EBF3 suppresses lung adenocarcinoma progression and immune evasion via transcriptional repression of CCL24

Abstract

Purpose

The transcription factor EBF3 is frequently downregulated in lung adenocarcinoma (LUAD), and its low expression is associated with poor patient prognosis. The functional significance and mechanistic basis of EBF3 in LUAD pathogenesis, particularly its potential impact on the tumor immune microenvironment, remain largely unexplored.

Methods

A series of in vitro and in vivo assays were performed. Cell proliferation and apoptosis were assessed using CCK-8 and flow cytometry. Signaling pathways were analyzed by Western blot. A syngeneic mouse model was established to evaluate tumor growth and immune cell infiltration by flow cytometry. Macrophage polarization was examined using conditioned medium co-culture assays. The key secreted factor was identified through cytokine screening and validated by rescue experiments.

Results

EBF3 overexpression significantly suppressed LUAD cell proliferation and induced apoptosis, while its knockdown promoted growth. Mechanistically, EBF3 inhibited the phosphorylation of AKT and P38. In vivo, EBF3 overexpression restricted tumor growth, reduced M2-like macrophage infiltration, and increased CD4⁺ and CD8⁺ T cell recruitment. This immunomodulation was mediated through transcriptional repression of CCL24. Conditioned medium from EBF3-expressing cells inhibited M2 macrophage polarization, and this effect was reversed by exogenous CCL24. Crucially, CCL24 administration rescued the tumor-suppressive and immune-modulatory effects of EBF3 in vivo.

Conclusions

Our study identifies EBF3 as a pivotal tumor suppressor in LUAD that operates through a dual mechanism: direct suppression of tumor cell growth via AKT/P38 signaling and remodeling of the immune microenvironment via CCL24 repression. The EBF3-CCL24 axis represents a promising therapeutic target for LUAD treatment.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-026-01212-7.

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide, with its main pathological types comprising non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) [1–3]. NSCLC accounts for approximately 85% of all lung cancer cases and can be further classified into subtypes such as lung adenocarcinoma and lung squamous cell carcinoma [4]. Current standard clinical treatments for lung adenocarcinoma include surgical resection, radiotherapy, and chemotherapy [5]. However, due to widespread issues such as tumor heterogeneity and the development of chemoresistance, the overall prognosis for patients remains poor, with a discouraging five-year survival rate [6]. Thus, there is an urgent need to elucidate the molecular mechanisms underlying the initiation and progression of lung adenocarcinoma and to identify novel therapeutic targets.

Accumulating evidence has highlighted the critical roles of transcription factors in tumorigenesis, progression, and treatment resistance [7, 8]. For instance, the classic tumor suppressor p53, known as the “guardian of the genome,” can induce cell cycle arrest, promote DNA repair, or initiate apoptosis to prevent the proliferation of damaged cells [9]. Under specific contexts, Krüppel-like factor 4 (KLF4) can upregulate various tumor suppressor genes and inhibit the acquisition of stem-like properties, while aberrant expression of KLF5 has been associated with the development of several solid tumors and poor patient prognosis [10–12]. PTEN, a key negative regulator of the PI3K/AKT signaling pathway, inhibits cell growth and promotes apoptosis [13]. Moreover, the Early B-cell Factor (EBF) family has garnered increasing attention for its role in cancer regulation [14]. Among its members, EBF3 has been reported to suppress the activity of the EGR1/EZH2/HDAC9 complex in nasopharyngeal carcinoma, thereby promoting vimentin expression and enhancing metastatic capacity [15]. Another study revealed that SNORA47 influences cancer stemness and chemosensitivity in Luminal A breast cancer via the EBF3/RPL11/c-Myc axis [16]. These findings collectively indicate that the role of EBF3 in cancer is highly context-dependent. Emerging evidence highlights the diverse and complex functions of EBF family members across various solid tumors. For instance, EBF1 has been reported to suppress colorectal cancer progression by modulating the Wnt/β-catenin pathway, yet it can also promote the progression of triple-negative breast cancer by regulating the HIF1α pathway, and inhibit the malignant progression of thyroid cancer [17–19]. Meanwhile, EBF2 interacts with estrogen receptor signaling to influence breast cancer differentiation, cooperatively suppresses the proliferation, migration, and invasion of pancreatic ductal adenocarcinoma (PDAC) cells by upregulating KLLN, and is downregulated in bladder cancer [20, 21]. However, the expression pattern and function of EBF3 in lung cancer, particularly in lung adenocarcinoma (LUAD), remain poorly defined. Preliminary bioinformatics analyses suggest that EBF3 is significantly downregulated in LUAD tissues and correlates with unfavorable patient prognosis. Nevertheless, its precise biological role and regulatory mechanisms in LUAD pathogenesis are yet to be elucidated.

The tumor microenvironment (TME) also plays a pivotal role in lung cancer progression and therapeutic resistance [22–25]. Tumor-associated macrophages (TAMs), as key cellular components of the TME, contribute to multiple processes including tumor growth, angiogenesis, immune regulation, metastasis, and chemoresistance [26, 27]. The polarization of TAMs is finely regulated by various cytokines within the TME, generally classifying them into anti-tumor M1-like and pro-tumor M2-like phenotypes [28, 29]. M2-type TAMs are widely recognized for promoting malignant progression through the secretion of immunosuppressive and pro-angiogenic factors [30–32]. To date, whether EBF3 influences TAM polarization and functional regulation remains unclear.

Based on this background, our study aims to systematically investigate the biological functions and underlying molecular mechanisms of EBF3 in the progression of lung adenocarcinoma. Furthermore, we will explore its potential role in TAM polarization and remodeling of the tumor microenvironment, with the goal of providing new theoretical insights and candidate targets for targeted therapy and immunomodulatory strategies in lung adenocarcinoma.

Materials and methods

Cell Lines and cell culture

All human lung adenocarcinoma cell lines (A549, RRID: CVCL_0023; H1299, H1993, PC9) and normal human bronchial epithelial cell lines (HBE, BEAS-2B) were obtained from the American Type Culture Collection (ATCC) or the Chinese Academy of Sciences Cell Bank. The mouse lung adenocarcinoma cell line LLC (RRID: CVCL_4358) was purchased from ATCC. Cells were cultured in DMEM or RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere with 5% CO₂. All cell lines were authenticated by STR profiling and regularly tested for mycoplasma contamination.

Animal studies

Female C57BL/6 mice aged 6–8 weeks were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. Mice were housed under specific pathogen-free (SPF) conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC).

Human clinical specimens

Human lung adenocarcinoma tissues and paired adjacent normal tissues were obtained from 30 patients who underwent surgical resection at Shanghai Chest Hospital between 2020 and 2023. The cohort included 16 males and 14 females, with a median age of 62 years (range 45–78). Tumor stages were distributed as follows: Stage I (n = 12), Stage II (n = 10), Stage III (n = 8). Tumor size, lymph node involvement, and TNM staging data were collected from pathological reports. Informed consent was obtained from all patients, and the collection and use of specimens were approved by the Hospital Ethics Committee.

Plasmid construction and stable cell line establishment

The full-length human EBF3 cDNA (NM_001015049.2) was cloned into the GV492 vector (Ubi-MCS-SV40-puro) to construct the EBF3 overexpression plasmid. shRNA targeting human EBF3 (shEBF3#1: 5′-GCTGACAACTTCGACTACAAA-3′) was cloned into the pLKO.1 vector for knockdown experiments. Lentiviruses were packaged using the pSPAX2 and pMD2.G system in 293T cells. Target cells were transduced with the virus and selected with 2 µg/ml puromycin to establish stable lines.

RNA extraction and quantitative real-time PCR (qPCR)

Total RNA was extracted from cells using TRIzol reagent. Reverse transcription was performed using the Hifair^® III 1st Strand cDNA Synthesis Kit (Yeasen). qPCR was carried out using SYBR Green Master Mix (Yeasen) on a QuantStudio 5 Real-Time PCR System. Primer sequences were as follows:

EBF3-F: 5′-CTGGAGCTGGAAGAAGCTG-3′

EBF3-R: 5′-GCTGTTGCTGCTGTAGTAGG-3′

CCL24-F: 5′-AGCTGCCTTATGGTGGTG-3′

CCL24-R: 5′-TGGTAGTGGTGGTGGTGG-3′

β-actin was used as the internal control.

Western blot analysis

Cells were lysed using RIPA lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein (20–30 µg) from three independent biological replicates were separated by SDS-PAGE and transferred to PVDF membranes. After blocking, membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using ECL substrate. Band intensity was quantified using ImageJ software (RRID: SCR_003070). Antibodies used: Anti-EBF3 (RRID: AB_2797973), Anti-p-AKT Ser473 (RRID: AB_329825), Anti-p-AKT Thr308, Anti-p-P38 (RRID: AB_331641), Anti-p-PI3K p85, Anti-β-actin (RRID: AB_306371), Anti-mTOR, Anti-p-STAT3, Anti-total STAT3, Anti-p-P65, Anti-total P65, Anti-p-ERK1/2, Anti-total ERK1/2, Anti-p-JNK, Anti-total JNK.

Cell proliferation and apoptosis assays

CCK-8 Assay: Cells were seeded in 96-well plates (3 × 10³ cells/well). Each group was set up with six replicate wells. After treatment, 10 µl of CCK-8 reagent was added to each well. After incubation for 2 h, the absorbance at 450 nm was measured. Experiments were repeated independently three times.

Apoptosis Detection

An Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) was used according to the manufacturer’s instructions. Cells from three independent experiments were analyzed using a BD FACS Celesta flow cytometer, with each sample analyzed in duplicate.

Signaling pathway analysis

To identify the core downstream pathway of EBF3 in LUAD, we first performed a systematic screening of 5 key LUAD-related oncogenic pathways (MAPK family, PI3K/AKT, NF-κB, STAT3, mTOR) via Western blot. The screening was performed in validated EBF3-OE/EV A549, H1299 and LLC cells, with 3 independent biological replicates. We detected both phosphorylated and total protein levels of each pathway’s core effector, and defined a pathway as regulated by EBF3 only if its phosphorylation was consistently altered in all three cell lines.The screening results showed that only the p38 MAPK and PI3K/AKT axes were specifically and significantly regulated by EBF3, thus we focused on these two pathways for in-depth validation. Western blot was performed to detect the phosphorylated and total forms of p38 and AKT (Ser473), with the full protocol described in the Western Blot Analysis section. The screening results of other pathways are provided in Supplementary Fig. 1.

Macrophage polarization assay

The human monocytic THP-1 cell line (RRID: CVCL_0006) was purchased from ATCC, authenticated by STR profiling, and regularly tested for mycoplasma contamination. THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.05 mM β-mercaptoethanol, at 37 °C with 5% CO₂. Cells were passaged every 2–3 days to maintain a density of 2 × 10⁵–1 × 10⁶ cells/ml, and only cells within 10 passages were used for experiments.

THP-1 cells were seeded at 1 × 10⁶ cells/well in 6-well plates, and differentiated into M0 macrophages with 100 ng/ml PMA for 24 h. Successful M0 differentiation was validated by flow cytometry, with > 95% CD14-positive cells confirmed in 3 independent replicates. After washing, M0 macrophages were polarized to the M2 phenotype with 20 ng/ml IL-4 and IL-13 for 48 h. M2 polarization was validated by: (1) flow cytometry for M2 marker CD206 and M1 marker CD86; (2) qPCR for M2 markers (CD206, IL-10, TGF-β, CXCL1) and M1 markers (iNOS, TNF-α).

For co-culture experiments, tumor cells were seeded in 0.4 μm Transwell inserts at a macrophage: tumor cell ratio of 1:2, and co-cultured with differentiated macrophages for 48 h in RPMI-1640 with 2% FBS. This cell ratio was selected based on classic published LUAD-macrophage crosstalk protocols, and the 48 h duration was determined by pre-experimental time gradient tests. For conditioned medium (CM) assays, CM was collected from 48 h tumor cell cultures, filtered, and applied to M2 macrophages for 24 h before analysis. CD206-positive cell proportion was detected by flow cytometry, and M2/M1 marker expression was assessed by qPCR. All assays were performed in 3 independent biological replicates, with each condition tested in triplicate.

Cytokine screening

The levels of 12 cytokines (including IL-6 and CCL24) in tumor cell conditioned media were quantified using the LEGENDplex™ Human Inflammation Panel multiplex assay kit (BioLegend) according to the manufacturer’s protocol. Samples were analyzed in duplicate from three independent collections.

Mouse tumor models

LLC cells (5 × 10⁵) stably expressing control vector or EBF3 were resuspended in 100 µl PBS and subcutaneously injected into the right flank of C57BL/6 mice. Each experimental group consisted of 4–5 mice. Tumor volume was measured every 3 days using calipers and calculated using the formula: Volume = (Length × Width²) / 2. For the CCL24 treatment group, recombinant mouse CCL24 (100 ng per mouse, R&D Systems) was administered intraperitoneally every 3 days starting from day 7 post-implantation. Mice were euthanized at day 18, and tumors were harvested for weight measurement and further analysis.

Flow cytometric analysis of tumor-infiltrating immune cells

Tumor tissues were minced and digested with collagenase IV (1 mg/ml) and DNase I (100 µg/ml) at 37 °C for 30 min to generate single-cell suspensions. Cells were stained with the following antibody panels:

Macrophages: CD45.2-FITC, Mac-1-pecy5.5, F4/80-PE, CD86- eFluor450, CD206-APC.

T cells: CD45.2-FITC, CD3-biotin- eFluor450, CD4-PE, CD8-APC

NK cells: CD45.2-FITC, NK1.1-PE-Cy7

MDSCs: CD45.2-FITC, CD11b-APC, LY6G-Pecy7,LY6C-PerCP

Data were acquired on a BD FACS Celesta and analyzed using FlowJo software (version 10, RRID: SCR_008520). Single-cell suspensions from three individual tumors per group were analyzed.

Bioinformatics analysis

To comprehensively assess the role of EBF3 in cancer, we conducted a series of bioinformatic analyses using public databases. First, the expression profile of EBF3 across multiple cancer types, including lung adenocarcinoma, was analyzed via the TIMER2.0 web server (http://timer.cistrome.org/). We further utilized GEPIA2 (http://gepia2.cancer-pku.cn/) to specifically examine EBF3 mRNA expression levels in LUAD tissues compared to normal lung tissues and to analyze its expression across pathological stages (I–IV). To evaluate the clinical prognostic significance of EBF3 and CCL24, survival data for LUAD patients were retrieved from the Kaplan–Meier plotter database (KM plotter, https://kmplot.com/analysis/). Overall survival (OS), progression-free survival (PFS), and post-progression survival (PPS) curves were generated based on the mRNA expression levels of EBF3 and CCL24. Statistical significance of survival differences was assessed using the log-rank test. Finally, the correlation between EBF3 and CCL24 mRNA expression in LUAD was analyzed using the “Correlation” module in TIMER2.0.

Luciferase reporter assay

To investigate whether EBF3 transcriptionally represses CCL24 by directly binding to its promoter, a luciferase reporter assay was performed. A DNA fragment containing the putative promoter region of the human CCL24 gene (approximately − 1500 to + 200 bp relative to the transcription start site) was cloned into the pGL3-Basic vector (Promega) to generate the CCL24-promoter luciferase reporter plasmid (pGL3-CCL24-pro). A549 or H1299 cells were co-transfected with the pGL3-CCL24-pro reporter plasmid, a Renilla luciferase control plasmid (pRL-TK, for normalization), and either an EBF3 overexpression plasmid or an empty control vector using Lipofectamine 3000 (Invitrogen). At 48 h post-transfection, firefly and Renilla luciferase activities were measured sequentially using the Dual-Luciferase Reporter Assay System (Promega) on a microplate luminometer. The relative luciferase activity was calculated as the ratio of firefly to Renilla luminescence. Experiments were performed in triplicate and repeated independently three times.

Immunohistochemistry (IHC) and TUNEL assay

To validate the effects of EBF3 on tumor proliferation, apoptosis, and immune cell infiltration at the tissue level, IHC and TUNEL assays were performed on formalin-fixed, paraffin-embedded (FFPE) tumor sections from the mouse xenograft model. For IHC, sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After blocking endogenous peroxidase and nonspecific binding, sections were incubated overnight at 4 °C with primary antibodies against Ki67 (for proliferation), CD4, or CD8 (for T cell infiltration). Following incubation with appropriate HRP-conjugated secondary antibodies, signals were developed using a DAB substrate kit and counterstained with hematoxylin. For the TUNEL assay, which detects apoptotic cells, sections were processed using the In Situ Cell Death Detection Kit, TMR red (Roche) according to the manufacturer’s instructions. Nuclei were counterstained with DAPI. All stained sections were scanned using a digital slide scanner (Pannoramic 250, 3DHISTECH). For IHC, the number of positive cells (Ki67+, CD4+, CD8+) in five randomly selected high-power fields (HPF, 400x) per sample was counted manually in a blinded manner. For TUNEL, the percentage of TUNEL-positive (apoptotic) nuclei relative to total DAPI-positive nuclei was quantified using ImageJ software. Three tumors per experimental group were analyzed.

Quantification and statistical analysis

All data are presented as the mean ± standard deviation (SD). Comparisons between two groups were performed using Student’s t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Survival analysis was performed using the Kaplan-Meier method with the log-rank test. Correlation analysis was performed using Spearman’s rank correlation. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 9 software (RRID: SCR_002798).

Results

EBF3 is downregulated in lung adenocarcinoma and its low expression correlates with poor patient prognosis

The role of EBF3 in lung adenocarcinoma (LUAD) remains unclear. Integrated multi-database analysis revealed that EBF3 was significantly downregulated in lung adenocarcinoma (Fig. 1A), and its expression level was markedly lower in LUAD tissues compared to normal lung tissues (Fig. 1B). Further analysis showed that EBF3 expression was negatively correlated with the clinical stage of lung adenocarcinoma, with an increase observed only in stage IV (Fig. 1C). To investigate its clinical relevance, we analyzed patient data from the Kaplan–Meier database and found that high expression of EBF3 was associated with better overall survival (OS), progression-free survival (PFS), and post-progression survival (PPS) in LUAD patients (Fig. 1D–F), suggesting a potential protective role of EBF3 in LUAD progression.

Fig. 1.

EBF3 is downregulated in lung adenocarcinoma and its low expression correlates with poor patient prognosis. (A) Analysis of EBF3 expression across multiple tumor types based on the TIMER database. The red circle indicates the expression level of EBF3 in lung adenocarcinoma (LUAD).(B) Comparison of EBF3 expression between normal lung tissues and lung adenocarcinoma tissues in the GEPIA3 database.(C) Analysis of EBF3 expression across different clinical stages (I–IV) of lung adenocarcinoma. (D-F) Kaplan-Meier survival analysis of LUAD patients from the KM database. High EBF3 expression is significantly associated with longer (D) overall survival (OS), (E) progression-free survival (PFS), and (F) post-progression survival (PPS).(G-H) EBF3 protein expression is significantly lower in LUAD tumor tissues (T) compared to adjacent normal tissues (N) as demonstrated by representative Western blot images (G) and quantification (H). (I-J) Western blot analysis (I) and its quantification (J) confirming the downregulation of EBF3 protein in a panel of human LUAD cell lines (A549, H1299, H1993, PC9) compared to normal human bronchial epithelial cells (HBE, BEAS-2B).(K) qRT-PCR analysis validates the reduction of EBF3 at the mRNA level in LUAD cell lines. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001

We further evaluated EBF3 expression in clinical specimens and found that its levels were significantly lower in LUAD tumor tissues than in normal lung tissues from healthy individuals (Fig. 1G–H). This downregulation was consistently reproduced in vitro across a panel of lung adenocarcinoma cell lines (A549, H1299, H1993, PC9), all of which exhibited reduced EBF3 expression compared to the normal bronchial epithelial cell lines HBE and BEAS-2B (Fig. 1I–J). Moreover, transcriptional analysis also confirmed the downregulation of EBF3, with significantly decreased EBF3 mRNA levels in LUAD cell lines (Fig. 1K). To further explore the clinical relevance of EBF3 in LUAD, we analyzed the correlation between EBF3 protein expression levels and the clinicopathological characteristics of the 30 enrolled LUAD patients. Patients were divided into EBF3 high-expression and low-expression groups based on the median expression level of EBF3 in tumor tissues. As shown in Table 1, low EBF3 expression was significantly associated with larger tumor size (P = 0.018), positive lymph node metastasis (P = 0.009), and advanced TNM stage (P = 0.012). No significant correlation was found between EBF3 expression and age or gender (both P > 0.05). These results further confirmed that downregulation of EBF3 is closely associated with the malignant progression of LUAD in clinical settings.

Table 1.

Correlation between EBF3 protein expression and clinicopathological characteristics in 30 LUAD patients

Clinicopathological characteristics	Total (n = 30)	EBF3 high expression (n = 15)	EBF3 low expression (n = 15)	P value
Age (years), mean ± SD	62 ± 9.7	60.8 ± 10.2	63.2 ± 9.3	0.506
Gender, n (%)				0.725
Male	16 (53.3%)	8 (53.3%)	8 (53.3%)
Female	14 (46.7%)	7 (46.7%)	7 (46.7%)
Tumor size, n (%)				0.018*
< 3 cm	13 (43.3%)	10 (66.7%)	3 (20.0%)
≥ 3 cm	17 (56.7%)	5 (33.3%)	12 (80.0%)
Lymph node metastasis, n (%)				0.009**
Negative	18 (60.0%)	12 (80.0%)	6 (40.0%)
Positive	12 (40.0%)	3 (20.0%)	9 (60.0%)
TNM stage, n (%)				0.012*
I-II	22 (73.3%)	14 (93.3%)	8 (53.3%)
III	8 (26.7%)	1 (6.7%)	7 (46.7%)

*P < 0.05, **P < 0.01. Statistical analysis was performed by Student’s t-test (age) and Chi-square test (gender, tumor size, lymph node metastasis, TNM stage)

In summary, these results indicate that EBF3 is frequently downregulated in lung adenocarcinoma in both clinical samples and cellular models, and its loss may be associated with disease progression and poor clinical outcomes.

EBF3 suppresses proliferation and induces apoptosis in lung adenocarcinoma cells

To elucidate the functional role of EBF3 in lung adenocarcinoma, we first validated its overexpression and knockdown in human (A549, H1299) and mouse (LLC) lung adenocarcinoma cell lines using Western blot analysis (Fig. 2A and B). Based on these validated models, EBF3 was stably overexpressed in these cell lines (Fig. 2C). CCK‑8 proliferation assays demonstrated that EBF3 overexpression significantly suppressed proliferation in A549, H1299, and LLC cells (Fig. 2D and F), indicating its growth‑inhibitory function.

Fig. 2.

EBF3 suppresses proliferation and induces apoptosis in lung adenocarcinoma cells. (A) Representative Western blot images validating the overexpression of EBF3 in A549, H1299, and LLC cells. (B) Representative Western blot images validating the knockdown of EBF3 in A549, H1299, and LLC cells. (C) Western blot analysis confirming the efficient overexpression of EBF3 in human (A549, H1299) and mouse (LLC) LUAD cell lines. (D-F) Cell proliferation assessed by CCK-8 assays was significantly impaired upon EBF3 overexpression in A549 (D), H1299 (E), and LLC (F) cells. (G) qRT-PCR analysis showing successful knockdown of EBF3 in the indicated cell lines. (H-J) Knockdown of EBF3 significantly enhanced cell proliferation in A549 (H), H1299 (I), and LLC (J) cells as measured by CCK-8 assays.(K-P) Flow cytometric analysis of apoptosis. Overexpression of EBF3 promoted apoptosis in A549 (K, N), H1299 (L, O), and LLC (M, P) cells. The bar graphs (N-P) show the quantification of apoptotic cells. Data are presented as mean ± SD from three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001

To further confirm this phenotype, EBF3 was knocked down in the corresponding cell lines (Fig. 2G). Consistent with the overexpression results, knockdown of EBF3 markedly enhanced cellular proliferation (Fig. 2H and J), providing complementary evidence that EBF3 acts as a negative regulator of cell growth.

In addition, flow cytometry‑based apoptosis analysis revealed that overexpression of EBF3 significantly increased the proportion of apoptotic cells in A549, H1299 (Fig. 2K, L, N and O), and LLC cells (Fig. 2M and P). Taken together, these findings demonstrate that EBF3 not only inhibits proliferation but also promotes apoptosis in lung adenocarcinoma cells, supporting its potential role as a tumor suppressor in this malignancy.

EBF3 negatively regulates the phosphorylation of P38 and AKT in lung adenocarcinoma cells

To further elucidate the molecular mechanism underlying the tumor-suppressive function of EBF3 in lung adenocarcinoma, we first performed a systematic screening of core oncogenic pathways that are frequently dysregulated and drive malignant progression in LUAD, including the MAPK family (P38, ERK, JNK), PI3K/AKT, NF-κB, STAT3, and mTOR signaling axes. The screening results revealed that among all the pathways examined, only the phosphorylation levels of P38 and AKT were consistently modulated by EBF3 overexpression, whereas no significant or reproducible changes were observed in the other pathways (Supplementary Fig. 1A-D).

Given the well-established roles of P38 and AKT signaling in regulating LUAD cell proliferation, survival, and tumor immune microenvironment remodeling [33–38], we further performed in-depth validation of this regulatory relationship. Our results confirmed that EBF3 significantly modulated the phosphorylation of P38 and AKT at Ser473. Specifically, genetic knockdown of EBF3 robustly enhanced the phosphorylation of both P38 and AKT (Ser473) in human A549 and H1299 LUAD cell lines (Figs. 3A–C), whereas stable overexpression of EBF3 markedly suppressed the activation of these two kinases (Figs. 3D–F). This regulatory pattern was further confirmed to be conserved across species in the mouse LLC lung adenocarcinoma model: EBF3 knockdown significantly increased (Figs. 3G, H), while its overexpression decreased, the phosphorylation levels of P38 and AKT (Ser473) (Figs. 3G, I).We further examined whether EBF3 regulates other key nodes within the PI3K/AKT pathway. Notably, neither knockdown nor overexpression of EBF3 produced a significant effect on the phosphorylation of AKT at Thr308 or on PI3K activation in A549, H1299, or LLC cells (Supplementary Figs. 2A–D). This result indicates that the regulatory effect of EBF3 on AKT signaling is phosphorylation site-specific, primarily targeting Ser473 rather than Thr308 or upstream PI3K.

Fig. 3.

EBF3 negatively regulates the phosphorylation of P38 and AKT in lung adenocarcinoma cells. (A-C) Western blot analysis (A) and quantitative results (B, C) demonstrate that knockdown of EBF3 in human LUAD cells (A549, H1299) enhances the phosphorylation levels of P38 and AKT (Ser473). GAPDH serves as a loading control. (D-F) Conversely, overexpression of EBF3 (D) significantly suppresses the phosphorylation of P38 and AKT (Ser473) in human LUAD cell lines (E, F).(G-I) Consistent with the findings in human cells, EBF3 knockdown enhances (G, H), while its overexpression inhibits (G, I), the phosphorylation of P38 and AKT (Ser473) in mouse LLC cells. Data are presented as mean ± SD from three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001

Collectively, these data demonstrate that EBF3 functions as a specific negative regulator of the P38 and AKT (Ser473) signaling axes. Its tumor-suppressive activity is likely mediated through the precise inhibition of these critical pro-survival and proliferative pathways, rather than through a broad suppression of the entire PI3K/AKT cascade.

EBF3 overexpression inhibits tumor growth and remodels the immunosuppressive microenvironment in vivo

Building upon the established role of EBF3 in suppressing LUAD progression in vitro, we sought to validate its tumor-suppressive function in an in vivo setting. C57BL/6 mice were subcutaneously implanted with LLC cells stably overexpressing EBF3. Consistent with our cellular findings, EBF3 overexpression markedly inhibited tumor growth in vivo (Fig. 4A), significantly reduced tumor volume (Fig. 4B), and decreased tumor weight (Fig. 4C) compared to the control group.

Fig. 4.

EBF3 overexpression inhibits tumor growth and remodels the immunosuppressive microenvironment in vivo. (A) Representative images of subcutaneous tumors from C57BL/6 mice implanted with control or EBF3-overexpressing LLC cells at the endpoint. (B) Tumor growth curves of xenografts from the indicated groups (n = 6–8 mice per group). Data are presented as mean ± SD. (C) Weights of excised tumors from the control and EBF3-overexpressing groups at the study endpoint. (D) Flow cytometry analysis showing the frequency of total tumor-infiltrating macrophages (CD11b⁺F4/80⁺) within CD45⁺ hematopoietic cells. (E) The proportion of immunosuppressive M2-like macrophages (identified as CD206⁺) within the total macrophage gate. (F) The frequency of immunostimulatory M1-like macrophages (identified as CD86⁺) within the total macrophage population. (G-H) Quantification of tumor-infiltrating CD4⁺ T cells (G) and CD8⁺ T cells (H) among CD45⁺ immune cells. (I) The percentage of natural killer (NK) cells (NK1.1⁺CD3⁻) within the CD45⁺ cell population. (J-K) Frequencies of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs, CD11b⁺Ly6G⁺Ly6Cᵉˡ) (J) and monocytic myeloid-derived suppressor cells (M-MDSCs, CD11b⁺Ly6G⁻Ly6Cʰⁱ) (K) among CD45⁺ cells. (L) Representative images of KI67 staining, Tunel assay, and immunohistochemical detection of CD4 and CD8 in subcutaneous xenograft tumors derived from EV‑ and EBF3‑overexpressing LLC cells. (M) Statistical analysis of KI67‑positive rate, Tunel‑positive rate, and the infiltration levels of CD4⁺ and CD8⁺ T cells in subcutaneous xenograft tumors from EV‑ and EBF3‑overexpressing LLC cells

To investigate whether EBF3 modulates the tumor immune microenvironment, we performed comprehensive flow cytometry analysis of tumor-infiltrating immune cells. Our results revealed that EBF3 overexpression significantly reduced the overall infiltration of macrophages (Fig. 4D). Further subset analysis demonstrated a specific decrease in the M2-like macrophage population (Fig. 4E), while the proportions of M1-like macrophages (Fig. 4F) and myeloid-derived suppressor cells (PMN/M-MDSCs) (Figs. 4J–K) remained unaltered. Concurrently, we observed a significant increase in tumor-infiltrating CD4⁺ and CD8⁺ T cells in the EBF3-overexpressing group (Figs. 4G–H). In contrast, the frequency of natural killer (NK) cells was not significantly affected (Fig. 4I).

Further histological examination of the xenograft tumors corroborated these findings. Representative images showed decreased Ki-67 staining and increased Tunel-positive signals in the EBF3-overexpressing group, along with enhanced infiltration of CD4⁺ and CD8⁺ T cells (Fig. 4L). Quantitative analysis confirmed a significant reduction in proliferation, a marked increase in apoptosis, and elevated levels of CD4⁺ and CD8⁺ T cell infiltration (Fig. 4M).

Collectively, these in vivo findings confirm the growth-inhibitory function of EBF3 in LUAD and uncover its role in reshaping the tumor immune landscape. The data suggest that EBF3-mediated tumor suppression involves a dual mechanism: direct inhibition of cancer cell proliferation and apoptosis induction, coupled with the orchestration of an anti-tumor immune response characterized by reduced M2 macrophages and enhanced T cell infiltration.

EBF3 reprograms the tumor cell secretome to inhibit M2 macrophage polarization in vitro

Guided by our in vivo observation that EBF3 reshapes the tumor immune microenvironment by reducing M2-like macrophage infiltration, we sought to mechanistically dissect this phenomenon using a reductionist in vitro approach. We employed a well-established model wherein THP-1 human monocytes are differentiated into M2-polarized macrophages (Fig. 5A). These macrophages were then treated with conditioned medium (CM) collected from control or EBF3-overexpressing tumor cells to simulate paracrine signaling.Flow cytometric analysis confirmed successful M2 polarization, and revealed that CM from EBF3-overexpressing cells significantly inhibited this process, as evidenced by a marked reduction in the CD206-positive cell population (Fig. 5B, C). This suppression of the M2 phenotype was further corroborated at the transcriptional level by qPCR, showing decreased CD206 mRNA expression (Fig. 5D). Furthermore, EBF3 CM downregulated the mRNA levels of other pivotal M2-associated factors, including IL-10, TGF-β, and CXCL1 (Fig. 5E-G).To gain a comprehensive, protein-level overview of how EBF3 alters the tumor cell secretome, we profiled a panel of inflammatory cytokines and chemokines in the CM via ELISA. Among the factors analyzed (IL-6, IL-12, IL-23, CCL10, CCL22, CCL24, CXCL10, CXCL11, CXCL16), the secretion of CCL24 and IL-6 was specifically and significantly diminished in the EBF3 group (Fig. 5H, I).Collectively, these in vitro data not only validate the immunomodulatory role of EBF3 on macrophages observed in vivo but also pinpoint a specific reprogramming of the tumor cell secretome. The identified downregulation of IL-6 and CCL24 provides a plausible mechanistic link for how EBF3-expressing tumor cells counteract M2 polarization within the tumor microenvironment, unveiling a novel facet of EBF3 as a regulator of cancer cell-driven immune evasion.

Fig. 5.

EBF3 reprograms the tumor cell secretome to inhibit M2 macrophage polarization in vitro. (A) Schematic diagram of the experimental workflow for assessing the effect of tumor cell-secreted factors on macrophage polarization using a conditioned medium (CM) model. (B) Representative flow cytometry plots showing CD206 expression on M2 macrophages following treatment with CM from control or EBF3-overexpressing tumor cells. (C) Quantitative analysis of the percentage of CD206-positive cells from flow cytometry experiments (n = 3 independent experiments). (D) qPCR analysis of CD206 mRNA expression in M2 macrophages treated with the indicated CM.(E-G) qPCR analysis of mRNA expression for M2-associated genes IL-10 (E), TGF-β (F), and CXCL1 (G) in treated macrophages. (H-I) ELISA-based systematic profiling of the tumor cell secretome identified CCL24 and IL-6 as key cytokines selectively suppressed by EBF3 overexpression. Among the nine inflammatory cytokines and chemokines analyzed, these two factors showed significant reduction while others remained unchanged. A549(H) and H1299 (I)

CCL24 functions as a critical downstream effector of EBF3-mediated tumor suppression

Building upon our discovery that EBF3 modulates macrophage polarization through CCL24, we sought to determine whether CCL24 serves as a critical downstream effector of EBF3-mediated tumor suppression. Bioinformatic analysis showed that high expression of CCL24 was associated with poor prognosis in lung adenocarcinoma (Fig. 6A), and its expression was negatively correlated with EBF3 levels in tumor samples (Fig. 6B). A luciferase reporter assay confirmed that EBF3 transcriptionally suppressed CCL24 expression (Fig. 6C).

Fig. 6.

CCL24 functions as a critical downstream effector of EBF3-mediated tumor suppression. (A) Prognostic analysis of CCL24 expression in lung adenocarcinoma patients based on the Kaplan-Meier (KM) database, showing that high CCL24 expression is associated with poor prognosis. (B) Correlation analysis between CCL24 and EBF3 expression in lung adenocarcinoma using the TIMER3 database. (C) Luciferase reporter assay demonstrating that EBF3 transcriptionally suppresses CCL24 expression. (D) qPCR analysis of CCL24 mRNA expression in A549 and H1299 cells following EBF3 knockdown. (E) qPCR analysis of CCL24 mRNA expression in A549 and H1299 cells with EBF3 overexpression. (F-G) Rescue of proliferation capacity by recombinant CCL24 protein in EBF3-overexpressing A549 (F) and H1299 (G) cells, as determined by CCK-8 assays. (H-I) Apoptosis analysis by flow cytometry showing that recombinant CCL24 protein rescues the enhanced apoptosis induced by EBF3 overexpression in A549 (H) and H1299 (I) cells. (J) Representative western blot images showing the restoration of P38 and AKT (Ser473) phosphorylation by recombinant CCL24 protein in EBF3-overexpressing A549 and H1299 cells.(K-L) Quantitative analysis of p-P38 and p-AKT (Ser473) protein levels from western blot experiments. A549(K), H1299(L)

We then validated this regulatory relationship in lung adenocarcinoma cells. Knockdown of EBF3 significantly upregulated CCL24 mRNA levels in both A549 and H1299 cells (Fig. 6D), whereas EBF3 overexpression potently suppressed its transcription (Fig. 6E), further establishing EBF3 as a transcriptional repressor of CCL24.

To determine the functional necessity of CCL24 in EBF3-mediated tumor suppression, we performed rescue experiments. CCK-8 assays demonstrated that the addition of exogenous recombinant CCL24 protein significantly reversed the proliferation inhibition induced by EBF3 overexpression in both A549 and H1299 cells (Figs. 6F, G). Correspondingly, flow cytometric analysis revealed that CCL24 supplementation effectively counteracted the pro-apoptotic effect of EBF3 overexpression (Figs. 6H, I).

Investigating the underlying signaling mechanisms, we found that CCL24 addition similarly reversed the EBF3-mediated suppression of P38 and AKT (Ser473) phosphorylation (Figs. 6J-L). These consistent findings establish that the downregulation of CCL24 is essential for EBF3’s tumor-suppressive signaling.

CCL24 rescues the tumor-suppressive effects of EBF3 in a syngeneic mouse model

To validate the functional significance of the EBF3–CCL24 axis identified in vitro, we established a syngeneic mouse model. C57BL/6 mice were subcutaneously implanted with LLC cells stably overexpressing EBF3 and then treated intraperitoneally with recombinant CCL24 protein (Fig. 7A). While EBF3 overexpression potently suppressed tumor growth, subsequent CCL24 administration substantially rescued this tumor‑suppressive phenotype (Fig. 7B), as consistently reflected in final tumor weight (Fig. 7C) and longitudinal tumor volume measurements (Fig. 7D). These results demonstrate that CCL24 functionally counteracts EBF3‑mediated growth inhibition in vivo.

Fig. 7.

CCL24 rescues the tumor-suppressive effects of EBF3 in a syngeneic mouse model. (A) Schematic representation of the experimental design. C57BL/6 mice were subcutaneously implanted with LLC cells overexpressing EBF3 and treated with recombinant CCL24 protein via intraperitoneal injection. (B) Representative images of excised tumors from the four experimental groups at the study endpoint.(C-D) Final tumor weights (C) and tumor growth curves (D) demonstrating that CCL24 administration rescues the growth inhibition induced by EBF3 overexpression .(E-G) Flow cytometric analysis of tumor-infiltrating immune cells: frequencies of total TAMs (CD11b⁺F4/80⁺) (E), M2-type TAMs (CD206⁺) (F), and M1-type TAMs (CD86⁺) (G) among CD45⁺ cells.(H-I) Frequencies of tumor-infiltrating CD4⁺ T cells (H) and CD8⁺ T cells (I) among CD45⁺ cells, showing that CCL24 reverses the T cell enhancement mediated by EBF3. (J) Representative images of Ki67 staining, Tunel assay, and immunohistochemical staining for CD4 and CD8 in tumor tissues from the EV, EBF3‑OE, EV‑NC, and EBF3‑OE + CCL24 groups. (K) Quantitative analysis showing the Ki67‑positive rate, Tunel‑positive rate, and infiltration levels of CD4⁺ and CD8⁺ T cells in tumor tissues across the indicated groups

To delineate the underlying immunomodulatory mechanisms, we performed comprehensive flow cytometry analysis of tumor‑infiltrating immune cells. CCL24 treatment effectively restored the diminished infiltration of total tumor‑associated macrophages (TAMs, Fig. 7E) and specifically recovered the M2‑TAM subset that had been reduced by EBF3 overexpression (Fig. 7F), whereas the M1‑TAM population remained unaltered (Fig. 7G). Concurrently, the enhanced infiltration of CD4⁺ (Fig. 7H) and CD8⁺ T cells (Fig. 7I) induced by EBF3 was significantly attenuated by CCL24 supplementation.

Further histological examination corroborated these findings. Representative images showed that CCL24 treatment reversed the reduction in Ki‑67‑positive cells and the increase in Tunel‑positive signals caused by EBF3 overexpression, while also diminishing the enhanced infiltration of CD4⁺ and CD8⁺ T cells (Fig. 7J). Quantitative analysis confirmed that CCL24 significantly rescued the proliferation inhibition, apoptosis induction, and elevated T‑cell infiltration mediated by EBF3 (Fig. 7K).

Collectively, our findings establish CCL24 as the pivotal downstream effector of EBF3‑mediated tumor suppression in vivo, orchestrating both direct growth control and reprogramming of the tumor immune microenvironment. This mechanistic insight into the EBF3–CCL24 axis advances our understanding of LUAD pathogenesis and highlights a potential therapeutic target for intervention.

Discussion

Lung adenocarcinoma continues to pose significant clinical challenges, characterized by high rates of chemoresistance, recurrence, and metastasis [39, 40]. This grim reality underscores the urgent need to identify novel and potent therapeutic targets. Transcription factors, as master regulators of gene expression networks, represent a promising yet underexplored frontier in this endeavor [41, 42]. Our study sheds light on the pivotal role of the transcription factor EBF3, revealing its frequent downregulation in LUAD and establishing its potent tumor-suppressive function. The growth-inhibitory and pro-apoptotic effects observed upon EBF3 overexpression position it as a critical guardian against tumorigenesis, potentially functioning in a manner analogous to the well-characterized tumor suppressor p53 [43]. While our work delineates the downstream consequences of EBF3 loss, an intriguing question for future investigation is whether genetic alterations, such as mutations in the EBF3 gene itself, contribute to its silencing during LUAD pathogenesis.

At the molecular level, we delineated a key mechanism through which EBF3 exerts its tumor-suppressive effects: the direct inhibition of the MAPK and AKT signaling cascades. Specifically, we demonstrated that EBF3 potently suppresses the phosphorylation of P38 and AKT, two central nodes in oncogenic signaling that are known to drive cell proliferation, survival, and inflammatory responses within the tumor milieu [35, 44]. This places EBF3 upstream of critical pathways that fuel cancer progression, providing a mechanistic explanation for its ability to curb tumor growth.

Beyond its cell-autonomous effects, our research elucidates a novel and sophisticated dimension of EBF3 function: the active reprogramming of the tumor immune microenvironment (TIME). Tumor-associated macrophages (TAMs), particularly those polarized towards an M2 phenotype, constitute a dominant pro-tumorigenic stromal component in LUAD, fostering an immunosuppressive niche that facilitates angiogenesis, extracellular matrix remodeling, and T-cell dysfunction [45–50]. Our results demonstrate that EBF3 serves as a pivotal regulator of this crosstalk. By transcriptionally repressing key secreted factors—most notably the chemokine CCL24 and the cytokine IL-6—EBF3 fundamentally rewires the tumor cell secretome. This reprogramming disrupts the paracrine signaling between malignant cells and immune constituents, thereby impairing M2 macrophage polarization. The consequent remodeling of the immune landscape, marked by a reduction in immunosuppressive M2-TAMs and a concurrent increase in tumor-infiltrating CD4⁺ and CD8⁺ T cells, collectively establishes an immunological context less permissive to tumor progression.

The delineation of CCL24 as a critical downstream effector of EBF3 constitutes a central finding of our study, forging a direct molecular link between the loss of a tumor-suppressive transcription factor and the establishment of an immune-evasive microenvironment. This axis not only advances the mechanistic understanding of LUAD pathogenesis but also reveals a tractable therapeutic vulnerability. The consistent reversal of EBF3-mediated tumor suppression and immune modulation by exogenous CCL24, both in vitro and in vivo, positions CCL24 as a candidate therapeutic target. Consequently, strategies aimed at neutralizing CCL24 activity—such as monoclonal antibodies—emerge as a translationally promising approach. Targeting the EBF3-CCL24 axis holds potential to alleviate immune suppression and may synergize with established treatment modalities, offering a rational combinatorial strategy for LUAD management.

While our study establishes a functional and mechanistic role of the EBF3-CCL24 axis, several limitations should be acknowledged. First, the clinical correlation analysis was conducted in a single-center cohort of limited size (30 patients); validation in larger, multi-center clinical cohorts is necessary to strengthen the prognostic and clinical relevance of EBF3 and CCL24 in LUAD. Second, although we demonstrated that EBF3 transcriptionally represses CCL24 via luciferase reporter assay, the precise cis-regulatory elements in the CCL24 promoter that directly bind to EBF3, as well as the potential transcriptional co-factors involved in this process, remain to be mapped and validated by ChIP-seq and EMSA assays. Third, while our in vitro and in vivo rescue experiments identified CCL24 as the critical downstream effector of EBF3, the contribution of other EBF3-regulated secreted factors (such as IL-6) to its immunomodulatory function warrants further in-depth investigation. Fourth, our in vivo functional experiments were mainly performed in a subcutaneous syngeneic tumor model, which cannot fully recapitulate the pathological process of spontaneous LUAD in humans; future studies using genetically engineered mouse models of spontaneous LUAD are needed to further validate the role of the EBF3-CCL24 axis in tumor initiation and progression. Finally, the therapeutic potential of CCL24 blockade, though conceptually promising, requires rigorous preclinical evaluation in immunocompetent LUAD models to assess its in vivo efficacy, potential resistance mechanisms, and synergistic effect with immune checkpoint inhibitors.

Conclusions

In conclusion, our study delineates EBF3 as a multifunctional tumor suppressor in lung adenocarcinoma, which executes a dual‑pronged strategy against tumor progression: directly restraining cancer cell proliferation and survival, and indirectly subverting the immunosuppressive niche through transcriptional repression of CCL24. These findings substantially deepen our mechanistic understanding of LUAD pathogenesis and highlight the therapeutic promise of modulating the EBF3‑CCL24 axis—either by restoring EBF3 activity or by targeting its key effector CCL24—thereby offering a rational foundation for developing novel immunotherapeutic combinations.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualisation: L.L.Y.YR and C.CM; Methodology: L.L. Y.YR and C.CM; Data acquisition and analysis: L.L., C.CM., and M.XM; Writing original draft and revisions: L.L., C.CM, M.XM W.JX. and Y.J; Writing – review & editing, Project administration, Funding acquisition, Conceptualization, Supervision.L.L L.D and Y.J.

Funding

This work was supported by Shanghai Chest Hospital (2023YHTCQ105). This work was also supported by the Shanghai Key Laboratory of Thoracic Tumor Biotherapy. This project was funded by the National Natural Science Foundation of China(82573530, 8250104597).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Informed consent and approval were obtained from all the patients and the Ethics Committee of Shanghai Chest Hospital. All the animal studies were performed following the Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Shanghai Chest Hospital.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.