Anlotinib reverses osimertinib resistance via inhibiting epithelial-to-mesenchymal transition and angiogenesis in non-small cell lung cancer

Introduction

Non-small cell lung cancer (NSCLC), exhibiting a high incidence rate, poses a serious threat to human health^[1]. Currently, treatment strategies for advanced NSCLC have entered a phase dominated by targeted therapy^[2] and immunotherapy^[3]. The predominant gene mutations of NSCLC include EGFR mutations, ALK fusions, and KRAS mutations, while the less prevalent mutations include MET amplification, ROS1 fusions, NTRK translocations, and RET mutations^[4]. The mutation rate of EGFR is about 50% within lung adenocarcinoma patients^[5]. However, the majority of NSCLC patients inevitably develop drug resistance to osimertinib over time, and the reported possible mechanisms predominantly involve EGFR-dependent (C797X)^[6] and EGFR-independent pathways, including bypass activation, downstream signaling pathway activation, epithelial-to-mesenchymal transition (EMT), and small cell transformation^[5,7]. The drug-resistant patients are generally advised to undergo a second biopsy for additional mutation detection, and the corresponding combination therapies are used for those experiencing extensive progression of drug resistance^[8]. Therefore, understanding individual resistance mechanisms and formulating rational treatment approaches might provide novel therapeutic options for such patients.

EMT, recognized as the driving force behind tumor progression, leads to both innate and acquired resistance to the EGFR-tyrosine kinase inhibitor (TKI) treatment^[9]. Upon acquiring EMT, tumor cells lose the polar characteristics of epithelial cells and fail to adhere to neighboring cells^[10]. Furthermore, the migratory and metastatic properties of tumor cells are enhanced, resulting in the acquisition of more invasive characteristics^[7]. In NSCLC patients, the occurrence of EMT-related resistance to EGFR-TKIs is estimated to be approximately 2%^[5]. Thus, the identification of EMT occurrence and the subsequent determination of treatment strategies may have clinical benefits for patients. Recent advancements have indicated that a combination therapy involving anti-tumor angiogenesis and TKIs prolong the median progression-free survival (mPFS) and median overall survival (mOS) of patients exhibiting resistance to EGFR-TKIs^[10]. Anlotinib, a multi-target TKI that is employed as a last-line treatment for advanced or metastatic NSCLC patients, exerts anti-angiogenic and tumor growth inhibitory effects by targeting the vascular endothelial growth factor receptor (VEGFR), the platelet-derived growth factor receptor (PDGFR), the fibroblast growth factor receptor (FGFR), the stem cell factor receptor (c-Kit), and other kinases^[11–12]. The ALTER 0303 trial compared anlotinib with placebo in advanced NSCLC patients who received at least two lines of treatment, and showed that anlotinib effectively extended the mOS by 3.30 months (9.60 months vs. 6.30 months, P < 0.05), compared with the placebo^[13], highlighting promising clinical applications for anlotinib in treating NSCLC.

Recently, clinical observations have indicated that the addition of anlotinib provides clinical benefits to patients becoming resistance to osimertinib^[14]. However, few studies have pointed out the specific mechanism of reversal of osimertinib resistance by anlotinib. The present study aimed to explore whether anlotinib could reverse the EMT-induced osimertinib resistance, which might offer a novel clinical diagnostic and therapeutic strategy for the management of osimertinib resistance in NSCLC patients.

Materials and methods

Cell lines

The EGFR exon 19 deletion (EGFR-19del) cell lines, including PC9 and HCC827, were cultured in the 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 5% CO₂, at 37 ℃. The osimertinib-resistant (Osi-R) cells were generated by gradually adding osimertinib (Cat. #S7297, Selleck, Houston, Texas, USA) to PC9 and HCC827 cells ranging from 1 nmol/L to 1 μmol/L concentrations. Following a period of three to four months of induction, the IC₅₀ value of osimertinib exceeded 1 μmol/L, signifying the successful induction of Osi-R cell lines.

Western blotting assay

The treated cells were lysed and subjected to 10% SDS-PAGE electrophoresis and transferred to 0.45 mm membranes. The membranes were then incubated overnight with primary antibodies against E-cadherin (1∶1000, Cat. #3195, CST, Danvers, MA, USA), N-cadherin (1∶1000, Cat. #22018-1-AP, Proteintech, Wuhan, China), vimentin (1∶1000, Cat. #5741, CST), vascular endothelial growth factor A (VEGFA; 1∶1000, Cat. #19003-1-AP, Proteintech), and β-actin (1∶5000, Cat. #66009-1-Ig, Proteintech), respectively. Then, the membranes were incubated with indicated secondary antibodies, followed by exposure to horseradish peroxidase (HRP)-luminescent solution.

Enzyme-linked immunosorbent assay (ELISA)

A human VEGFA ELISA kit (FCMACS, Nanjing, China) was used to determine the VEGFA levels in the cell culture supernatants of HCC827-Osi-R (HCC827-OR) cells. The cell supernatant was collected with the cell fragments removed by centrifugation at 1500 rpm for 10 min. It was stored at −80 ℃ for subsequent experiments, following the manufacturer's instructions.

RNA extraction and real-time reverse transcription-PCR (qRT-PCR)

The total RNA from treated cells was extracted using TRIzol reagents. The RNA was then reverse-transcribed into cDNA using a transcription kit (Vazyme, Nanjing, China) following the manufacturer's protocol. Subsequently, the qRT-PCR assay was performed using SYBR Green Master Mix (Vazyme). The relative expression levels of the target genes were analyzed using the 2^−∆∆Ct method. The primer sequences for qRT-PCR were as follows: VEGFA forward: 5′-CCCACTGAGGAGTCCAACAT-3′, VEGFA reverse: 5′-TCCCTTTCCTCGAACTGATT-3′, GAPDH forward: 5′-GAAGGTGAAGGTCGGAGTC-3′, and GAPDH reverse: 5′-GAAGATGGTGATGGGATTTC-3′.

3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay

PC9, HCC827, PC9-Osi-R (PC9-OR), and HCC827-OR cells were seeded into 96-well plates at a density of 3 000–5 000 cells/well. The next day, the cells were cultured with varying concentrations of osimertinib and anlotinib (Cat. #S8726, Selleck). After 48 h of incubation, the cells were incubated with a culture medium containing MTT for 2–4 h. Subsequently, formazan was dissolved using dimethyl sulfoxide (DMSO). Finally, the absorbance was measured at 490 nm, and the IC₅₀ value was calculated.

Clone formation

PC9, HCC827, PC9-OR, and HCC827-OR cells in the log phase were seeded into six-well plates at a density of 1 000 cells/well. The next day, the cells were cultured with a medium containing osimertinib and anlotinib if required for 10–14 days. Upon the formation of clone spots exceeding 50 cells/clone, the cells were fixed with methanol for 15 min, stained with Giemsa (Beyotime, Shanghai, China) for 45 min, and visualized under a microscope.

Wound-healing assay

The treated PC9, HCC827, PC9-OR, and HCC827-OR cells were seeded in 6-well plates. A 100-μL pipette was used to scratch the cell monolayer, when the cells grew to around 80% confluence. In the wound-healing assay, the scratches were recorded at 0 h and 24 h by using a microscope, and the wound-healing conditions were analyzed after 24 h of culture.

Transwell assay

The PC9, HCC827, PC9-OR, and HCC827-OR cells in the logarithmic growth phase were seeded in the Transwell chamber at a density of 5 × 10⁴ cells/well. The 1640 basic medium was added in the upper chamber, while the lower chamber was supplemented with 1640 medium containing 20% FBS for 48 h. The number of cells penetrating the membrane was observed by using a microscope and photographed. When needed, 1 μmol/L osimertinib and 4 μmol/L anlotinib were added.

Tubular formation experiment

A total of 50 μL precooled Matrigel (Corning, USA) was added to 96-well plates and incubated at 37 ℃ for 30 min. The supernatant of the indicated cells was then extracted, and a mixture of the supernatant and human umbilical vein endothelial cells (HUVECs) at a density of 1.5 × 10⁴ cells was added to each well. The cells were then incubated at 37 ℃ for 6 h. Subsequently, the tube-like structures were observed under a microscope.

Immunofluorescence assay

The treated cells (PC9, PC9-OR, HCC827, and HCC827-OR cells) were fixed with 4% paraformaldehyde and permeabilized with Triton X-100. Then, the cells were blocked and incubated overnight at 4 ℃ with the indicated primary antibodies. On the next day, the cells were incubated with Fluorescein (FITC)-conjugated Affinipure Goat anti-Rabbit IgG antibody (1∶200, Cat. #SA00003-2, Proteintech) at 37 ℃ for 1 h and counterstained with DAPI (Cat. #C1006, Beyotime) for 5 min. Subsequently, the fluorescence signals were detected by using a fluorescence microscope (Zeiss, Oberkochen, Germany).

For cells stained with phalloidin, they were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, incubated with phalloidin (Yeasen, Cat. #40735ES75, Shanghai, China) for 30 min, and counterstained with DAPI for 10 min. Then, the fluorescence signals were detected using Nikon A1R confocal microscope (Nikon, Japan).

Patient information

Three NSCLC patients exhibiting resistance to osimertinib were selected from the Affiliated Jinling Hospital, Nanjing Medical University. The experiments involving these patients were approved by the Ethics Committee of the Affiliated Jinling Hospital, Nanjing Medical University (No. 2023DZGZR-030).

Xenograft experiment

A total of 20 male nude mice (BALB/c) aged 6–8 weeks were procured from GemPharmatech (Nanjing, China) for xenograft experiments. The well-grown PC9-OR cells (3 × 10⁶ cells) were resuspended in 50 μL PBS, mixed with 50 μL of Matrigel, and inoculated on the inner side of the left upper limb of the nude mice. When the transplanted tumors reached the dimensions of 5 × 5 mm² on the fifth day, these nude mice were evenly divided into four groups based on the tumor size, with five mice in each group. Each group underwent daily intragastric administration of either PBS, osimertinib (5 mg/kg), anlotinib (3 mg/kg), or a combination of osimertinib and anlotinib for 14 days. The mice were weighed and measured for tumor sizes every three days. The tumor volume was calculated according to the formula: L (length) × W (width)²/2. The nude mice were euthanized after 14 days of administration. The tumors were excised, weighed, fixed with paraformaldehyde, and embedded with paraffin for subsequent IHC experiments. Animal experiments were approved by the Animal Ethics Committee of the Affiliated Jinling Hospital, Nanjing Medical University (No. 2023JLHGZRDWLS-00032).

Immunohistochemistry (IHC) assay

Paraffin sections were used for IHC experiments on both samples from three patients and twenty mice. The slides were antigen-retrieved and incubated overnight with E-cadherin (1∶200, Cat. #3195, CST), N-cadherin (1∶1000, Cat. #66219-1-Ig, Proteintech), vimentin (1∶200, Cat. #5741, CST), VEGFA (1∶500, Cat. #19003-1-AP, Proteintech), and Ki-67 (1∶300, Cat. #ab15580, Abcam, Cambridge, UK), respectively. Then, the slides were incubated with HRP-conjugated secondary antibodies, and the indicated proteins were visualized using diaminobenzidine (DAB, Cat. #K3468, Dako, Denmark). The sections were counterstained with hematoxylin and dehydrated with gradient ethanol before being sealed for microscopic observations. The IHC staining density was scored as negative or weakly positive (score 1), moderately positive (score 2), or strongly positive (score 3). The percentage of positive cancer cells was classified as 1 (< 25%), 2 (25%–50%), 3 (50%–75%), and 4 (> 75%). An H-score was calculated by multiplying the intensity score and the proportion score to quantitatively evaluate the protein expression.

TdT-mediated dUTP nick end labeling (TUNEL) assay

For the TUNEL experiment, The paraffin sections of mouse samples were deparaffinized and subsequently processed following the manufacturer’s instructions (Cat. #A112, Vazyme). After DAPI counterstaining, the apoptotic signals were observed using a microscope and photographed for subsequent statistical analysis.

Statistical analysis

The value of the Western blot band was quantized using the Image J software. The Student’s t-test was used to statistically analyze the quantitative data. Two-way ANOVA was used to compare differences among more than two groups. The data were calculated as the means±SD. Moreover, GraphPad Prism software was used to draw the graphs. A significant difference was indicated as ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. All experiments were performed in triplicates.

Results

Anlotinib reversed clinical osimertinib resistance and EMT formation in osimertinib-resistant patients

It is known that cancer patients may develop resistance to osimertinib over time. In a case study, a 57-year-old male was diagnosed with lung cancer at the stage of T4N0M1a, and underwent a lung biopsy in August 2017. The patient exhibited EGFR-19del revealed by the next-generation sequencing and took gefitinib orally for targeted therapy. The patient switched to osimertinib in November 2020 due to the emergence of the EGFR T790M mutation. However, the patient encountered recurrence in March 2022, and the treatment regime was modified to a combination of osimertinib and anlotinib (Fig. 1A). The computed tomography imaging revealed a notable reduction in tumor size in the lungs in August 2022, compared with that in March 2022, particularly on the left side (Fig. 1B). This demonstrated synergistic anti-tumor effects of the osimertinib and anlotinib combination therapy in the case of osimertinib resistance.

Figure  1.  Combination therapy of osimertinib and anlotinib was effective for osimertinib-resistant (Osi-R) patients.

A: A patient with EGFR-T790M mutation gained osimertinib resistance and benefited from the combination therapy with anlotinib. B: The computed tomography imaging of the patient before osimertinib treatment and after osimertinib resistance. C and D: Representative images of the IHC staining of E-cadherin and vimentin (C) as well as VEGFA (D) in relapsed tissues of osimertinib-acquired resistant patients. H-scores are listed individually. Scale bar = 100 μm.

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Increasing studies are focusing on the mechanism of resistance to Osimertinib, while the mechanism and treatment options targeting EMT, one of the bypass mechanisms of osimertinib resistance, often receive less attention from investigators, compared with other resistance mechanisms. To explore the specific metastatic mechanisms in lung cancer patients with osimertinib resistance, three patients who underwent a secondary lung biopsy in the Affiliated Jinling Hospital, Nanjing Medical University, were recruited. Paraffin sections of lung tissues were collected for IHC experiments. The results indicated a decrease in the expression levels of E-cadherin but an increase in the expression levels of vimentin in the lung tissues of these three patients upon developing resistance (Fig. 1C). Furthermore, neovascularization is a crucial process during oncogenesis, in which VEGFA enhances the formation of blood vessels. In the present study, therefore, the expression of VEGFA was also upregulated in the recurrent slides (Fig. 1D). All the H-scores are provided in Fig. 1C and 1D.

Establishment and identification of PC9-OR and HCC827-OR cells

To elucidate the mechanisms and clinical treatment strategies associated with the EMT-induced osimertinib resistance, we performed a series of experiments by stimulating PC9 and HCC827 cells with osimertinib starting from 1 nmol/L concentration. Upon observing cell viability even at 1 μmol/L osimertinib, the PC9, PC9-OR, HCC827, and HCC827-OR cells were further treated with osimertinib at various concentrations. The 48-h MTT assay results showed that the IC₅₀ values of osimertinib in PC9 and PC9-OR cells were 8.54 nmol/L and 2.10 μmol/L, respectively. Similarly, the IC₅₀ values of osimertinib were 33.59 nmol/L and 2.58 μmol/L in the HCC827 and HCC827-OR cells, respectively (Fig. 2A), indicating the successful induction of osimertinib-resistant cell lines. Furthermore, cell clone formation experiments demonstrated that PC9 and HCC827 parental cells failed to form cell clones when treated with 200 nmol/L osimertinib, while the clone formation ability of PC9-OR and HCC827-OR cells remained unaffected by the same concentration of osimertinib (Fig. 2B). This observation suggested that PC9-OR and HCC827-OR cells exhibited resistance to osimertinib. Surprisingly, gradual acquisition of EMT phenotypes was observed in the PC9-OR and HCC827-OR cells during the induction of Osi-R cells, indicating an increased tendency towards mesenchymalization (Fig. 2C). Besides, the characteristics of morphological changes were identified using phalloidin staining and confocal microscopy. Accordingly, the results showed that the filopodia of PC-OR and HCC827-OR cells were longer than that of the parental cells, suggesting a higher invasive ability of the resistant cells (Fig. 2D).

Figure  2.  Establishment of PC9-OR and HCC827-OR cells from parental cells.

A: PC9, PC9-OR, HCC827, and HCC827-OR cells were treated with osimertinib at the concentrations of 0, 1, 10, 100, 1,000, and 10,000 nmol/L for 48 h. The MTT assay was performed to identify the IC₅₀ values of osimertinib. B: PC9 and HCC827 cells as well as their resistant cells were treated with 200 nmol/L osimertinib for 10–14 days. Clone formation experiments were performed to evaluate the osimertinib resistance. The number of clones was statistically analyzed by Student's t-test. ^*P < 0.05 and ^***P < 0.001. C: Morphological changes in the parental and resistant cells of PC9 and HCC827. Scale bar = 100 μm. D: Parental and Osi-R cells were stained with phalloidin. Red indicates phalloidin staining; blue indicates nucleus. Scale bar = 50 μm. The experiments were performed in triplicates. Abbreviations: OR and Osi-R: osimertinib resistance.

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EMT formation of PC9-OR and HCC827-OR cells

The EGFR-independent pathways include bypass activation, small cell transformation, and EMT formation^[8]. To verify the EMT formation in the Osi-R cells, we performed the wound-healing and Transwell assays, and found an enhanced migration ability of the PC9-OR and HCC827-OR cells, compared with the parental cells (Fig. 3A and 3B). Furthermore, the immunofluorescence experiment indicated a stronger fluorescence signal of E-cadherin but a weaker fluorescence signal of vimentin in parental cells than in resistant cells (Fig. 3C). Moreover, Western blot experiments showed decreased expression levels of E-cadherin but increased expression levels of N-cadherin and vimentin in PC9-OR and HCC827-OR cells than in their parental cells (Fig. 3D). These findings indicated that the PC9-OR and HCC827-OR cells might enhance their migration ability through EMT, ultimately leading to osimertinib resistance.

Figure  3.  PC9-OR and HCC827-OR cells exhibited EMT properties.

A and B: Wound-healing (A) and Transwell assays (B) were performed to identify the migrative ability of Osi-R cells. Scale bar = 200 μm. C and D: Immunofluorescence (C) and Western blot (D) experiments showed the expression of EMT markers in parental and resistant cells. Scale bar = 100 μm. The experiments were performed in triplicates. The Student's t-test was used to statistically analyze the quantitative data. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. Abbreviations: OR and Osi-R, osimertinib resistance ; E-cad, E-cadherin; Vim, vimentin.

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Anlotinib exhibited a synergistic effect in overcoming osimertinib resistance in PC9-OR and HCC827-OR cells

To further explore whether anlotinib reverses the osimertinib resistance by inhibiting EMT, we performed the MTT assays in PC9-OR and HCC827-OR cells using various concentrations of osimertinib with or without anlotinib. The results showed that the IC₅₀ value of anlotinib was 4.199 μmol/L in PC9-OR cells and 4.775 μmol/L in HCC827-OR cells. Moreover, the IC₅₀ value of osimertinib in PC9-OR cells significantly decreased from 2.15 μmol/L to 0.43 μmol/L after the addition of anlotinib. Similarly, the IC₅₀ value of osimertinib in HCC827-OR cells decreased from 3.41 μmol/L to 0.43 μmol/L with anlotinib treatment (Fig. 4A). Moreover, the colony formation experiments revealed that osimertinib treatment alone failed to inhibit the colony formation of PC9-OR and HCC827-OR cells; however, when combined osimertinib with anlotinib, the colony formation of resistant cells was significantly inhibited (Fig. 4B). The results of MTT and colony formation experiments collectively supported that anlotinib could effectively reverse osimertinib resistance in PC9-OR and HCC827-OR cells. To further validate the synergistic effect of osimertinib and anlotinib, we performed the MTT assay with six individual concentrations of osimertinib and anlotinib, and analyzed the data using Combenefit, an interactive platform for analyzing and visualizing drug combinations, which revealed a consistent synergistic effect of osimertinib and anlotinib under several concentrations (Fig. 4C). Therefore, 1 μmol/L osimertinib combined with 4 μmol/L anlotinib was used for the subsequent experiments.

Figure  4.  Combination therapy with anlotinib reversed osimertinib resistance.

A: Resistant cells were treated with anlotinib (0, 1, 5, 10, 20, and 40 μmol/L), or osimertinib (0, 0.1, 1, 2, 5, and 10 μmol/L) and anlotinib (1 μmol/L) for 48 h. The MTT assays were performed to detect the IC₅₀ values of osimertinib and anlotinib. B: PC9-OR and HCC827-OR cells were cultured with osimertinib at a concentration of 200 nmol/L in the presence or absence of 1 μmol/L anlotinib for 10–14 days. Clone formation experiments were used to verify the combination effect. C: PC9-OR and HCC827-OR cells were treated with osimertinib and anlotinib for 48 h. The MTT assays were performed to detect the cell viability. Combenefit was used to verify the synergistic effect of osimertinib and anlotinib. The experiments were performed in triplicates. Two-way ANOVA was used to compare differences among more than two groups. ^***P < 0.001. Abbreviations: OR and Osi-R, osimertinib resistance.

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Anlotinib reversed EMT and tubular formation ability of Osi-R cells

To investigate whether the combination of anlotinib could inhibit the migration ability of resistant cells, we performed wound-healing and Transwell assays. The results revealed that the combination of osimertinib with anlotinib effectively inhibited the migration ability of PC9-OR and HCC827-OR cells, ultimately enhancing the sensitivity of osimertinib (Fig. 5A-5D). Furthermore, the immunofluorescence assays revealed a significant upregulation of E-cadherin and downregulation of vimentin in the presence of combination treatment (Fig. 5E). To validate these results, we treated the two Osi-R cells with 1 μmol/L osimertinib with or without 4 μmol/L anlotinib for 24 h. Western blot analysis indicated that the combined strategy significantly inhibited the expression levels of N-cadherin and vimentin while upregulating E-cadherin expression. This finding strongly suggested that the addition of anlotinib to osimertinib inhibited EMT formation in resistant cells (Fig. 5F and 5G). Thus, the underlying mechanism of anlotinib reversing osimertinib resistance in PC9-OR and HCC827-OR cells involved the inhibition of EMT formation.

Figure  5.  Combination of osimertinib and anlotinib reversed the EMT formation in resistant cells.

A–D: PC9-OR and HCC827-OR cells were treated with 1 μmol/L osimertinib and 4 μmol/L anlotinib. Wound-healing and Transwell assays were used to identify the migration ability. Scale bar = 200 μm. E: Immunofluorescence experiments were performed to stain E-cadherin and vimentin. Scale bar = 100 μm. F and G: Western blot experiments were performed to identify the expression levels of the EMT markers. The experiments were performed in triplicates. The Student's t-test was used to statistically analyze the quantitative data. Two-way ANOVA was used to compare differences among more than two groups. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. Abbreviations: OR and Osi-R, osimertinib resistance; E-cad, E-cadherin; Vim, vimentin.

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The mechanism of anlotinib in NSCLC involves anti-angiogenesis^[15]. To further investigate whether anlotinib could reverse resistance via anti-angiogenesis in the Osi-R cells, we examined the expression levels of VEGFA in these resistance cells. The results showed that VEGFA was highly induced upon osimertinib resistance, compared with the parental cells (Fig. 6A). The resistant cells were then treated with 1 μmol/L osimertinib and 4 μmol/L anlotinib for 24 h. Subsequently, the supernatants and cells were collected for ELISA and qRT-PCR assays, respectively. The results showed that the expression of VEGFA was significantly restrained in both resistant cells by the combined treatments, compared with osimertinib or anlotinib treatment alone (Fig. 6B and 6C). Moreover, tubular formation experiments also showed that tube-like structures were scarce in the combination group than in other groups (Fig. 6D). Besides, the Western blot experiment indicated that the protein expression levels of VEGFA were lower in the combination group than in other groups (Fig. 6E and 6F). The results also showed that anlotinib did not reduce the phosphorylation level of P-AKT, which further validated that the mechanistic effects of osimertinib combined with anlotinib might be because of the inhibition of EMT and VEGFA pathways. These results suggested that the combination therapy inhibited both EMT formation and angiogenesis, ultimately contributing to the reversal of osimertinib resistance.

Figure  6.  VEGFA was highly expressed in Osi-R cells, and combined therapy inhibited VEGFA expression and tubular formation.

A: Western blot experiments showed the high expression levels of VEGFA in resistant cells. B: qRT-PCR detected the mRNA levels of VEGFA in resistant cells. C: ELISA assays detected the expression of VEGFA in HCC827-OR cells. D: Tubular formation experiments showed tube-like structures in the presence or absence of osimertinib and anlotinib in resistant cells. Scale bar = 200 μm. E and F: Western blot experiments showed the protein expression of VEGFA with indicated treatment in the resistant cells. The experiments were performed in triplicates. The Student's t-test was used to statistically analyze the quantitative data. Two-way ANOVA was used to compare differences among more than two groups. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. Abbreviations: OR and Osi-R, osimertinib resistance; E-cad, E-cadherin; Vim, vimentin; NS, no significance.

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Reversal of osimertinib resistance by combination of osimertinib and anlotinib in vivo

We further investigated whether osimertinib in combination with anlotinib could synergistically prevent NSCLC progression using xenograft experiments in mice (Fig. 7A). The growth curves showed an increased inhibition of Osi-R xenograft growth in the mice treated with the combination of osimertinib and anlotinib, compared with the osimertinib or anlotinib monotherapy (Fig. 7B and 7C). The biochemical indicators were assessed, which demonstrated the safety of the combinational strategy for mice, as no obvious impairments in hepatic and renal functions were observed in the indicated treatment groups (Fig. 7D).

Figure  7.  Anlotinib reversed osimertinib resistance in vivo.

Nude mice were divided into four groups, with five mice in each group, and treated with 5 mg/kg osimertinib or 3 mg/kg anlotinib alone or together for 14 days. A: Photograph of mice in four groups. B: The growth curves of tumors for mice in each group. C: Tumor volume and statistics of tumor weight for mice in each group. D: The levels of aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and Creatinine (Cr) in the serum were measured to determine the hepatic and renal function of mice. E: Representative images of IHC staining of Ki-67, E-cadherin, N-cadherin, vimentin, and VEGFA in the syngeneic tumors. Scale bar = 100 μm. F: The TUNEL assay for cell apoptosis was used to evaluate synergistic effect. Representative images are shown. Scale bar = 100 μm. Student's t-test was used for statistically analysis. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. Abbreviations: NC, negative control; OR, osimertinib resistance; osi, osimertinib; anlo, anlotinib.

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Moreover, IHC experiments with the tumor tissues revealed that the positive signal of E-cadherin was the strongest in the combination group, while those of the N-cadherin and vimentin were the weakest among the four groups, suggesting that combination therapy effectively inhibited the EMT formation in tumor tissues in vivo, consequently reversing drug resistance. Simultaneously, this combined approach inhibited the expression levels of VEGFA, thereby restraining the angiogenesis of tumors (Fig. 7E). Moreover, TUNEL experiments showed that the combined group exhibited the highest proportion of tumor apoptosis among the four groups, providing further evidence that the combination therapy exerted anti-tumor effects (Fig. 7F).

Discussion

Targeting the EGFR pathway in anti-tumor therapies has demonstrated significant improvements in the survival rates of NSCLC patients^[16]. Upon binding EGF to the extracellular domains of EGFR, the receptor dimerization occurs, which mediates self-phosphorylation, thereby activating multiple downstream signaling pathways, including PI3K/AKT and MAPK/ERK^[17–18]. Abnormal EGFR activation sustainably activates genes associated with tumor proliferation and differentiation, thereby initiating and promoting tumor formation and progression^[19]. Therefore, clinical therapies targeting EGFR encompass three generations of drugs. After an initial course of treatment with gefitinib or erlotinib, approximately half of the patients develop T790M mutations^[20]. The sequential administration of osimertinib can effectively inhibit this mutational activity^[21]. With the advancement of clinical research, osimertinib has now been promoted to the frontline, significantly prolonging both the mOS and mPFS of the patients^[22]. In the FLAURA study, a randomized and double-blind phase III clinical study that aimed to assess the efficacy and safety of osimertinib in previously untreated advanced NSCLC with EGFR mutations, it was found that compared with the first-generation EGFR-TKIs, the first-line treatment with osimertinib significantly prolonged mPFS by 8.7 months, and extended mOS to 38.6 months, thereby reducing the mortality risk of the patients by 20%^[23]. The results from the “ADAURA” clinical study further emphasized the clinical use of osimertinib in resected EGFR-mutated NSCLC patients, revealing a 2-year PFS rate of 89% in the osimertinib group, compared with only 52% in the placebo group^[24]. These findings underscore the significance of osimertinib in the management of EGFR-mutated NSCLC.

Recently, a series of studies have focused on osimertinib resistance, to provide an effective clinical treatment for osimertinib-resistant patients^[25]. While the fourth generation of EGFR-TKIs is still in the development and not yet utilized in clinical practice, combining therapies after osimertinib resistance has shown a promising potential in mitigating resistance^[26]. Anlotinib, by inhibiting targets such as c-Kit (proliferation), RET (proliferation), FGFR (proliferation/metastasis), and c-Met (metastasis), has been demonstrated the ability to control tumor cell proliferation and metastasis^[11]. The mechanisms of osimertinib resistance include the activation of pathways, such as c-Met and FGFR, as well as the formation of EMT^[27]. Moreover, anlotinib also exhibits inhibitory effects on these pathways^[28]. A retrospective and exploratory study conducted by Chinese scholar Baohui Han revealed a survival benefit of anlotinib in T790M-positive NSCLC patients with acquired osimertinib resistance^[29]. Lei et al. proposed that anlotinib could reverse osimertinib resistance by inhibiting the c-MET/MYC/AXL pathway^[14]. Furthermore, another study focusing on the third-generation drugs indicated that anlotinib could reverse acquired resistance to gefitinib^[30]. However, investigations lack exploration into whether anlotinib could reverse osimertinib resistance specifically by inhibiting the EMT pathway. The present study addressed this gap by revealing that tumor tissues of osimertinib-resistant patients tended to exhibit enhanced migration ability and elevated mesenchymal indicators. Therefore, we established resistant cell lines (PC9-OR and HCC827-OR) to demonstrate the reinforced metastatic abilities using wound-healing and Transwell assays. Western blot analysis also showed that the mesenchymal phenotype indicators, such as N-cadherin and vimentin, were highly expressed in the resistant cells, while the expression of E-cadherin was reduced, compared with the parental cells. Thus, subsequent experiments were performed based on these resistant cells, to explore the potential of anlotinib in inhibiting and reversing osimertinib resistance.

EMT is a process involved in the loss of cellular polarity and adhesion, resulting in a shift towards a mesenchymal phenotype with enhanced migration capabilities^[31]. Importantly, EMT serves as a resistance mechanism against various treatments, including targeted drugs, cytotoxic drugs, and radiotherapy^[32–33]. The EMT process activates AXL through the PI3K/AKT pathway, leading to the loss of E-cadherin, thereby causing metastasis of NSCLC^[34]. Furthermore, the increased expression of EMT-transcription factors (TFs) and ZEB1 expression may also lead to the EGFR-TKIs resistance^[35]. The co-inhibition of these pathways, along with the EGFR pathway, holds the potential to reverse the EMT-mediated resistance. Therefore, targeting EMT represents a promising strategy to effectively kill tumor cells and overcome resistance to EGFR-TKIs in NSCLC. In the present study, we observed that the combination of osimertinib and anlotinib effectively reversed osimertinib resistance induced by EMT in vitro. This finding was further validated in thymic nude mice, where tumors in the combined treatment group exhibited the smallest size. The IHC results showed that E-cadherin was upregulated in the combination group, while the expression levels of N-cadherin and vimentin were reduced, indicating the transformation of tumor cells into mesenchymal-to-epithelial transition. Based on these observations, we concluded that anlotinib effectively reversed osimertinib resistance, extending the duration of osimertinib treatment, and may consequently prolong the OS of patients by inhibiting the EMT occurrence. The current understanding of whether EMT resistance arises from the selection of pre-existing EMT clones or the secondary acquisition of stromal phenotype by epithelial cells after EGFR-TKI treatment remains unclear. Future investigations targeting this mechanism and developing targeted treatment strategies might enhance treatment efficacy, offering clinical benefits to the patients.

Briefly, the development of osimertinib resistance in NSCLC may be partly attributed to the occurrence of EMT. Anlotinib inhibits the migration of resistant cells, offering a potential solution to combat osimertinib resistance. The combination of osimertinib and anlotinib emerges as a promising clinical strategy for the patients, holding the potential to extend their survival time.