modulates tumor–macrophage interactions within the tumor microenvironment.

However, this study has certain limitations that should be acknowledged. Although A549‐CM‐induced TAM‐like macrophages were generated and used in the present conditioned‐media workflow, detailed phenotypic characterization of 4‐FPAC‐treated TAM‐like macrophages was not performed within the same experimental series. Accordingly, the present findings are best interpreted as evidence that 4‐FPAC modifies the functional activity of TAM‐derived conditioned media, while detailed macrophage phenotypic reprogramming within this specific experimental workflow remains to be further validated. Previous macrophage polarization studies from our group provide useful mechanistic background showing that 4‐FPAC can promote M1‐associated features and suppress M2‐associated characteristics in THP‐1‐derived macrophages [17]; however, future studies should directly examine macrophage polarization markers, cytokine profiles, and secretome‐level changes in 4‐FPAC‐treated A549‐CM‐induced TAM‐like macrophages within the same experimental workflow. In addition, the current study utilized a single NSCLC cell line, A549, together with a THP‐1‐derived TAM model, which may not fully reflect the heterogeneity of patient‐derived tumors and macrophage populations. Although our previous study demonstrated that A549 cells exhibit marked sensitivity to 4‐FPAC compared with additional NSCLC cell lines [17], further validation using diverse NSCLC models, primary macrophages, patient‐derived systems, and in vivo approaches will be necessary to establish the broader translational relevance of these findings.

The strong association of IL8 with CD163 expression suggests a critical role for IL8 in driving M2‐like, immunosuppressive macrophage polarization and enhancing macrophage infiltration within the LUAD tumor microenvironment. Elevated IL8 levels, together with poor survival outcomes, indicate its function as a key mediator linking genomic alterations to immune suppression and disease progression. The observed tumor‐driven morphological and phenotypic reprogramming of macrophages further underscores the dynamic crosstalk between lung cancer cells and TAMs, providing a mechanistic basis for their pro‐tumorigenic activity and highlighting IL8 signaling as a potential therapeutic target.

Despite these considerations, the present findings highlight 4‐FPAC as a promising therapeutic candidate for tumor microenvironment (TME)‐targeted intervention in NSCLC. By modulating TAM‐derived conditioned‐media activity and disrupting macrophage‐associated oncogenic signaling, 4‐FPAC effectively attenuates key processes associated with tumor progression, including proliferation, metastasis, angiogenesis, and stemness, through a macrophage‐centered mechanism. Importantly, these macrophage‐associated effects complement our previously reported findings demonstrating the direct cytostatic, pro‐apoptotic, and anti‐metastatic activities of 4‐FPAC against NSCLC cells [17]. Collectively, the ability of 4‐FPAC to simultaneously exert direct anti‐tumor effects and modulate tumor‐supportive macrophage‐derived signaling underscores its potential as a multifaceted therapeutic strategy for NSCLC management.

5. Conclusion

This study identifies 4‐FPAC as a promising tumor microenvironment‐targeted therapeutic candidate for NSCLC by demonstrating its ability to disrupt tumor‐supportive signaling mediated through tumor‐associated macrophage‐conditioned media. In the MTT assay, direct exposure of A549 cells to 4‐FPAC in the presence of TAM‐CM reduced cell viability in a dose‐dependent manner, indicating that 4‐FPAC retains cytotoxic activity under TAM‐CM‐supported conditions. Conditioned media derived from 4‐FPAC‐treated TAMs significantly suppressed A549 cell proliferation, migration, invasion, angiogenesis, and stemness, all hallmark features commonly reinforced by M2‐like macrophages within the NSCLC microenvironment. These functional alterations were accompanied by reversal of epithelial–mesenchymal transition (EMT), suppression of AKT‐associated survival signaling, inhibition of cell‐cycle progression, and activation of p53‐dependent apoptotic pathways, together with reduced OCT3/4 and NANOG expression, indicating attenuation of cancer stem cell‐associated characteristics. Importantly, when interpreted alongside our previous studies demonstrating the direct cytostatic and macrophage‐polarizing effects of 4‐FPAC, the present findings suggest that the compound exerts both direct anticancer activity and macrophage‐associated tumor suppression. Collectively, these observations demonstrate that 4‐FPAC modulates TAM‐derived conditioned‐media activity, shifting NSCLC cells toward a less aggressive, growth‐arrested, and apoptosis‐prone phenotype. Although additional macrophage phenotyping within the same experimental workflow, broader NSCLC models, and in vivo validation are warranted, the dual‐action nature of 4‐FPAC highlights its strong translational potential as a multifaceted therapeutic strategy capable of targeting tumor cells and tumor–macrophage interactions within the supportive tumor microenvironment.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Anjali Singh: data curation, writing – original draft, methodology, investigation, formal analysis, validation. Dhanush Danes: data curation, writing – review and editing. Suresh Balakrishnan: conceptualization, funding acquisition, project administration, supervision, resources, writing – review and editing.

Supporting information

Supporting File 1

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.