The inhibitory effect of M2 macrophage-derived exosomes on gefitinib resistant lung adenocarcinoma cells through the MIF/TIMP1/CD74 axis

Abstract

Lung adenocarcinoma (LA) ranks among the most common malignant tumors worldwide. M2 macrophage-derived exosomes have been implicated in regulating LA progression and drug resistance. However, whether proteins encapsulated in these exosomes contribute to gefitinib resistance in LA remains to be elucidated. This study investigates the role of tissue inhibitor of metalloproteinase 1 (TIMP1) in mediating LA resistance to gefitinib. We demonstrated that M2 macrophages-derived exosomes enhanced the sensitivity of gefitinib-resistant LA cell lines (P < 0.05). Bioinformatics analyses validated the correlation of TIMP1 expression with LA progression and patient survival. Depletion of TIMP1 in M2 macrophage-derived exosomes suppressed the proliferation of gefitinib-resistant LA cells. Additionally, we confirmed that TIMP1 interacts with cluster of differentiation 74 (CD74), a process linked to the proliferation and migration of gefitinib-resistant LA cells. Additionally, this study validated that TIMP1 mediates the interaction between CD74 and macrophage migration inhibitory factor (MIF), potentially activating the PI3K/AKT signaling pathway. Collectively, these findings demonstrate that TIMP1 in M2 macrophage-derived exosomes facilitate the binding of MIF to CD74 in LA cells, thereby attenuating gefitinib resistance.

Introduction

Lung cancer ranks among the most prevalent malignant tumors globally, with its incidence and mortality rates topping those of urban malignancies. Non-small cell lung cancer (NSCLC) constitutes the major histological type, among which lung adenocarcinoma (LA)-a subtype of NSCLC-accounts for approximately 43% of all lung cancer cases¹. Over the past decade, the incidence of LA has shown a continuous upward trend, attributable to risk factors such as smoking and environmental pollution². Surgical resection represents the conventional treatment for LA. However, in patients with advanced-stage lung cancer, conventional therapies fail to eradicate metastatic lesions completely. Currently, chemotherapy remains a mainstay treatment modality for LA patients³among which gefitinib is widely employed as a first-line agent in LA chemotherapy. Clinical investigations have confirmed that gefitinib is indicated for locally advanced or metastatic NSCLC patients who have received prior chemotherapy^4,5. Regrettably, the development of acquired drug resistance substantially restricts the clinical utility and patient prognosis of gefitinib^5,6. Thus, elucidating the molecular mechanisms underlying gefitinib resistance is critical for improving the prognosis of LA patients.

Macrophages exhibit two well-characterized polarization subtypes: classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages)⁷. Tumor-associated macrophages (TAMs), defined as macrophages infiltrating the tumor stroma, represent the most abundant inflammatory cell population within the tumor microenvironment^7,8. During tumor progression, TAMs predominantly polarize toward an M2-like phenotype, underscoring the critical role of M2 macrophages in tumor regulation. Substantial evidence from preclinical and clinical studies has established a robust association between M2 macrophage polarization and the initiation and progression of LA^{9, 10–11}. Elucidating the precise molecular mechanisms by which M2 macrophages modulate LA development may provide innovative insights for clinical diagnosis and therapeutic intervention in LA.

Exosomes are small extracellular vesicles (EVs) with a typical diameter ranging from 30 to 100 nanometers. Derived from intracellular endosomes, they form within multivesicular bodies (MVBs) through endosomal sorting complexes required for transport (ESCRT)-dependent or -independent pathways¹². Exosomes encapsulate diverse cargoes, among which exosomal proteins serve as critical mediators of intercellular communication, regulating various biological processes¹³. Recombinant tissue inhibitor of metalloprotein 1 (TIMP1) is a well-characterized protein that specifically inhibits metalloproteinases (MMP) activity, modulating extracellular matrix (ECM) remodeling and cell-matrix interactions. TIMP1 is implicated in multiple biological processes, including the regulation of tumor cell proliferation, migration, and apoptosis in malignancies such as prostate cancer, pancreatic cancer, and gastric cancer^{14, 15–16}. Dysregulated TIMP1 expression in certain diseases suppresses downstream target gene activity, leading to ECM accumulation and subsequent alteration of cancer cell physiological functions. Cluster of differentiation 74 (CD74), a transmembrane glycoprotein, functions as a molecular chaperone mediating intracellular protein transport. Additionally, CD74 functions as a cell surface receptor for cytokines such as macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase^17,18. However, the exact regulatory mechanisms and molecular pathways through which TIMP1, CD74, and MIF mediate drug resistance in LA remain poorly defined.

The objectives of this study were to assess the effect of M2 macrophages on LA cells, and elucidate the specific role of aberrantly expressed genes in M2 macrophages in mediating gefitinib resistance in LA cells, and investigate the underlying regulatory mechanisms, thereby providing potential therapeutic strategies for managing drug resistance in lung cancer.

Materials and methods

Clinical specimens

A total of 46 LA patients treated with gefitinib at The Fourth Hospital of Hebei Medical University were enrolled. Serum samples were collected from patients with disease progression (PD) following gefitinib therapy and from those who achieved complete response (CR) or partial response (PR). Patient responses to gefitinib were evaluated using the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Patients with CR or PR were categorized as the sensitive group, whereas those with PD were assigned to the resistant group. All serum samples were stored at -80 °C. Written informed consent was obtained from all participating patients or their legal guardians. This study was approved by the Medical Ethics Committee of The Fourth Hospital of Hebei Medical University (approval number: [FH-2024-H-0316]) and conducted in accordance with the World Medical Association Declaration of Helsinki.

Cell culture

Peripheral blood mononuclear cells (PBMCs), A549, and PC9 cell lines were obtained from the ATCC Cell Resource Center. All cells were cultured under standardized conditions (5% CO₂, 37 ℃) in Dulbecco’s modified Eagle’s medium (DMEM) complete culture medium, composed of DMEM base medium, fetal bovine serum (FBS), and penicillin/streptomycin at a ratio of 89:10:1. Gefitinib-resistant LA cell lines derived from A549 and PC9 cells (designated as A549/Ge and PC9/Ge, respectively) were established following the protocols described by Yuan et al. and Sun et al.^19,20. Cells were maintained in complete medium containing 12% FBS, 10 µg/mL gefitinib, 100 U/mL streptomycin, and 100 U/mL penicillin. Upon reaching approximately 85% confluence, cells were passaged using 0.25% (w/v) trypsin-EDTA solution.

A transwell co-culture system was established using polycarbonate membrane inserts. M2 macrophages were seeded in the upper chamber, whereas LA cells were plated in the lower compartment. The exosome secretion inhibitor GW4896 (5 mM, UMIBIO, Shanghai, China) was applied to the culture system. As a control, LA cells were co-cultured with DMEM medium alone.

Induction of M2 macrophages

To generate M2 macrophages, PBMCs were cultured, and adherent cells were isolated following removal of non-adherent cells. Human PBMCs were then stimulated with 100 ng/mL macrophage colony-stimulating factor (M-CSF) for 7 days to promote differentiation into M2 macrophages. M2 macrophage identity was confirmed by quantitative real-time PCR (qRT-PCR) and western blot analysis.

Isolation, identification, and fluorescent dye labeling of exosomes

Following 72 h culture of cells in the logarithmic growth phase, 1 mL of culture medium was collected and centrifuged at 2,000 × g for 15 min to obtain the supernatant. Exosomes were extracted using ExoQuick exosome precipitation solution (System Biosciences, Beijing, China).

Transmission electron microscopy (TEM) was used for exosome imaging. Exosomes pellets were fixed with 4% glutaraldehyde solution and stored at 4 ℃ prior to analysis. An aliquot of the fixed sample was applied to a carbon-coated copper grid, stained with 2% phosphotungstic acid, and washed with distilled water before air-drying. Exosome morphology was observed by TEM at an accelerating voltage of 80 kV.

Nanosight tracking analysis (NTA) was performed to characterize the size distribution of exosomes. Exosomes were resuspended in 50 µL phosphate-buffered saline (PBS) and analyzed using the ZetaView Particle Metrix system (Ammersee, Germany) according to the manufacturer’s protocols.

Exosomes were labeled with the fluorescent dye using the PKH67-Membrane EVs Labeling & Purification Kit (Yanjiang Bio, Shanghai, China). The TIMP1 gene in gefitinib-resistant LA cells was tagged with red fluorescent protein (RFP). Briefly, 20 µL of diluted PKH67 dye was added to exosomes resuspended in PBS, followed by incubation at 25 ℃ for 3 min. The labeled exosomes were collected by centrifugation, and the cells were further cultured in fresh medium. The samples were fixed with paraformaldehyde (4%) and observed using a STELLARIS 5 fluorescence microscope (Leica, Wetzlar, Germany).

qRT-PCR

Total RNAs were extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Genomic DNA was removed by treating RNA samples with gDNA Eraser (TaKaRa, Liaoning, China). Reverse transcription of total RNA was performed using SuperScript IV (ThermoFisher). qRT-PCR was conducted with the One Step SYBR^® PrimeScript RT-PCR Kit (TaKaRa) on a ProFlex™ PCR system (ThermoFisher). The PCR primer sequences are provided below.

TIMP1:

Forward: 3’-CTTCTGCAATTCCGACCTCGT-5’;

Reverse: 3’-ACGCTGGTATAAGGTGGTCTG-5’.

CD74:

Forward: 3’-GATGACCAGCGCGACCTTATC-5’;

Reverse: 3’-GTGACTGTCAGTTTGTCCAGC-5’.

MIF:

Forward: 3’-CCACGGGGATTGTCAGTGAAG − 5’;

Reverse: 3’-CTGTGCAGGTTTGTCTGTTCC-5’.

GAPDH:

Forward: 3’-ACACCATGTATTCCGGGTCAAT-5’;

Reverse: 3’-GGGCACGAAGGCTCATCATT-5’.

GAPDH is the internal control.

Enzyme-linked immunosorbent assay (ELISA)

TIMP1 and MIF ELISA kits were obtained from Biovision (California, USA), and the assays were performed according to the manufacturer’s protocols.

Transwell

The migration and invasion capacities of LA cells were evaluated using Transwell assays. The Transwell system employed iP-TEC^® Cell Transport Containers (Whatman, Shanghai, China). Following centrifugation and resuspension, cells were seeded into the upper chamber of the Transwell device. The upper chamber was filled with serum-free culture medium, whereas the lower chamber contained complete culture medium. Following 24 h cell culture, cells were fixed with 4% paraformaldehyde (Merck, Darmstadt, Germany) for 10 min, then stained with 0.5% crystal violet (Macklin, Shanghai, China). After washing the membrane, cell counting was performed using an Olympus microscope. For invasion assays, the upper chamber of the Transwell device was pre-coated with Matrigel (Merck), with all other steps identical to the migration detection protocol.

CCK-8

Cell viability was assessed using the (Beyotime) according to the manufacturer’s instructions. Transfected LA cells were incubated with gefitinib (0, 5, 10, 15, or 20 µM). Afterwards, 10 µL of CCK-8 solution in 100 µL of DMEM medium supplemented with FBS were added to the LA cells and incubated for 4 h. Absorbance was measured at 450 nm.

Plate colony formation assay

LA cells were diluted and seeded into 6-well plates at a density of 1000 cells per well. Plates were incubated in a humidified incubator (37 °C, 5% CO₂) until visible cell colonies formed. Culture medium was aspirated, and cells were fixed with 2 mL of methanol for 30 min. Following fixation, colonies were stained with crystal violet for 3 min, imaged under a microscope, and counted.

Flow cytometry apoptosis assay

Pyroptosis was detected using the Annexin V-eGFP/PI Kit (Genenode, Wuhan, China). Cells were washed with PBS, and 100 µL of cell suspension was transferred into a 5 mL flow tube. Following the addition of 5 µL Annexin V-eGFP, samples were incubated at room temperature for 5 min in the dark. Subsequently, 10 µL PI and 400 µL PBS were added, and samples were analyzed by FACSVERSE flow cytometry (BD Biosciences, CA, USA). Data were acquired and analyzed using Flowjo software (BD Biosciences).

Western blot

Cells and mouse tissues were lysed using RIPA Buffer Concentrate (Cayman Chemical, State of Michigan, USA). Total protein extracts were separated by SDS-PAGE under constant pressure for 120 min. Proteins were then electrophoretically transferred to an Immobilon-E-PVDF membrane (Merck, Darmstadt, Germany). The membrane was incubated with primary antibodies at 4 ℃ overnight (12 h), followed by secondary antibody incubation for 2 h. Protein bands were visualized using enhanced chemiluminescence (ECL) detection. The antibodies utilized in our study are detailed below: anti-Caspase-3 antibody (ab184787, Abcam, Cambridge, UK), anti-Caspase-9 antibody (ab184786, Abcam), anti-TIMP1 antibody (ab179580, Abcam), anti-CD74 antibody (ab215898, Abcam), anti-MIF antibody (ab187064, Abcam), and anti-GADPH antibody (ab9485, Abcam).

Co-immunoprecipitation (Co-IP)

Co-IP was performed using Pierce’s classic magnetic bead IP/Co-IP kit (ThermoFisher) to assess the interaction between TIMP1 and CD74, followed by Western blot analysis. Cells were lysed using Co-IP lysis buffer, and cell lysates were incubated with specific antibodies overnight at 4 ℃ to form immunoprecipitation complexes. Pre-treated protein A/G beads were then added to each tube and incubated with the complexes at room temperature for 1 h. The supernatant containing the target antigen was collected using a magnetic separator and subjected to Western blot analysis.

Pull-down

A protein pull-down assay was performed using the Pierce™ GST Protein Interaction Pull Down Kit (Thermo Fisher). Biotin-labeled CD74 (GTS-CD74) and biotin-labeled TIMP1 (TIMP1-His) were mixed, and 50 mM Binding Buffer was added. The protein mixture was co-incubated at 4 ℃ for 2.5 h, following by centrifugation to collect the pellet. The product was analyzed by Western blot.

Bioinformatic analysis

The GSE5060 dataset (subseries GSE5056) was obtain from the Gene Expression Omnibus (GEO) database. Differential expression analysis of TIMP1-related genes were performed using the limma R package, with data normalization conducted via GeneSpring version 6.2 software (Agilent Technologies). Abnormally expressed genes from GSE5056 were visualized using heat maps and volcano plots generated by the Gene Set Cancer Analysis tool (http://bioinformatics.psb.ugent.be/webtools/Venn/).

Animal model

Subcutaneous xenografts of drug-resistant LA (A549/Ge cells) were established in 6-8-week-old female BALB/c nude mice (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) according to a previously described protocol. The mice were randomly divided into 5 groups (n = 6 per group), including PBS, M2-CM, M2-exo, M2-CM depleted, and M2-CM GW4869 groups. All mice were sacrificed by cervical dislocation at 28th day after the subcutaneous injections of tumor cells. All animal experiments were performed according to ARRIVE guidelines and approved by the Animal Care Ethics Committee of The Fourth Hospital of Hebei Medical University. And all methods were carried out in accordance with relevant guidelines and regulations.

Statistical analysis

Data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using Student’s t-test for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple-group comparisons. Progression-free survival (PFS) curves were constructed and analyzed using Kaplan-Meier (K-M) method. All experiments were independently repeated three times. Statistical significance was defined as a p-value < 0.05.

Results

M2 macrophages inhibit the proliferation of LA cells

To explore the mechanism by which macrophages influence LA, bioinformatics analyses were performed to assess the expression of macrophage markers and related factors in clinical LA from public databases. First, immunohistochemical expression of the M2 macrophage marker mannose receptor, cluster of differentiation 206 (CD206) and cluster of differentiation 68 (CD68) was evaluated in clinical LA samples using The Human Protein Atlas database. As shown in Figure S1A, immunohistochemical staining with CD206 antibody demonstrated higher positivity in the cancer tissue of LA patient 4553 than in adjacent normal tissues (Figure S1A). Similarly, CD68 antibody staining revealed increased cellular positivity in the cancer tissue of LA patient 1907 compared to adjacent normal tissues (Figure S1B). Subsequently, the expression of M1 macrophage markers (intracellular interferon regulatory factor 5 (IRF5) and tumor necrosis factor-alpha (TNF-α)) and M2 macrophage markers (arginase-1 (Arg1) and intracellular interferon regulatory factor 4 (IRF4)) in clinical samples was evaluated using the Gene Set Cancer Analysis database. Compared with IRF5 and TNF-α, Arg1 and IRF4 showed more significant abnormal expression (Fig. 1A-D). Finally, the effect of M2 macrophages on drug resistance in LA cell lines were investigated by establishing gefitinib-resistant cell lines. Compared with parental A549 and PC9 cells, the viability of gefitinib-resistant A549/Ge and PC9/Ge cell lines was significantly increased in the presence of gefitinib. However, the presence of M2 macrophages suppressed the viability of A549/Ge and PC9/Ge cell lines (Figs. 1E-F). Collectively, these findings demonstrate that macrophages, particularly the M2 subtype, are significantly involved in the gefitinib resistance process of LA.

Fig. 1 [Images not available. See PDF.]

M2 macrophages inhibit the activity of drug-resistant LA cells in gefitinib medium. (A-D) Markers of M1 macrophages (IRF5 and TNF-α) The expression of M2 macrophage markers (Arg1 and IRF4) in clinical samples. (E-F) CCK-8 was used to detect the cell viability of A549, PC9, and their gefitinib-resistant cells in gefitinib medium. ***P < 0.001.

Effects of M2 macrophage-derived exosomes on proliferation of gefitinib resistant A549/Ge and PC9/Ge cells

To elucidate the specific role of M2 macrophages in the gefitinib resistance mechanism of LA cells, the effect of M2 macrophage-derived exosomes on lung cancer cells was investigated. First, monocyte-derived macrophages were stimulated with M-CSF. Inverted microscope analysis showed the presence of spindle-shaped cells, confirming successful induction of M2 macrophages (Fig. 2A). Subsequently, exosomes were isolated from M2 macrophages and characterized using TEM, NTA, and Western blot. TEM imaging revealed the presence of double-layered circular vesicles (Fig. 2B), a morphological hallmark of exosomes. NTA analysis showed that the vesicles had a diameter of approximately 100 nm (Fig. 2C), falling within the typical size range for exosome identification. Western blot analysis confirmed the expression of exosome-specific marker proteins cluster of differentiation 63 (CD63), cluster of differentiation 81 (CD81), and TSG101 in the isolated exosomes, whereas Calnexin showed negative expression (Fig. 2D). To investigate the interaction between M2 macrophage-derived exosomes and LA cells, exosomes were labeled with PKH67 and their uptake by A549/Ge and PC9/Ge cells was visualized. Immunofluorescence analysis showed PKH67-positive signals within A549/Ge and PC9/Ge cells, indicating that these cells internalize exosomes via endocytosis (Fig. 2E). Finally, we established culture systems for M2 macrophages (M2-CM) derived from gefitinib-resistant LA cells, M2 macrophage-derived exosomes (M2-exo), exosome-depleted M2 macrophages, and M2-CM treated with the exosome inhibitor GW4896 (M2-CM GW4896). The aforementioned cell lines were co-cultured with A549/Ge and PC9/Ge cells. As shown in Fig. 2E, the number of gefitinib-resistant LA cells was significantly reduced in the M2-CM and M2-exo groups compared with the control group. By contrast, the number of gefitinib-resistant LA cells was significantly higher in the exosome-depleted M2 macrophages and M2-CM GW4896 groups than in the M2-CM and M2-exo groups. These results suggest that exosomes derived from M2 macrophage inhibit the proliferation of gefitinib-resistant LA cells (Fig. 2F). Next, the in vivo role of M2-exo was investigated using a tumor-bearing mouse model. Mice were administered with different groups, and tumor volumes were measured weekly (Fig. 2G-H). Results showed that M2-CM and M2-exo significantly inhibited tumor growth compared to controls, but the tumor growth was significantly higher in the exosome-depleted M2 macrophages and M2-CM GW4896 groups than in the M2-CM and M2-exo groups (Fig. 2G-H). No significant changes in body weight were observed across all treatment groups (Fig. 2I). Collectively, these findings demonstrate that M2 macrophage-derived exosomes promote the restoration of gefitinib sensitivity in gefitinib-resistant LA cells.

Fig. 2 [Images not available. See PDF.]

Collection and identification of exosomes secreted by tumor associated macrophages. (A) M2 cells under inverted microscope (400 ×, Scale bars, 50 mm). (B) TEM imaging of separated extracellular vesicles (20000 ×; Scale bars, 100 nm). (C) Measure the parameters of extracellular vesicles through NTA analysis. (D) Western detection of the expression of extracellular vesicle marker proteins CD81, CD63, TSG101, and Calnexin. (E) PKH67 fluorescent labeling was performed to determine the level of exosome uptake by A549/Ge and PC9/Ge cells. (F) Detection of proliferation ability of LA cell lines A549 and PC9 by colony formation assay in gefitinib medium. (G) Tumor volume changes in mice from 7–28 days. (H) Statistical plot of tumor weights in mice. (I) Graph of body weight changes in mice from 7–28 days. *** P < 0.001, ^@@@P < 0.001, ^###P < 0.001.

TIMP1 localization in M2 macrophage-derived exosome of LA

To explore the specific molecular mechanism by which M2 macrophage-derived exosomes influence LA cells, pan-cancer analysis of TIMP1 was performed using the GSCA online analysis platform. TIMP1 was found to be overexpressed in multiple cancer types, including Bladder Urothelial Carcinoma (BLCA), Breast invasive carcinoma (BRCA), Cholangiocarcinoma (CHOL), Colon adenocarcinoma (COAD), Esophageal carcinoma (ESCA), Glioblastoma multiforme (GBM), Head and Neck squamous cell carcinoma (HNSC), Chromophobe (KICH), Kidney renal clear cell carcinoma (KIRC), Kidney renal papillary cell carcinoma (KIRP), Lung adenocarcinoma (LUAD), Lung squamous cell carcinoma (LUSC), Rectum adenocarcinoma (READ), Sarcoma(SARC), Stomach adenocarcinoma (STAD), Thyroid carcinoma (THCA), and Thymoma (THYM) (Fig. 3A). Analysis of the GSE5056 dataset identified 265 differentially expressed genes (Fig. 3B). A heatmap revealed overexpression of TIMP1 and SLC23A2 in early-stage lung cancer patients (Fig. 3C). Kaplan-Meier survival analysis showed that aberrant TIMP1 expression was associated with reduced survival in LA patients (Fig. 3D). Exosome staining experiments confirmed TIMP1 enrichment in M2 macrophage-derived exosomes from LA samples (Fig. 3E).

Fig. 3 [Images not available. See PDF.]

TIMP1 is an important protein in M2 macrophages secreting exosomes. (A) Pan-cancer analysis of TIMP1. (B-C) Abnormal expression genes in the GSE5056 database. (D) The Kaplan-Meier survival curve was used for TIMP1 survival analysis. (E) Observation of the distribution of exosomes and TIMP1 in LA cells using laser confocal microscopy.

Low expression of TIMP1 in M2 macrophage-derived exosome inhibits the proliferation of gefitinib-resistant LA cells

To investigate the correlation between TIMP1 dysregulation and gefitinib resistance in LA, we first conducted clinical validation. Serum exosomes were extracted from gefitinib-sensitive and -resistant patients. qRT-PCR analysis showed that TIMP1 expression in serum exosomes from the resistant group was significantly higher than that in the sensitive group (Fig. 4A). In vitro validation experiments were then performed. ELISA analysis revealed significant overexpression of TIMP1 in the supernatant of gefitinib-containing culture medium (Fig. 4B). qRT-PCR results showed that TIMP1 expression was markedly upregulated in gefitinib-resistant A549/Ge and PC9/Ge cell lines compared to parental cells (Fig. 4C). Subsequently, TIMP1-knockdown and overexpressed A549/Ge and PC9/Ge cells were successfully generated (Fig. 4D). CCK-8 assay revealed that the IC50 values of gefitinib in A549/Ge-sh-TIMP1 and PC9/Ge-sh-TIMP1 cells were significantly lower than those in parental cells, indicating that TIMP1 knockdown suppressed gefitinib resistance in A549/Ge and PC9/Ge cells (Figs. 4E). Flow cytometry and Western blot analyses demonstrated that M2 macrophage-derived exosomes suppressed apoptosis in gefitinib-resistant LA cells, whereas TIMP1 knockdown reversed this effect (Fig. 4F and G). CCK-8 assays confirmed that TIMP1 knockdown abrogated the pro-proliferative effect of M2 macrophage exosomes (Fig. 4H). Finally, the proliferation, migration, and invasion capacities of the sh-TIMP1 LA resistant cell lines were evaluated using plate colony formation and Transwell assays. The results demonstrated that TIMP1 knockdown suppressed M2 tumor-associated macrophage exosome-promoted proliferation (Fig. 5A), migration (Fig. 5B), and invasion (Fig. 5C) of lung cancer cells. Collectively, these findings indicate that M2 macrophage-derived exosomes enhance gefitinib resistance in LA through TIMP1-mediated mechanisms.

Fig. 4 [Images not available. See PDF.]

TIMP1 in exosomes secreted by M2 macrophages is associated with gefitinib resistance in LA. (A) qRT-PCR analysis of TIMP1 mRNA levels in gefitinib-resistant LA clinical samples. (B) ELISA was used to analyze the expression levels of TIMP1 in A549 and PC9 cells in both conventional (control group) and gefitinib media (gefitinib group). (C) qRT-PCR was used to analyze the expression levels of TIMP1 in different cells. (D) Successful construction of sh-TIMP1 and lv-TIMP1 resistant cells. (E) CCK-8 assays were performed to determine the IC50 of gefitinib in gefitinib resistant cells after knockdown of TIMP1. (F) Flow cytometry was used to detect the level of cell apoptosis. (G) Western blot was used to detect the expression levels of caspase-3 and caspase-9 in gefitinib medium. (H) CCK-8 was used to detect the cell viability of A549, PC9, and their gefitinib-resistant cells in gefitinib medium. ***P < 0.001;^@@@P < 0.001.

Fig. 5 [Images not available. See PDF.]

M2 macrophage exosomes regulate lung cancer proliferation, migration, and invasion through TIMP1. (A) Detection of proliferation ability of LA cell lines A549 and PC9 by colony formation assay in gefitinib medium. Transwell detected the migration ability (B) and invasion ability (C) of A549 and PC9 cell lines in gefitinib medium. ***P < 0.001;^@@@P < 0.001.

CD74 is a targeted protein of TIMP1

To investigate the specific regulatory mechanisms of TIMP1 in LA cell resistance, the interaction between TIMP1 and CD74 was examined using co-IP and pull-down assays. Both TIMP1 and CD74 were detected in the Input group of both assays, whereas no bands were observed in the IgG group. In the TIMP1 group, distinct bands corresponding to both TIMP1 and CD74 were identified, indicating a direct interaction between the two proteins (Fig. 6A). Similarly, pull-down assays confirmed the interaction between TIMP1 and CD74 (Fig. 6B). To explore the correlation between TIMP1 and CD74 in gefitinib-resistant LA clinical samples, qRT-PCR analysis was performed, revealing that CD74 expression in serum exosomes from the resistant group was significantly lower than that in the sensitive group (Fig. 6C). Moreover, we found a negative correlation between the expression levels of TIMP1 and CD74 in gefitinib-resistant LA clinical samples (Fig. 6D). In vitro validation experiments were subsequently conducted. qRT-PCR analysis showed that CD74 expression was significantly downregulated in gefitinib-resistant A549/Ge and PC9/Ge cell lines compared to parental cells (Fig. 6E). Subsequently, CD74-overexpressing A549/Ge and PC9/Ge cell lines were successfully established (Fig. 6F).

Fig. 6 [Images not available. See PDF.]

The binding effect between TIMP1 and CD74. Co-IP (A) and pull-down (B) were used to detect the binding of TIMP1 to CD74. (C) qRT-PCR analysis of CD74 mRNA levels in gefitinib-resistant LA clinical samples. (D) The correlation between the expression of CD74 and TIMP1 in gefitinib-resistant LA clinical samples was analyzed using Pearson’s correlation coefficient. (E) qRT-PCR was used to analyze the expression levels of CD74 in different cells. (F) Successful construction of lv-CD74 resistant cells. ***P < 0.001.

TIMP1 affects the proliferation, migration, and invasion of LA cells through CD74 and PI3K/AKT pathway

Plate colony formation and Transwell assays were used to evaluate the proliferation, migration, and invasion capacities of lv-CD74 LA-resistant cell lines. The results indicate that CD74 overexpression inhibits the proliferation, migration, and invasion of gefitinib-resistant LA cells. The suppressive effect of CD74 was further enhanced by the addition of M2 macrophage-derived exosomes. Conversely, TIMP1 overexpression promoted the proliferation, migration, and invasion of gefitinib-resistant LA cells (Fig. 7A-C). The PI3K/AKT signaling pathway is central to EGFR-mediated regulation of cell proliferation, survival, and metastasis. To investigate the mechanistic basis of TIMP1-CD74-mediated acquired resistance to gefitinib, we assessed pathway activation via phosphorylation status. Western blot analysis revealed that CD74 overexpression significantly attenuated PI3K/AKT activation, as evidenced by reduced phosphorylation of key pathway components, in gefitinib-resistant LA cells. The suppressive effect of CD74 on PI3K/AKT activation was potentiated by M2 macrophage-derived exosomes. In contrast, TIMP1 overexpression rescued PI3K/AKT signaling (Fig. 7D). Analogously, CD74 overexpression upregulated apoptotic proteins (caspase-3 and caspase-9) in LA cells, an effect abrogated by TIMP1 overexpression (Fig. 7D). Collectively, these findings identify CD74 as a direct target of TIMP1, through which TIMP1 activates the PI3K/AKT signaling pathway to promote the progression of gefitinib-resistant LA cells.

Fig. 7 [Images not available. See PDF.]

M2 macrophage exosomes regulate lung cancer proliferation, migration, and invasion through CD74. (A) Detection of proliferation ability of LA cell lines A549 and PC9 by CCK-8 in gefitinib medium. Transwell detected the migration ability (B) and invasion ability (C) of A549 and PC9 cell lines in gefitinib medium. (D) Western blot was used to detect the levels of phosphorylated PI3K and AKT proteins, as well as the expression levels of caspase-3 and caspase-9 in gefitinib culture medium. **P < 0.01; ***P < 0.001; ^@@@P < 0.001.

TIMP1 affects the proliferation of LA cells through MIF/CD74

To further explore the regulatory role of TIMP1 targeting CD74 in LA resistance, the CD74 target gene MIF was examined. Using qRT-PCR, we first analyzed the mRNA levels of MIF in serum exosomes from gefitinib-sensitive and -resistant patients. The results demonstrated that MIF expression was significantly higher in the resistant group than in the sensitive group (Fig. 8A). Additionally, a negative correlation was observed between CD74 and MIF expression in gefitinib-resistant LA clinical samples, whereas TIMP1 expression was positively correlated with MIF expression in the same cohort (Fig. 8B-C). ELISA analysis revealed significant overexpression of MIF in the supernatant of gefitinib-containing culture medium (Fig. 8D). qRT-PCR results showed that MIF expression was markedly upregulated in gefitinib-resistant A549/Ge and PC9/Ge cell lines compared to parental cells (Fig. 8E). Additionally, qRT-PCR analysis showed that MIF expression was significantly upregulated in TIMP1-overexpressing cells and markedly downregulated in TIMP1-knockdown cells (Fig. 8F). Furthermore, co-IP assays were performed to investigate the binding of TIMP1 to the MIF/CD74 complex. The results demonstrated enhanced interaction between CD74 and MIF in PC9/Ge cells with TIMP1 overexpression (Fig. 8G). As shown in Fig. 8D, the interaction between CD74 and MIF was reduced in TIMP1-knockdown cells, whereas this binding was enhanced in TIMP1-overexpressing cells (Fig. 8H). Collectively, these results indicate that M2 macrophage-derived exosomes modulate gefitinib resistance in LA cells by targeting the TIMP1/MIF/CD74 axis.

Fig. 8 [Images not available. See PDF.]

TIMP1 in exosomes affects the binding between CD74 and MIF. (A) qRT-PCR analysis of MIF mRNA levels in gefitinib-resistant LA clinical samples. (B) The correlation between the expression of CD74 and MIF in gefitinib-resistant LA clinical samples was analyzed using Pearson’s correlation coefficient. (C) The correlation between the expression of TIMP1 and MIF in gefitinib-resistant LA clinical samples was analyzed using Pearson’s correlation coefficient. (D) ELISA was used to analyze the expression levels of MIF in A549 and PC9 cells in both conventional (control group) and gefitinib media (gefitinib group). (E) qRT-PCR was used to analyze the expression levels of MIF in different cells in gefitinib medium. (F) qRT-PCR was used to analyze the expression levels of MIF in cells transfected with sh-TIMP1 or lv-TIMP1. (G-H) Co-IP was used to detect the binding of CD74 and MIF. ***P < 0.001.

Discussions

Macrophages have emerged as key regulators of tumor progression, with accumulating evidence highlighting the critical role of M2 macrophages in shaping the tumor microenvironment. M2-polarized macrophages have been shown to suppress anti-tumor immune responses and promote tumor progression by modulating angiogenic factors and extracellular matrix remodeling²¹. Concomitantly, growing research has implicated M2 macrophages in mediating tumor resistance to gefitinib, a phenomenon closely linked to altered chemosensitivity and disease progression in LA^22,23.

Clinical studies have demonstrated a significant correlation between M2 macrophage infiltration and tumor cell invasiveness in lung cancer patients²⁴. M2 macrophages exert pro-tumor effects by modulating immune homeostasis in the lung cancer microenvironment, with their elevated abundance and diffuse distribution particularly evident in patients with LA. Tan et al. demonstrated that M2 macrophage polarization promotes ovarian cancer progression²⁵while Sungu et al. revealed that M2 macrophages enhance breast tumor proliferation and mediate drug resistance²⁶. Jiang et al. reported that M2 macrophages facilitate colorectal cancer cell progression under hypoxic conditions²⁷. Consistent with previous findings, our study confirms that macrophages, particularly the M2 subtype, are crucially involved in LA pathogenesis.

Exosomes are small (≈ 100 nm diameter), bilayered membranous vesicles actively secreted by various cell types²⁸. These nanovesicles are cargo-rich carriers of regulatory molecules that traffic between cells, modulating immune surveillance and tumor progression²⁹. Notably, M2 macrophage-derived exosomes have been shown to promote malignant tumor growth and angiogenesis³⁰positioning them as promising targets for novel antitumor therapeutic strategies. Xu et al.’s demonstrated that M2 macrophage-derived exosomal LINC01001 promotes NSCLC progression by regulating METTL3³¹. Guan et al. confirmed that M2 macrophage-derived exosomes carrying miR-1911-5p enhance cell migration and invasion of LA cells by downregulating CELF2 to activate ZBTB4³². Building on these findings, our study reveals that M2 macrophage-derived exosomes suppress the progression of gefitinib-resistant LA cells.

TIMP1, a ubiquitously expressed glycoprotein, shows dysregulated expression in most tumors¹⁴. Dantas et al. identified TIMP1 as a potential biomarker for early detection and prognosis of lung cancer³³. Pietrzak et al. further confirmed the correlation between TIMP1 expression, therapeutic response, and survival outcomes in NSCLC patients³⁴. Accumulating evidence suggests that TIMP1 may contribute to drug resistance across multiple disease contexts³⁵. Guccini et al. demonstrated that TIMP1 deficiency is associated with paclitaxel resistance in prostate cancer patients¹⁴. Wang et al. further confirmed the correlation between TIMP1 expression and sorafenib resistance in colorectal cancer³⁶. Gefitinib, a pyrimidine-derived tyrosine kinase inhibitor, has been implicated in TIMP1-mediated resistance mechanisms in preclinical studies³⁷. Notably, TIMP1 has been identified as a key cargo of tumor-derived exosomes, highlighting its potential role in intercellular communication during drug resistance³⁸. Hu et al. confirmed the high expression of TIMP1 in extracellular vesicles derived from human dermal fibroblasts^39,40. Lin et al. demonstrated TIMP1 overexpression in exosomes secreted by lung cancer and colorectal cancer cells⁴¹. This study for the first time reveals that TIMP1 packaged in M2 macrophage-derived exosomes contributes to the gefitinib resistance of LA cells. Ebert et al. recently predicted an interaction between TIMP1 and CD74 using sequence alignment and computational protein docking analyses⁴². This predicted interaction was shown to modulate the binding affinity between CD74 and MIF. This study experimentally validates computational predictions in LA, demonstrating thatTIMP1 dysregulation directly modulates the CD74-MIF interaction, with potential activation of the PI3K/AKT signaling pathway. The CD74-MIF interaction has been implicated in lung cancer pathogenesis, with prior studies showing its role in tumor progression^43,44. De Azevedo et al. demonstrated that the MIF-CD74 axis mediates drug resistance in melanoma⁴⁵. This study for the first time reveals that TIMP1 expressed in M2 macrophage-derived exosomes modulates the MIF-CD74 interaction in LA, a mechanism directly linked to gefitinib resistance.

Conclusion

Collectively, this study elucidates the regulatory role of M2 macrophage-derived exosomes in LA progression. Specifically, TIMP1 depletion in M2 macrophage exosomes disrupts the CD74-MIF interaction, thereby suppressing proliferation and migration of gefitinib-resistant LA cells. These findings provide mechanistic insights into the role of M2 macrophage exosomes in LA and identify potential therapeutic targets for clinical intervention in gefitinib-resistant LA.

Author contributions

Wenxia Hu contributed to the study concept and design; Jingcui Peng performed the main experiments and wrote and revised the manuscript; Yan Zhang, Bin Li, Xin He and Cuimin Ding partly contributed to the experiments, data analysis, and interpretation. All authors read and approved the final manuscript.

Funding

This study was supported by Medical Science Research Project of Hebei (No. 20150777).

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study was approved by the institutional review board of The Fourth Hospital of Hebei Medical University (approval number: [FH-2024-H-0316]), and written informed consent was obtained from all participants.

Research ethics and patient consent

The present study does not involve human or animal experiments.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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