Graphical Abstract [50]graphic file with name 12575_2025_295_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12575-025-00295-0. Keywords: Lung cancer, Circulating tumor cell clusters, Metastasis, Osimertinib resistance, C-MET inhibitor Highlights λ Lung cancer CTC clusters resist osimertinib, driving treatment resistance.​. λ HGF/c-MET signaling activates in CTC clusters, revealing a resistance mechanism.​. λ Tivantinib, a c-MET inhibitor, outperforms HGF inhibitor SRI in suppressing CTC cluster survival.​. λ Tivantinib plus osimertinib exerts additive inhibition on CTC cluster survival and metastasis, offering a potential anti-metastasis strategy. Supplementary Information The online version contains supplementary material available at 10.1186/s12575-025-00295-0. Introduction Lung cancer ranks first in both incidence and mortality among malignant tumors worldwide [[51]1]. Surgical resection is the cornerstone of treatment for early-stage lung cancer patients; however, postoperative recurrence and metastasis remain the primary causes of mortality [[52]2]. Adjuvant therapies, including chemotherapy and molecular targeted therapy, have been shown to significantly extend disease-free survival (DFS) and overall survival (OS) [[53]3, [54]4]. Despite these advances, a substantial proportion of lung cancer patients experience disease progression within 1–3 years of adjuvant treatment, mainly attributed to treatment resistance [[55]5]. Therefore, elucidating the mechanisms underlying lung cancer metastasis and disease progression during adjuvant therapy is essential for improving patient outcomes. For non-small cell lung cancer (NSCLC) patients harboring EGFR gene mutations, EGFR-tyrosine kinase inhibitors (EGFR-TKIs) represent the standard-of-care treatment. Osimertinib, a third-generation EGFR-TKI, has demonstrated remarkable efficacy in prolonging DFS, progression-free survival (PFS), and OS for both early- and advanced-stage NSCLC patients with EGFR mutations [[56]4, [57]6]. Nevertheless, treatment resistance inevitably emerges, primarily due to EGFR gene mutations, activation of bypass signaling pathways, and phenotypic transformation of lung cancer cells. Among these, MET amplification or overexpression is a well-recognized mechanism of acquired resistance to osimertinib [[58]7]. Combination therapy with MET inhibitors and EGFR-TKIs has shown promise in overcoming MET-mediated resistance [[59]8], highlighting the importance of targeting alternative pathways.​ Tumor metastasis is facilitated by the dissemination of tumor cells from the primary site to distant organs. Circulating tumor cells (CTCs), regarded as the “seeds” of primary tumor dissemination, play a critical role in this process. The quantity and state of CTCs are strongly correlated with metastasis and prognosis in early-stage lung cancer patients. Detectable CTCs, particularly CTC clusters, are associated with poorer DFS and OS, regardless of the treatment stage (pre-surgery, post-surgery, or adjuvant therapy) [[60]9, [61]10]. This is largely due to the enhanced metastatic potential of CTC clusters [[62]11, [63]12]. Strategies aiming to dissociate CTC clusters into single cells or inhibit their survival may offer new approaches for preventing lung cancer metastasis [[64]13]. Additionally, CTCs serve as valuable biomarkers for predicting the response to osimertinib treatment and elucidating tumor characteristics and resistance mechanisms [[65]14]. Despite the established significance of CTCs in lung cancer progression, the role of CTC clusters in mediating resistance to EGFR-TKIs remains unclear. In this study, using a human lung adenocarcinoma-derived CTC line (CTC-TJH-01) established in our laboratory, we aimed to investigate whether CTC clusters contribute to EGFR-TKI resistance and to elucidate the underlying molecular mechanisms. Our findings revealed that CTC-TJH-01 cell clusters exhibit resistance to osimertinib, accompanied by activation of the HGF/c-MET pathway and downregulation of EGFR protein expression. Moreover, the c-MET inhibitor Tivantinib, either alone or in combination with osimertinib, effectively suppressed the survival and metastasis of CTC-TJH-01 cell clusters. These results suggest that CTC clusters may mediate resistance to EGFR-TKI treatment, and c-MET inhibitors could be potential therapeutic agents for targeting CTC clusters to inhibit lung cancer metastasis. Materials and methods Reagents and antibodies Osimertinib dimesylate (O981804), Tivantinib (T873788) and SRI (S881106) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd (Shanghai, China). Antibodies against EGFR, p-EGFR, HGF, c-MET, p-c-MET, Cleaved caspase-3 and Vimentin were obtained from Affinity Biosciences Pty Ltd (Cincinnati, OH, USA). Ki-67 and Survivin antibodies were procured from Proteintech (Wuhan, China). Cell culture The human lung adenocarcinoma circulating tumor cell line CTC-TJH-01 was isolated from the peripheral blood of a female patient with stage IIa lung adenocarcinoma and subsequently established as a stable passaging cell line in our laboratory ^[15, 16]. H1975 and A549 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). For cell cultivation, CTC-TJH-01 cells and A549 cells were cultured in F12K medium supplemented with 10% fetal bovine serum (FBS, Vivacell BIOSCIENCES, Germany) and 1% penicillin-streptomycin solution (Gibco Life Technologies, Carlsbad, CA, USA). H1975 cells were maintained in RPMI-1640 medium containing the same concentration of FBS and penicillin-streptomycin. All cell cultures were incubated at 37 °C in a humidified atmosphere with 5% CO[2]. To establish a CTC suspension culture system for generating cell clusters, ultra-low attachment culture dishes (Corning Cellgro, USA) were employed. Cells were seeded at a density of 1 × 10^5 cells per well in 6 - well ultra-low attachment plates and cultured for 48 h, during which time cell clusters spontaneously formed. Animals Twenty-four specific-pathogen-free (SPF) grade, six-week-old male NOD-SCID mice, weighing 18–22 g, were procured from Shanghai Bikai Keyi Biotechnology Co., Ltd. The mice were housed in the SPF-grade animal facility of the Experimental Animal Centre at the Shanghai Tenth People’s Hospital, where the environmental conditions were strictly controlled: room temperature ranged from 20 to 26 °C, humidity was maintained at 40% − 60%, and the animals had free access to autoclaved water and standard rodent chow. All animal experiments were conducted in strict accordance with the regulations of the Experimental Animal Ethics Committee of the Shanghai Tenth People’s Hospital, and the study protocol was approved (Ethics number: SHDSYY-2023-T7885), adhering to the ethical guidelines of the Basel Declaration. Cell viability analysis Logarithmically-growing CTC-TJH-01 and A549 cells were seeded into 96-well plates at a density of 3 × 10^3 cells per well. After cell seeding, the cells were treated with various concentrations of osimertinib, Tivantinib, and SRI for 48 h. Subsequently, 10 µL of CCK-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well, and the plates were incubated for an additional 2 h. Finally, the absorbance values were measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA), and the cell viability was calculated based on the absorbance data. LDH cytotoxicity assay Logarithmically-growing CTC-TJH-01 and A549 cells were seeded into 96-well plates or ultra-low attachment 96-well culture plates. Following cell seeding, the cells were treated with different concentrations of osimertinib, Tivantinib, and SRI for 48 h. Then, 120 µL of the supernatant from each well was carefully transferred to the corresponding well of a new 96-well plate. The LDH assay working solution was freshly prepared according to the manufacturer’s instructions, and 60 µL of the working solution was added to each well of the new plate. After gentle mixing, the plate was incubated at room temperature (approximately 25 °C) in the dark for 30 min. The absorbance was measured at 490 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) to quantify the released LDH, which reflects cell membrane damage and cytotoxicity. Calcein AM/EthD-1 fluorometric detection Logarithmically-growing CTC-TJH-01 and A549 cells were inoculated into 24-well plates or ultra-low attachment 24-well culture plates. After cell seeding, the cells were treated with various concentrations of osimertinib, Tivantinib, and SRI for 48 h. To assess cell viability and membrane integrity, 1 µL of Calcein AM (500×, CellBiolabs, Inc, USA) and 1 µL of EthD-1 (500×, CellBiolabs, Inc, USA) were added to each well of the 24-well plates. The plates were then incubated at 37 °C for 30–60 min. Subsequently, the cells were observed and imaged using a fluorescence microscope with appropriate excitation and emission filters. Calcein AM stains live cells with green fluorescence, while EthD-1 stains dead cells with red fluorescence, enabling visual assessment of cell viability. Cell apoptosis assay CTC-TJH-01 and A549 cells were seeded into the 6-well plate at a density of 1 × 10^5 cells per well. After cell attachment, the cells were treated with osimertinib, Tivantinib, or a combination of osimertinib and tivantinib for 48 h. Following treatment, the cells were harvested, washed twice with cold phosphate - buffered saline (PBS), and stained with Annexin V-PE and caspase 3-FITC (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocols. Briefly, the cells were resuspended in binding buffer, incubated with Annexin V-PE and caspase 3-FITC for 10 min in the dark at room temperature, and then analyzed by flow cytometry (BD FACSCalibur, BD Biosciences, San Jose, CA, USA). Transcriptomics analysis Briefly, CTC-TJH-01 cells were collected after adherent and suspension culture, then sent to Sinotech Genomics (Shanghai, China) for RNA sequencing. KEGG signaling pathway enrichment analysis was performed on genes with a fold change greater than 2. Real-time quantitative real time polymerase chain reaction (qRT-PCR) analysis Cells were collected, and total RNA was extracted using a total RNA extraction kit (TaKaRa, Japan). RNA concentration was determined, and total RNA was reverse-transcribed into cDNA using RNA reverse transcription reagents according to the manufacturer’s instructions (TaKaRa, Japan). The PCR system was prepared for amplification under the following conditions: predenaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. Relative mRNA expression was calculated using the 2^(-ΔΔCt) method. Primers were synthesized by Shanghai Generay Biotech Co., Ltd., and their sequences are listed in Table [66]1. Table 1. Primers used for RT-qPCR analysis Gene Primers Sequence Forword (5’−3’) Reverse (5’−3’) EGFR AAGAAGACATGGACGACG CAGAGGCTGATTGTGATAGAC HGF TGGTAAAGGACGCAGCTACA GCGTACCTCTGGATTGCTTG c-MET CCCACCCTTTGTTCAGTGTG AGTCAAGGTGCAGCTCTCAT GAPDH CCCATCACCATCTTCCAG CATCACGCCACAGTTTCC [67]Open in a new tab Western blot analysis After treating cells with different drugs, the medium was discarded, and cells were washed three times with pre-cooled PBS. Protein lysate was added, and cells were incubated on ice for 15 min. Cells were scraped, collected, and centrifuged at 12,000 rpm for 15 min; the supernatant was harvested for protein quantification. Proteins were separated by SDS-PAGE and transferred to PVDF membranes, which were then blocked at room temperature for 2 h. Membranes were washed three times with PBST (5 min per wash), incubated with the corresponding primary antibodies (1∶1,000) at 4 °C overnight, and then washed again three times with PBST. Horseradish peroxidase (HRP)-conjugated secondary antibody (rabbit IgG) was added, and membranes were incubated at room temperature for 1 h, followed by three additional washes with PBST. Finally, ECL chemiluminescence reagent (Bio-Rad, CA, USA) was applied for development, and images were captured using a chemiluminescence imaging system. Lung metastasis assays After digestion and centrifugation, CTC-TJH-01 cells were resuspended and cultured for 24 h. The cell density was adjusted to 1 × 10^6 cells/mL. NOD-SCID mice were allowed a 1-week adaptation period, after which 0.1 ml of the cell suspension was injected via the tail vein to establish the lung cancer metastasis model. Subsequently, the 24 mice were randomly divided into four groups of six mice each. The next day, the mice in each group received intraperitoneal injections of either osimertinib (20 mg/kg/d), tivantinib (2 mg/kg/d), a combination of osimertinib and tivantinib, or an equivalent volume of normal saline. Treatments were administered three times a week for 11 weeks. At the end of the treatment period, the mice were euthanized, and their lungs were harvested. Lung metastatic nodules were visually inspected, counted, and photographed using an anatomical microscope. H&E staining and immunohistochemistry were performed on the lung samples. Hematoxylin-eosin (H&E) staining Lung metastasis sections were stained following the instructions of the H&E staining kit. The lung metastatic tissues were first fixed with paraformaldehyde, decalcified with EDTA, dehydrated through a series of gradient-concentration alcohols, and then made transparent with xylene. Subsequently, the tissues were embedded in paraffin and cut into 4-µm-thick paraffin sections. The paraffin sections were deparaffinized with xylene, rehydrated through gradient-concentration alcohols, stained with hematoxylin, and differentiated with hydrochloric acid alcohol. After eosin staining, the sections were dehydrated again with gradient-concentration alcohols, made transparent with xylene, sealed with neutral tree gum, and then observed and photographed under a light microscope. Immunohistochemistry assays Paraffin-embedded metastatic tissue specimens were sectioned, and antigen retrieval was performed. The sections were then blocked with BSA and incubated with Vimentin, Ki-67, and cleaved caspase-3 antibodies at 4 °C overnight. After washing, the specimens were incubated with secondary antibodies, stained with 3,3’-Diaminobenzidine (DAB), counterstained with hematoxylin, sealed with neutral mounting medium, and observed under a microscope. The total number of cells and positive-staining cells in each image were counted separately using ImageJ software. Statistical analysis Statistical analyses were conducted using SPSS 23.0 software. All experiments were performed in triplicate (n = 3). Data are presented as the mean ± standard deviation (SD) or the standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used for comparisons among multiple groups. For pairwise comparisons, the Tukey post hoc test was applied when the variances were homogeneous, and Tamhane’s T2 post hoc test was used for data with heterogeneous variances. Statistical significance was set at *P < 0.05, **P < 0.01, and ***P < 0.001. Results Lung cancer CTC clusters exhibit resistance to osimertinib Targeted inhibition of CTC cluster survival represents a therapeutic strategy for effectively preventing and treating lung cancer metastasis [[68]17]. EGFR-TKIs are effective therapeutic agents for lung cancer patients with EGFR mutations. The CTC-TJH-01 cell line harbors a synonymous single nucleotide variant (Q787Q) in exon 20 of EGFR. We found that adherent single CTC-TJH-01 cells, similar to A549 cells with wild-type EGFR, exhibit higher resistance to osimertinib (Fig. [69]1A), with IC[50] values of 5.36 µM and 5.61 µM, respectively (Fig. [70]1A). Next, we treated single CTC-TJH-01 cells and suspended CTC-TJH-01 cell clusters with osimertinib at concentrations of 2.5 µM and 5 µM. Osimertinib inhibited the proliferation and survival of both single CTC-TJH-01 cells and CTC-TJH-01 cell clusters (Fig. [71]1B-D). Notably, suspended CTC-TJH-01 cell clusters displayed greater resistance to osimertinib compared to single CTC-TJH-01 cells (Fig. [72]1B-D). Similar results were observed in human lung adenocarcinoma A549 cells: suspended A549 cell clusters showed reduced sensitivity to osimertinib (Fig. [73]S1). Furthermore, the H1975 cell line—a human non-small cell lung cancer cell line harboring the T790M mutation—was sensitive to osimertinib under adherent culture conditions, but suspended H1975 cell clusters exhibited robust resistance, with IC[50] values of 1.11 µM and 9.94 µM, respectively (Fig. [74]S2). These findings indicate that CTC cluster formation enhances resistance to EGFR-TKI (osimertinib) therapy. Fig. 1. [75]Fig. 1 [76]Open in a new tab Lung cancer CTC clusters are resistant to osimertinib. A Adherent CTC-TJH-01 cells were treated with various concentrations of osimertinib for 48 h, and cell viability was measured using the CCK-8 assay (n = 3). B and C Adherent and suspended CTC-TJH-01 cells were treated with osimertinib at different concentrations (0, 2.5, 5 µM) for 48 h, and cell viability was measured by CCK-8 and LDH assays (n = 3). D Adherent and suspended CTC-TJH-01 cells were treated with osimertinib at different concentrations (0, 2.5, 5 µM) for 48 h, and cell viability was detected by Calcein AM/EthD-1 fluorometric analysis (n = 3). Scale bars, 100 μm. Data are presented as mean ± SD. Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001 Activation of the HGF/c-MET signaling pathway in lung cancer CTC clusters To investigate the mechanism underlying the enhanced osimertinib resistance of lung cancer CTC clusters, we analyzed transcriptome sequencing data comparing single CTC-TJH-01 cells and CTC-TJH-01 cell clusters. HGF expression was significantly upregulated in CTC-TJH-01 cell clusters (Fig. [77]2A). We hypothesized that lung cancer CTC clusters may activate the c-MET signaling pathway via autocrine or paracrine HGF secretion, thereby promoting their survival. We therefore examined the expression of HGF, c-MET, and EGFR at both the mRNA and protein levels in CTC-TJH-01 cell clusters. HGF mRNA was significantly upregulated, while c-MET mRNA remained unchanged (Fig. [78]2B). Interestingly, EGFR mRNA was also significantly downregulated in CTC-TJH-01 cell clusters (Fig. [79]2B). At the protein level, HGF and phosphorylated c-MET (p-c-MET) were significantly upregulated, whereas EGFR and phosphorylated EGFR (p-EGFR) were significantly downregulated (Fig. [80]2C). These results were recapitulated in A549 cell clusters (Fig. S3). Collectively, these data indicate that the EGFR signaling pathway is inactivated in lung cancer CTC clusters, while the HGF/c-MET signaling pathway is activated—likely contributing to their enhanced osimertinib resistance. Fig. 2. [81]Fig. 2 [82]Open in a new tab Activation of the HGF/c-MET signaling pathway in lung cancer CTC clusters. A Transcriptome analysis of differentially expressed genes involved in the focal adhesion pathway in CTC-TJH-01 cells cultured under adherent and suspension conditions. B and C Expression levels of EGFR, HGF, and c-MET mRNA and protein in CTC-TJH-01 cells under adherent and suspension culture conditions were analyzed by RT-qPCR and western blot (n = 3). Data are presented as mean ± SD. Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001 Tivantinib, a c-MET inhibitor, but not the HGF inhibitor SRI, effectively suppresses the survival of lung cancer CTC clusters Given the activation of the HGF/c-MET pathway in lung cancer CTC clusters, we hypothesized that inhibitors of this pathway may effectively eliminate CTC clusters. We therefore treated CTC-TJH-01 cell clusters with the HGF inhibitor SRI or the c-MET inhibitor tivantinib. Tivantinib inhibited CTC-TJH-01 cell proliferation in a dose-dependent manner (Fig. [83]3A), whereas SRI had no inhibitory effect (Fig. [84]3D). Compared to osimertinib, tivantinib at 0.5 µM and 1 µM significantly inhibited the survival of both single CTC-TJH-01 cells and CTC-TJH-01 cell clusters, with clusters exhibiting greater sensitivity to tivantinib (Fig. [85]3B-C). In contrast, SRI had no significant effect on the survival of CTC-TJH-01 cells or clusters (Fig. [86]3E-F). Similar results were observed in A549 cells: A549 clusters were more sensitive to tivantinib, while SRI had no effect on A549 cell or cluster proliferation/survival (Fig. S4). In addition, western blot analysis revealed that tivantinib significantly downregulated the protein expression of p-c-MET in CTC-TJH-01 cell clusters, whereas SRI had no significant effect (Fig. S5). These findings demonstrate that the c-MET inhibitor tivantinib, but not the HGF inhibitor SRI, exerts significant cytotoxic effects on lung cancer CTC clusters. Fig. 3. [87]Fig. 3 [88]Open in a new tab The c-MET inhibitor tivantinib, but not the HGF inhibitor SRI, effectively suppresses the survival of lung cancer CTC clusters. A and D Adherent CTC-TJH-01 cells were treated with various concentrations of tivantinib and SRI for 48 h, and cell viability was measured using the CCK-8 assay (n = 3). B and E Adherent and suspended CTC-TJH-01 cells were treated with tivantinib (0, 0.5, 1 µM) and SRI (0, 2.5, 5 µM) for 48 h, and cell viability was measured by LDH assay (n = 3). C and F Adherent and suspended CTC-TJH-01 cells were treated with tivantinib (0, 0.5, 1 µM) and SRI (0, 2.5, 5 µM) for 48 h, followed by detection of cell apoptosis via Calcein AM/EthD-1 fluorometric analysis (n = 3). Scale bars, 100 μm. Data are presented as mean ± SD. Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001 Tivantinib enhances the ability of osimertinib to restrain lung cancer CTC cluster survival Bypass pathway activation, such as the HGF/c-MET pathway, is a known mechanism of EGFR-TKI resistance in lung cancer [[89]18]. Given the activation of HGF/c-MET signaling in lung cancer CTC clusters, we investigated whether combined EGFR and c-MET inhibition could more effectively suppress CTC cluster survival. We treated CTC-TJH-01 cell clusters with tivantinib in combination with osimertinib. Both 5 µM osimertinib and 0.5 µM tivantinib significantly inhibited CTC-TJH-01 cluster survival (Fig. [90]4A-C) and upregulated cleaved caspase-3 expression while downregulating Survivin expression (Fig. [91]4D). Notably, the combination of tivantinib and osimertinib exerted a stronger inhibitory effect on CTC-TJH-01 cluster survival (Fig. [92]4A-D). These results were confirmed in A549 cell clusters (Fig. S6), indicating that tivantinib enhances the ability of osimertinib to suppress lung cancer CTC cluster survival. Fig. 4. [93]Fig. 4 [94]Open in a new tab Tivantinib enhances the ability of osimertinib to restrain the survival of lung cancer CTC clusters. A Cell viability was measured by LDH assay (n = 3) using 5 µM osimertinib and 0.5 µM tivantinib. B Cell apoptosis was detected by Calcein AM/EthD-1 fluorometric detection (n = 3) using 5 µM osimertinib and 0.5 µM tivantinib. Scale bars, 100 μm. C Cell apoptosis was measured by Annexin V-FITC/caspase-3 assay (n = 3) using 5 µM osimertinib and 0.5 µM tivantinib. D Protein expression levels of cleaved caspase-3 and survivin in CTC-TJH-01 clusters were determined by western blotting (n = 3) using 5 µM osimertinib and 0.5 µM tivantinib. Data are presented as mean ± SD. Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001 Tivantinib enhances the inhibitory effect of osimertinib on lung cancer CTC cluster metastasis To further explore whether tivantinib combined with osimertinib inhibits lung cancer metastasis by suppressing CTC cluster survival, we established a lung metastasis model via tail vein injection of CTC-TJH-01 cell clusters into NOD/SCID mice. Both tivantinib and osimertinib significantly reduced the number of lung metastatic nodules, with the combination treatment yielding the greatest reduction (Fig. [95]5A). Neither treatment affected mouse body weight (Fig. [96]5B). Lung metastasis burden was also significantly reduced in the tivantinib, osimertinib, and combination groups (Fig. [97]5C). In metastatic tumor tissues, Ki-67 and vimentin expression were significantly downregulated, while cleaved caspase-3 expression was significantly upregulated in all treatment groups (Fig. [98]5D). Taken together, these results demonstrate that tivantinib effectively suppresses lung cancer CTC cluster metastasis and enhances the antimetastatic effect of osimertinib when used in combination. Fig. 5. [99]Fig. 5 [100]Open in a new tab Tivantinib enhances the inhibitory effect of osimertinib on metastasis of lung cancer CTC clusters. A A lung metastasis model was established by tail vein injection of CTC-TJH-01 cell clusters into NOD-SCID mice. Mice were treated with osimertinib (20 mg/kg), tivantinib (2 mg/kg), or their combination via intraperitoneal injection for 11 weeks. Representative lung images and quantification of pulmonary metastatic nodules in each group (n = 6). B Body weights of NOD-SCID mice. C H&E staining was performed to observe the morphology of pulmonary metastatic nodules (0.5×, 4×, 20×) (n = 3). D Immunohistochemistry was used to detect the expression of vimentin, Ki-67, and cleaved caspase-3 in pulmonary metastatic nodules (n = 3). Scale bars, 100 μm. Data are presented as mean ± SD. Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001 Discussion In this study, we investigated the role of lung cancer CTC clusters in acquired resistance to EGFR-TKIs. Our findings demonstrate that the HGF/c-MET signaling pathway is activated in lung cancer CTC clusters, contributing to osimertinib resistance. Additionally, the c-MET inhibitor tivantinib enhances the efficacy of osimertinib, effectively suppressing the survival and metastasis of lung cancer CTC clusters. These results provide a novel therapeutic strategy for lung cancer patients with detectable CTC clusters and high metastatic risk. CTCs, often referred to as the “seeds” of tumor metastasis, are widely used as biomarkers for assessing metastatic risk, monitoring treatment efficacy, evaluating prognosis, and guiding therapy in early postoperative lung cancer patients [[101]10, [102]14, [103]19]. Accumulating evidence indicates that CTC clusters exhibit higher metastatic potential than single CTCs [[104]20], and patients with detectable CTC clusters in peripheral blood have shorter DFS and OS [[105]21, [106]22]. This may be attributed to the quiescent state of CTC clusters, their cancer stem cell-like properties, and intrinsic drug resistance [[107]23]. For example, Dashzeveg et al. found that breast cancer CTC clusters are quiescent and resistant to chemotherapy [[108]12], prompting efforts to prevent metastasis by dissociating CTC clusters into single cells [[109]13]. In our study, we utilized the human lung adenocarcinoma CTC line CTC-TJH-01, which harbors a synonymous single nucleotide variant (Q787Q) in exon 20 of EGFR [[110]15]. We also incorporated A549 (EGFR WT) and H1975 (EGFR L858R/T790M mutant) cells to examine whether osimertinib resistance in CTC clusters is contingent upon EGFR mutation status. We noted that both A549 cells (intrinsically resistant) and H1975 cells (initially sensitive) exhibited enhanced resistance when clustered. These findings indicate that CTC cluster-mediated osimertinib resistance is independent of EGFR genotype, likely through a universal adaptive mechanism. These results may partially account for the reduced survival and poorer prognosis observed in patients with detectable CTC clusters [[111]21, [112]22]. CTC clusters evade anoikis by activating survival pathways [[113]24]. Bypass pathway activation is a critical mechanism of acquired osimertinib resistance, including PI3K/AKT/mTOR pathway activation due to MET or HER2 amplification [[114]25, [115]26]. We therefore compared adherent single CTC-TJH-01 cells with suspended CTC-TJH-01 clusters, finding that HGF mRNA and protein expression were significantly upregulated in clusters, while EGFR mRNA and protein were significantly downregulated. These data suggest that lung cancer CTC clusters may activate c-MET signaling via autocrine or paracrine HGF secretion, driving osimertinib resistance. Additionally, EGFR pathway inactivation likely reduces cluster sensitivity to osimertinib. MET amplification and overexpression are key mechanisms of acquired osimertinib resistance [[116]27], occurring in 15–20% of NSCLC patients with osimertinib resistance [[117]28]. Studies have confirmed that MET inhibitors improve survival in NSCLC patients with MET amplification or overexpression [[118]29], and combining MET inhibitors with EGFR-TKIs can overcome resistance by co-targeting EGFR and MET signaling [[119]8, [120]30]. In our experiments, the HGF inhibitor SRI had no effect on lung cancer CTC cluster proliferation or survival, whereas the c-MET inhibitor tivantinib markedly suppressed cluster survival. The lack of efficacy of the HGF inhibitor SRI in CTC-TJH-01 cells likely arises from its mechanism of action: SRI primarily blocks HGF-mediated c-MET activation but does not directly inhibit ligand-independent c-MET signaling or downstream pathway activity. In contrast, Tivantinib, as a direct c-MET kinase inhibitor, more effectively reduces p-c-MET levels regardless of HGF stimulation, which aligns with its observed anti-proliferative effects [[121]31]. This suggests that lung cancer CTC clusters are more sensitive to c-MET inhibition. Consistent with this, Yap et al. reported that the c-MET inhibitor ARQ 197 reduces CTC counts in the peripheral blood of lung cancer patients [[122]32]. Furthermore, we found that tivantinib enhanced osimertinib-mediated inhibition of CTC cluster survival and significantly reduced the metastatic potential of clusters in mice, with concomitant upregulation of cleaved caspase-3 in both CTC clusters and metastatic tumor tissues. Conclusions In conclusion, our study demonstrates that lung cancer CTC clusters exhibit resistance to osimertinib. Mechanistically, the HGF/c-MET signaling pathway is activated in lung cancer CTC clusters, accompanied by downregulated EGFR protein expression. Notably, our findings reveal that the c-MET inhibitor tivantinib—rather than the HGF inhibitor SRI—plays a critical role in suppressing the survival of lung cancer CTC clusters. Furthermore, tivantinib enhances the ability of osimertinib to inhibit the survival and metastasis of lung cancer CTC clusters. These results highlight tivantinib as a promising therapeutic agent for combating metastasis driven by lung cancer CTC clusters. Supplementary Information [123]Supplementary Material 1.^ (1.9MB, docx) Acknowledgements