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. 2025 Feb 4;116(4):1034–1047. doi: 10.1111/cas.70006

AXL‐Mediated Drug Resistance in ALK‐Rearranged NSCLC Enhanced by GAS6 From Macrophages and MMP11 Positive Fibroblasts

Takahiro Utsumi 1,2, Hayato Mizuta 1,3, Yosuke Seto 1, Ken Uchibori 1,4, Makoto Nishio 4, Isamu Okamoto 2, Ryohei Katayama 1,5,✉
PMCID: PMC11967273  PMID: 39904499

ABSTRACT

Anaplastic lymphoma kinase (ALK) rearranged non‐small cell lung cancer (NSCLC) shows marked tumor shrinkage by ALK‐tyrosine kinase inhibitors (TKIs). However, tumors almost inevitably relapse owing to the development of acquired resistance. Resistance mechanisms include secondary ALK mutations and the activation of bypass pathways, such as cMET, cKIT, or EGFR, though some remain unknown. In this study, we analyzed alectinib‐resistant patient samples and identified a significant increase in AXL expression in the tumor, and a high level of GAS6, the ligand for AXL, in the pleural effusion. AXL‐overexpressing H3122 ALK‐rearranged NSCLC cells exhibited partial resistance to alectinib, which was enhanced by GAS6 supplementation but could be overcome by the ALK/AXL inhibitor gilteritinib. Moreover, GAS6‐overexpressing NIH3T3 cells and AXL‐expressing H3122 cells were subcutaneously injected into the left and right sides of nude mice simultaneously, followed by alectinib treatment. The supply of GAS6 from NIH3T3 may have accelerated tumor relapse under alectinib treatment. However, even without GAS6‐overexpressing NIH3T3, AXL‐overexpressing H3122 tumor relapsed within 1 month possibly due to increased host mouse Gas6 expression. Single‐cell RNA sequencing revealed that specific cancer‐associated fibroblasts (CAFs) and a subset of tumor‐associated macrophages (TAMs) are the primary sources of Gas6 in the tumor microenvironment (TME). During alectinib treatment, TAMs increased their infiltration into the TME, whereas CAFs altered their expression patterns, substantially upregulating Mmp11. These findings suggest that AXL expression in resistant cancer cells, combined with increased Gas6 production in the TME, contributes to enhanced ALK‐TKI resistance.

Keywords: ALK, AXL, drug resistance, GAS6, sc‐RNA‐seq

Activation of the GAS6/AXL signaling pathway promotes resistance to ALK‐TKIs both in vitro and in vivo. Single‐cell RNA sequencing revealed that specific cancer‐associated fibroblasts (CAFs) and a subset of macrophages are the primary sources of Gas6 in the tumor microenvironment (TME). During alectinib treatment, macrophages exhibited increased infiltration into the TME, whereas CAFs significantly altered their expression patterns, particularly upregulating Mmp11.

graphic file with name CAS-116-1034-g007.jpg

Abbreviations

ALK

Anaplastic lymphoma kinase

AXL

AXL (Anexelekto) receptor tyrosine kinase

CAF

Cancer‐associated fibroblast

ERK

Extracellular signal‐regulated kinase

GAS6

Growth arrest specific 6

RTK

Receptor tyrosine kinase

TAM

Tumor‐associated macrophage

TKI

Tyrosine kinase inhibitor

1. Introduction

Lung cancer is the leading cause of cancer deaths worldwide [1], with non‐small cell lung cancer (NSCLC) accounting for approximately 85% of all cases. Anaplastic lymphoma kinase (ALK) gene rearrangement, which was initially identified by Soda et al. [2], is observed in 3%–5% of patients with NSCLC. At present, six US FDA‐approved ALK‐tyrosine kinase inhibitors (TKIs) are available as first‐line agents, all of which exhibit high response rates exceeding 70% [3, 4, 5, 6, 7, 8]. The advent of these ALK‐TKIs has greatly enhanced the prognosis of ALK‐rearranged lung cancer, with the latest report indicating a 5‐year overall survival (OS) rate of approximately 60% [9]. However, the majority of patients ultimately develop resistance to long‐term ALK‐TKI therapy, which remains as a significant challenge. The mechanisms of therapeutic resistance to ALK‐TKI can be classified into two categories: on‐target and off‐target. For on‐target resistance, the occurrence of one or more secondary mutations within the ALK gene sequence impedes ALK‐TKI binding, thereby conferring resistance. This type of resistance is observed in 50%–60% of patients undergoing second‐generation ALK‐TKI treatment [10]. Contrarily, off‐target resistance represents an ALK‐independent resistance mechanism and occurs in approximately half of the patients receiving second‐generation TKI treatment. Off target resistance includes activation of bypass pathways, such as cMET, cKIT, and EGFR [11, 12], as well as histological transformation, including small cell carcinoma, squamous cell carcinoma, and epithelial–mesenchymal transition (EMT) [13, 14, 15]. AXL, a receptor tyrosine kinase (RTK), is involved in many biological processes, such as cell proliferation, survival, migration, angiogenesis, efferocytosis, natural killer cell differentiation, and platelet aggregation [16]. The role of AXL activation in the development of therapeutic resistance has been increasingly recognized in the context of diverse malignancies, particularly in relation to bypass pathway activation and EMT [17, 18, 19, 20, 21]. In the context of ALK‐rearranged NSCLC, AXL overexpression was observed in experimentally generated ALK‐TKI‐resistant cells [22]. Furthermore, some patients with ALK‐rearranged NSCLC overexpressing AXL demonstrated poorer response to first‐generation ALK‐TKI crizotinib therapy than those with low AXL expression [22]. Meanwhile, it remains unclear how growth arrest‐specific protein 6 (GAS6), the primary ligand for AXL, contributes to ALK‐TKI therapeutic resistance; the source of GAS6 in this process is also unclear. In EGFR‐mutated NSCLC, there have been reports indicating that GAS6 plays a role in EGFR‐TKI resistance [23, 24]. Therefore, it is possible that GAS6 is also involved in ALK‐TKI resistance in ALK‐rearranged NSCLC.

In this study, we report an AXL‐overexpressing alectinib‐resistant case, with a high level of GAS6 in the relapsed pleural effusion. We conducted this study to determine the importance of the GAS6/AXL pathway and the regulatory mechanisms of GAS6, particularly in the host nontumor cells, with respect to treatment resistance in ALK‐rearranged NSCLC. In addition, we aimed to identify strategies for overcoming resistance. To achieve this, we overexpressed AXL in the ALK‐rearranged NSCLC cell line H3122 and evaluated the therapeutic response with or without GAS6 in vitro and in vivo. Moreover, we explored the source of GAS6 by examining the alterations in the tumor microenvironment (TME) in a mouse xenograft model with and without ALK‐TKI treatment.

2. Material and Methods

Detailed information is shown in the Supporting Information and methods (Data S1).

2.1. Patient

Clinical samples were obtained from patients with ALK‐rearranged NSCLC who had developed resistance to alectinib. The patients provided written informed consent for the genetic and cellular analyses, which were conducted in accordance with the protocols approved by the Institutional Review Board of the Japanese Foundation for Cancer Research (approval no. 2013‐1093).

2.2. Cell Lines

Patient‐derived JFCR‐028‐3 and JFCR‐028‐4 were established from pleural effusions obtained from the patient. H3122 human cancer cells were kindly provided by JA Engelman (Massachusetts General Hospital Cancer Center, Boston, MA, USA) and NIH3T3 mouse embryonic fibroblast cells by M Yoshida (Japanese Foundation for Cancer Research, Tokyo, Japan). 293FT human embryonic kidney cells were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

2.3. Reagents

Alectinib (CH5424802) was purchased from Active Biochem (Kowloon, Hong Kong) and gilteritinib (ASP2215) from Shanghai Biochempartner (Shanghai, China) and Biovision (Milpitas, CA, USA). In addition, human (885‐GSB‐050) and mouse (986‐GS‐025) recombinant Gas6 proteins were obtained from R&D Systems (Minneapolis, MN, USA).

2.4. Enzyme‐Linked Immunosorbent Assay (ELISA)

Enzyme‐linked immunosorbent assay (ELISA) was conducted using Human Gas6 DuoSet ELISA (R&D Systems) and DuoSet Ancillary Reagent Kit 2 (R&D Systems) according to the manufacturer's protocol.

2.5. Cell Viability Assay

Cell viability assay was performed as previously described [25] using the CellTiter‐Glo assay reagent (Promega, Madison, WI, USA) and TriStar LB941 microplate luminometer (Berthold Technologies, Bad Wildbad, Germany).

2.6. Flow Cytometry Analysis

The cells were treated with either a human Axl APC‐conjugated antibody (FAB154A, R&D Systems, Minneapolis, MN, USA) or an isotype control (400142, BioLegend, San Diego, CA, USA), then assayed using FACSMelody (BD Bioscience, San Jose, CA, USA). The data were analyzed using the FlowJo software (TOMY Digital Biology, Tokyo, Japan).

2.7. Immunoblotting

Cell lysis and immunoblotting were performed as previously described [25].

2.8. Quantitative Reverse Transcription‐Polymerase Chain Reaction (RT‐qPCR)

Quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) was conducted using FastStart Essential DNA Green Master (Roche) according to the manufacturer's protocol. The sequences of the primers are provided in Table S1.

2.9. Mouse Experiments

Female BALB/c‐nu/nu (nude) mice were purchased from Charles River Laboratories, Yokohama, Japan. All animal experiments were conducted in accordance with the protocols approved by the JFCR Animal Care and Use Committee.

2.10. RNA Sequencing Analysis (RNA‐Seq)

Total RNA was extracted using RNeasy Mini Kit (Qiagen, Venlo, Netherlands). All RNA samples were submitted to Macrogen Japan (Tokyo, Japan) for library preparation and sequencing in accordance with their established protocols. FASTQ format raw data were processed using the RaNA‐Seq [26] tool. Sample clustering, heatmap generation, extraction of differentially expressed genes (DEGs), and pathway enrichment identification were performed using the iDEP 2.01 tool [27] and the count data from RaNA‐Seq. The immune cell fractions of bulk RNA‐seq samples were estimated using CIBERSORTx [28]. The reference sample file was created by removing the T‐cell data from Chen et al.'s dataset [29].

2.11. Single‐Cell RNA Sequencing Analysis (Sc‐RNA‐Seq)

Frozen tumor samples were submitted to Takara Bio Inc. (Shiga, Japan) for library preparation and sequencing in accordance with their established protocols. Single‐cell expression analysis was conducted using Cell Ranger v7.1.0 (10x Genomics, Pleasanton, CA, USA). Furthermore, clustering and gene expression analyses were conducted using Loupe Browser v8.0.0 (10x Genomics).

2.12. Statistical Analysis

The data were analyzed using GraphPad Prism version 10.3.0 (GraphPad Software, Boston, MA, USA). The results were expressed as mean ± SEM. Statistical significance for comparisons among more than three groups was evaluated via one‐way analysis of variance, followed by Dunnett's or Bonferroni's multiple comparisons test. A two‐sided p‐value < 0.05 was considered statistically significant.

3. Results

3.1. AXL Activation Was Implicated in the Development of ALK‐TKI Resistance in a Patient With ALK‐Rearranged NSCLC

To estimate the mechanism of resistance to alectinib in a patient with ALK‐rearranged NSCLC, RNA‐seq was performed on two patient‐derived cell lines established from pleural effusion before and after treatment, which were designated as JFCR‐028‐3 and JFCR‐028‐4, respectively. The treatment course and timing of cell line establishment for this patient is illustrated in Figure S1A. Examination of the expression levels of 58 RTKs in the two cell lines showed that AXL was upregulated in JFCR‐028‐4 (Figure 1A and Table S2). Consistent with the results of the RNA‐seq analysis, flow cytometry analysis revealed that AXL was upregulated at the cell surface of JFCR‐028‐4 (Figure 1B). Furthermore, immunoblot analysis confirmed the increased total AXL expression in JFCR‐028‐4 cells, whereas the phosphorylated AXL expression, indicating AXL activation, was below the detection threshold (Figure 1C). GAS6 has been identified in human plasma at concentrations ranging from 13 to 23 ng/mL [30]. Moreover, the pleural effusion of the patient had a GAS6 concentration of approximately 60 ng/mL (Figure 1D). This suggests that AXL may have been activated in the patient's body by GAS6 derived from the plasma and pleural effusion. Based on these findings, it can be hypothesized that AXL activation may partially contribute to alectinib resistance in this patient.

FIGURE 1.

FIGURE 1

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AXL upregulation was observed in patient‐derived cell lines established after alectinib resistance. (A) Volcano plot of 58 receptor tyrosine kinase expressions in two patient‐derived cell lines (PDCs), JFCR‐028‐3 and JFCR‐028‐4, analyzed via RNA sequencing. (B) Histogram showing the AXL expression levels in the two PDCs analyzed via flow cytometry. The light blue and orange lines denote the isotype controls (mouse IgG, mIgG). (C) Immunoblot analysis of phosphor‐AXL (pAXL), and AXL in the two PDCs. (D) Bar graphs of enzyme‐linked immunosorbent assay (ELISA) showing the GAS6 concentrations (ng/mL) in the pleural effusion from which the two PDCs were established. Similar experiments were conducted twice (B, C) or thrice (D), and representative data are shown.

3.2. AXL Overexpression and the Presence of GAS6 Conferred Resistance to ALK‐TKI Therapy

To confirm that the increased expression of AXL conferred resistance to ALK‐TKI therapy, we overexpressed AXL in H3122 cells, an ALK‐rearranged NSCLC cell line (Figure 2A), and evaluated the sensitivity to alectinib. The AXL‐overexpressing H3122 cells exhibited slight resistance to alectinib compared with the parental cells, and the resistance was further enhanced by the addition of recombinant human GAS6 (hGAS6; Figures 2B and S2A). Subsequently, we established GAS6‐overexpressing NIH3T3 cells, a mouse fetal fibroblast cell line (Figure 2C), and confirmed GAS6 secretion in the conditioned medium (CM) from NIH3T3‐GAS6 cells (Figure 2D). AXL‐overexpressing H3122 cells cotreated in the CM from NIH3T3‐GAS6 cells also exhibited resistance to alectinib (Figures 2E and S2B). To determine whether AXL overexpression and the presence of GAS6 contributed to resistance to alectinib in vivo, we conducted experiments using a mouse xenograft model in which the parental or AXL‐overexpressing H3122 cells were subcutaneously implanted. To provide GAS6 in vivo, the GAS6‐overexpressing NIH3T3 was implanted subcutaneously on the opposite side of the tumor. The administration of alectinib to mice with H3122 parental cells resulted in the long‐term suppression of tumor growth. Contrarily, mice transplanted with AXL‐overexpressing cells exhibited tumor regrowth after approximately 2–3 weeks. Moreover, mice that inoculated NIH3T3‐GAS6 and H3122‐AXL cells demonstrated earlier tumor regrowth and a substantial increase in size on day 25 compared with the mice that inoculated control NIH3T3 without GAS6 overexpression (Figure 2F). Although plasma GAS6 concentrations in the mouse model were not evaluated, our findings suggest that GAS6 derived from NIH3T3 cells may have contributed to the acceleration of tumor relapse. These results indicated that AXL overexpression and the presence of GAS6 accelerated the acquired resistance to alectinib both in vitro and in vivo.

FIGURE 2.

FIGURE 2

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The overexpression of AXL and the presence of GAS6 were confirmed to have contributed to the development of resistance to alectinib, as evidenced by in vitro and in vivo studies. (A) Immunoblot analysis of phosphor‐AXL (pAXL) and AXL in H3122 parental and AXL‐overexpressing (AXL) cells. (B, E) Bar graphs showing the effects of alectinib (100 nmol/L) on the viability of H3122 parental (PT) and AXL‐overexpressing (AXL) cells. Recombinant GAS6 (400 ng/mL) (B) or conditioned medium from parental (PT) and GAS6‐overexpressing (GAS6) NIH3T3 single‐clone cells (no. 37) (E) was added 6 h before treatment. Cell viability was evaluated after 72 h using the CellTiter‐Glo Assay. (C) Immunoblot analysis of GAS6 in parental NIH3T3 cells as well as polyclonal (GAS6 poly) and single‐clone (GAS6 #37) GAS6‐overexpressing NIH3T3 cells. (D) Bar graphs of enzyme‐linked immunosorbent assay (ELISA) showing the concentrations (ng/mL) of GAS6 in the conditioned medium of parental (PT) NIH3T3 cells as well as polyclonal (GAS6 poly) and single‐clone (GAS6 no. 37) GAS6‐overexpressing NIH3T3 cells. An asterisk (*) indicates a value below the detection threshold. (F) Parental (PT) and AXL‐overexpressing (AXL) H3122 cells and parental (PT) and human GAS6‐overexpressing (GAS6) NIH3T3 polyclonal cells were subcutaneously transplanted into BALB/c nu/nu mice. The NIH3T3 cells were repeatedly transplanted every 3 days. Once the average tumor volume reached approximately 200 mm3, the mice were treated with vehicle or alectinib (30 mg/kg) once daily for 5 days per week via oral gavage (n = 6). *p < 0.05, ***p < 0.001, ****p < 0.0001 (one‐way analysis of variance following Bonferroni's multiple comparisons test). Similar experiments were conducted twice (A–C, E) or thrice (D), and representative data are shown.

3.3. Gilteritinib Is Effective in Overcoming Resistance to ALK‐TKI Treatment Caused by GAS6/AXL Signaling

We evaluated the means to overcome GAS6/AXL‐mediated ALK‐TKI resistance. Gilteritinib, a FLT3/AXL inhibitor, was approved in 2018 by the US Food and Drug Administration for the treatment of relapsed or refractory acute myeloid leukemia with FLT3 mutation. In a previous study, we reported that gilteritinib also functions as an ALK inhibitor, thereby providing a potential strategy for overcoming various types of treatment resistance, including AXL‐mediated resistance [25]. To test the hypothesis that gilteritinib could overcome the resistance of AXL‐overexpressing cells enhanced by GAS6 addition, we conducted an in vitro gilteritinib treatment study. The AXL‐overexpressing H3122 cells cultured with GAS6 supplementation exhibited sensitivity to gilteritinib in vitro (Figures 3A, and S2C). Similarly, the resistance induced by CM from NIH3T3‐GAS6 cells could be overcome with gilteritinib (Figures 3B and S2D). To confirm the mechanism of growth inhibition by gilteritinib, intracellular signaling alterations were evaluated via immunoblot analysis in the presence of GAS6. In AXL‐overexpressing cells, alectinib treatment decreased the levels of phosphorylated ALK. However, the sustained AXL phosphorylation preserved the activation of downstream oncogenic signaling pathways, including the MAPK and PI3K/AKT/mTOR pathways. Contrarily, gilteritinib treatment suppressed the phosphorylation of ALK and AXL, along with their associated downstream signaling pathways (Figure 3C). These findings indicated that gilteritinib is effective in overcoming resistance to ALK‐TKI treatment resulting from GAS6/AXL signaling. This overcoming of resistance was achieved through simultaneous suppression of the GAS6/AXL signaling pathway, which serves as a bypass signaling pathway, as well as signaling from the ALK fusion protein.

FIGURE 3.

FIGURE 3

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Gilteritinib was effective in overcoming resistance to ALK‐TKI treatment resulting from the activation of the GAS6/AXL signaling pathway. (A, B) Bar graphs showing the effects of gilteritinib (100 nmol/L) on the viability of H3122 parental (PT) and AXL‐overexpressing (AXL) cells. Recombinant GAS6 (400 ng/mL) (A) or conditioned medium from parental (PT) and single‐clone (#37) GAS6‐overexpressing (GAS6) NIH3T3 cells (B) was added 6 h before drug treatment. Cell viability was evaluated after 72 h using the CellTiter‐Glo Assay. (C) Immunoblot analysis of ALK, AXL, PARP, and the downstream pathways of ALK in parental (PT) and AXL‐overexpressing (AXL) H3122 cells. The cells were treated with the indicated concentrations of the drugs (Ale, alectinib; Gil, gilteritinib) for 6 h. Recombinant GAS6 (100 ng/mL) was added 1 h before the initiation of drug treatment.

3.4. The Levels of Host‐Derived Mouse Gas6 Were Elevated in the Tumor Microenvironment During the Initial Phase of Alectinib Treatment In Vivo

To elucidate the regulatory mechanisms of GAS6 in the TME, tumors were collected at each time point of alectinib treatment in a mouse xenograft model transplanted with AXL‐overexpressing H3122 cells (Figure 4A). In the RNA‐seq analysis of bulk tumor samples, the gene expression profiles of nontumor cells derived from mice were confirmed by mapping the mouse genome as a reference, whereas the profiles of tumor cells were confirmed by mapping the human genome as a reference. The results of the cluster analysis of gene expression indicated that the alectinib‐ and vehicle‐treated samples were classified into distinct clusters for both tumor cells and nontumor cells. The findings suggested that the gene expression profiles were altered by the alectinib treatment in a time‐dependent manner (Figure 4B,C). Enrichment analysis of DEGs revealed that cell cycle‐related genes were downregulated in both tumor cells and nontumor cells in the treated group. In addition, the “hematopoietic cell lineage” and “cytokine–cytokine receptor interaction” pathways were enriched in the tumor‐infiltrating cells of the treated group, indicating an increase in immune cell infiltration and activation of cell–cell interactions (Figure S3A–D). RT‐qPCR analysis of mouse Gas6 (mGas6) revealed a notable increase in expression levels at all time points after day 2 in alectinib‐treated samples compared with vehicle‐treated ones (Figure 4D). Moreover, the transcript per million values derived from the RNA‐seq results indicated an elevated expression of mGas6 in alectinib‐treated samples (Figure 4F). Contrarily, the expression level of hGAS6 derived from tumor cells was much lower than that of mGas6 (Figure 4F), and the change in the expression levels between vehicle and alectinib treatments was less pronounced (Figure 4E). Similar to the effect of hGAS6, mGas6 induced alectinib resistance in AXL‐overexpressing H3122 cells (Figure S2E). The collective findings indicated that Gas6, derived from host nontumor cells, was upregulated in the TME after alectinib treatment, thereby contributing to the emergence of therapeutic resistance.

FIGURE 4.

FIGURE 4

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Gas6 expression in the host nontumor cells was elevated from the early phase of alectinib treatment. (A) A schematic diagram of the methodology of the in vivo experiment. The tumor size at the specified time point is indicated by the line graphs. The timing of tumor collection is indicated by the arrows. BALB/c‐nu, BALB/c‐nude mice; H3122‐AXL, AXL‐overexpressing H3122 cells; RNA‐seq, RNA‐sequencing; RT‐qPCR, Quantitative reverse transcription‐polymerase chain reaction. (B, C) Hierarchical clustering with a heatmap of the bulk RNA sequencing (RNA‐seq) data, with the human (B) and murine (C) genomes used as the references. The timing of tumor collection (day) and the type of treatment (alectinib, ALE; vehicle, VEH) are indicated at the top. (D, E) Bar graphs of RT‐qPCR showing the expression levels of murine Gas6 (mGas6) and human GAS6 (hGAS6) in tumor samples obtained at various time points throughout the treatment period. hACTB, Human ACTB; mActb, Murine Actb. (F) Bar graphs of the transcript per million (TPM) value of mGas6 and hGAS6 determined via RNA‐seq analysis. Each data point represents a single TPM value derived from the bulk tumor sample collected on days 2–11. The number of samples at each time point is 3. *p < 0.05 (unpaired t‐test)

3.5. Macrophages and Fibroblasts Enhanced Gas6 Supply During Alectinib Treatment via Distinct Mechanisms

To identify the host‐derived cells supplying Gas6 in the TME, CIBERSORTx [31] was applied to the bulk RNA‐seq data to estimate the relative proportions of various immune cell populations. The results indicated that the proportion of macrophages increased in the samples at an early stage following alectinib treatment (Figure 5A). This increase in TAMs occurred concurrently with the increase in mGas6 within the TME. To gain further insight into the source of Gas6, sc‐RNA‐seq was performed on samples collected on days 8 and 11 following alectinib and vehicle treatments. To analyze the gene expression of host nontumor cells, the mouse genome was used as a reference. The t‐SNE plot showed that the majority of cells were clustered according to treatment, and some clusters were identified as a mixture of alectinib‐ and vehicle‐treated samples (Figure 5B, left panel). We classified the cells into 10 clusters using the k‐means method (Figure 5B, right panel) and annotated each cluster based on characteristic gene expression patterns (Figure S4A and Table S3) and the expression levels of cell type‐specific markers (Figure S4B). An elevated Gas6 expression was observed in Clusters 4, 5, and 6, indicating that some fractions of macrophages and fibroblasts are the primary sources of Gas6 (Figure 5C). Cluster 4, which comprised macrophages, exhibited an overlapping distribution of vehicle‐ and alectinib‐treated samples on the t‐SNE plot, indicating a high degree of gene expression similarity. The expression level of Gas6 was found to be similar between the two treatment groups. However, the number of cells within the clusters was greater in the alectinib‐treated samples (Figure 5D). These findings are consistent with the results of the CIBERSORTx analysis, which indicated that macrophages may contribute to increased Gas6 supply during alectinib treatment by promoting infiltration into the tumor sites. Contrarily, in Clusters 5 and 6, which consisted of fibroblasts, the overlap between vehicle‐ and alectinib‐treated samples on the t‐SNE plot was minimal, and gene expression appeared to differ between the two treatment groups. The expression level of Gas6 was higher in the alectinib‐treated cells than in the vehicle‐treated cells, and the number of cells within the clusters was similar between the two treatment groups (Figure 6A). A comparison of gene expression between the two treatment groups showed that Mmp11 was highly expressed in cells derived from alectinib‐treated samples (Figure 6B and Table S4). Furthermore, the majority of cells expressing Gas6 following alectinib treatment exhibited Mmp11 expression (Figure 6C). These findings implicate that macrophages enhance Gas6 availability by increasing the number of tumor‐infiltrating cells, whereas fibroblasts, characterized by Mmp11 expression, contribute to the increase in Gas6 levels through transcriptional alterations during alectinib treatment (Figure 6D).

FIGURE 5.

FIGURE 5

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Alectinib treatment resulted in an increase in the number of macrophages and resulting in elevated Gas6 levels in the tumor microenvironment (TME). (A) Bar plots showing the proportion of macrophages in samples collected from days 2 to 8 (left). Pie chart illustrating the proportion of immune cells in the day 8 samples. The mean proportions from triplicate samples are shown (right). B Cells Naïve, naïve B cells; DC Immature, immature dendritic cells; NK Activated, activated natural killer cells. (B) t‐SNE plots of 23,920 cells colored by sample origin (left) and 10 clusters determined using the k‐means method (right). Each cluster was annotated on the basis of differentially upregulated genes and expression levels of cell markers. “Veh_d8_1” represents replicate 1 of vehicle‐treated sample collected on day 8. Ale, alectinib; Veh, vehicle. (C) t‐SNE plots illustrating the expression patterns of Gas6 (left) and violin plots showing the Gas6 expression levels (log2 UMI count) in each cluster (right). (D) t‐SNE plots of cluster 4 colored by treatment group (left), violin plots showing the Gas6 expression (log2 UMI count) in each group (middle), and a pie chart illustrating the proportion of each group (right). *p < 0.05 (unpaired t‐test)

FIGURE 6.

FIGURE 6

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Alectinib treatment resulted in alterations in the expression of cancer‐associated fibroblasts, resulting in elevated Gas6 levels in the tumor microenvironment (TME). (A) t‐SNE plots of clusters 5 and 6 colored by treatment group (left), violin plots showing the Gas6 expression (log2 UMI count) in each group (middle), and a pie chart illustrating the proportion of each group (right). (B) Violin plots showing the Mmp11 expression (log2 UMI count) in each group. (C) t‐SNE plots of cluster 5 and 6 cells with high Gas6 expression (log2 UMI count > 1), colored by Mmp11 and Gas6 expression levels (log2 UMI count). (D) A schematic diagram of ALK‐tyrosine kinase inhibitor (ALK‐TKI) resistance by the increased GAS6 in the TME. CAFs, cancer‐associated fibroblasts.

4. Discussion

This study aimed to elucidate the significance of GAS6 and its regulatory mechanisms with regard to treatment resistance in ALK‐rearranged NSCLC. An additional important objective is to identify strategies to overcome such resistance. Indeed, the presence of GAS6 conferred significant resistance to ALK‐TKI in AXL‐overexpressing H3122 cells in in vitro and in vivo experiments. In a murine model, alectinib treatment significantly increased host‐derived Gas6 levels within the TME. The sources of Gas6 were tumor‐associated macrophages (TAMs), which increased infiltration into the TME, and cancer‐associated fibroblasts (CAFs), which altered gene expression, including Mmp11 upregulation. Moreover, GAS6/AXL signaling‐mediated treatment resistance was successfully overcome by gilteritinib as a single agent.

Many recent studies have demonstrated that AXL activation plays a role in the development of therapeutic resistance in various cancers [17]. Nakamichi et al. reported that in ALK‐TKI‐resistant cells generated through long‐term exposure to ALK‐TKIs, AXL overexpression, the EMT phenotype, and reduced dependence on ALK signaling were observed [22]. However, there is a lack of literature directly confirming the impact of GAS6 on the therapeutic efficacy of ALK‐TKIs, despite reports indicating that GAS6 induces resistance to EGFR‐TKIs in EGFR‐mutated NSCLC [23, 24]. In recent years, the concept of drug‐tolerant persisters (DTPs) has gained attention in relation to the limited long‐term efficacy of molecularly targeted therapies. DTPs are cells that survive the initial targeted therapy and, over time, acquire genetic mutations or other adaptations, ultimately becoming fully resistant [32]. In DTP cells, the upregulation of EMT‐related transcription factors, such as ZEB1, ZEB2, and SNAIL (SNAI2) [33], as well as the activation of the YAP signaling pathway [34] has been reported. JFCR‐028‐4, the alectinib‐resistant PDC analyzed in this study, also exhibited upregulation of these EMT‐related factors and activation of the YAP signaling pathway. However, typical EMT features, such as CDH2 or FN1 upregulation or CDH1 downregulation, were not observed (Figures S1B,C). In addition, the morphological change to a spindle shape was not pronounced (Figure S1D). These findings indicate that the EMT phenotype in this cell line is partial. Meanwhile, Nakamichi et al. reported that in ALK‐TKI‐resistant cells generated through long‐term exposure to ALK‐TKI, the dependence on ALK signaling was reduced and a full EMT phenotype was observed along with AXL overexpression [22]. JFCR‐028‐4 might have tolerated alectinib treatment as DTP cells through possible activation of the GAS6/AXL signaling pathway and may have subsequently acquired stable treatment resistance through mechanisms that appear distinct from EMT. This hypothesis is partly supported by our observation that the addition of conditioned medium from NIH3T3‐GAS6 cells induced partial resistance to alectinib in JFCR‐028‐4 cells, whereas no such effect was observed in JFCR‐028‐3 cells. In H3122, AXL overexpression and GAS6 supplementation increased the number of cells that survived the alectinib treatment. However, unlike the resistant cells described by Nakamichi et al., these cells appeared to be partially dependent on the ALK signaling pathway, as evidenced by the effectiveness of high concentrations of ALK inhibitors (Figure S2A–E). Furthermore, in a murine model where GAS6 was supplied within the body, AXL‐overexpressing H3122 cells showed resistance to alectinib treatment. However, a partial response was found during the early stages of therapy. These results suggest that GAS6/AXL signaling contributes to the reduced efficacy of ALK‐TKIs by increasing the number of DTP cells.

According to the documented significance of the GAS6/AXL pathway in a large number of cancers, numerous drugs targeting the signaling pathway are currently being developed [17]. In ALK‐rearranged NSCLC, preclinical data indicate that the AXL inhibitor R428 or the HSP90 inhibitor ganetespib in combination with ALK‐TKIs is an effective treatment for ALK‐TKI‐resistant cells overexpressing AXL [22]. We previously reported that gilteritinib overcomes resistance via ALK‐TKI‐resistant secondary mutation and AXL activation [25]. The in vitro experiments in this study confirmed the efficacy of gilteritinib in overcoming resistance to ALK‐TKI, even when treatment resistance was augmented by the presence of GAS6 (Figure 3A). As aforementioned, the GAS6/AXL pathway contributes to the increase in the number of DTP cells, suggesting that early use of gilteritinib in therapy is beneficial. Indeed, in our previous in vivo experiments, superior tumor control was achieved when gilteritinib was administered from the outset compared with its sequential use after tumor growth under alectinib treatment [25]. Further investigation through clinical trials is warranted to ascertain the efficacy of early gilteritinib administration in the treatment of ALK‐rearranged NSCLC.

GAS6 expression has been documented in several cell types within the TME, including cancer‐associated fibroblasts (CAFs) [35, 36] macrophages [37, 38], dendritic cells [38], and neutrophils [39], in addition to tumor cells [24]. In this study, the administration of alectinib to the AXL‐overexpressing H3122 mouse model increased the production of host‐derived Gas6 within the TME (Figure 4D,F). Macrophages and fibroblasts were identified as the primary sources of Gas6 (Figures 5 and 6). Protein S, another known ligand for AXL, exhibited low expression levels and no substantial alterations after alectinib treatment. In a colon cancer model, Loges et al. reported that tumor cells educated tumor‐infiltrating TAMs to upregulate GAS6 production, thereby promoting tumor growth [38]. The CIBERSORTx and sc‐RNA‐seq analyses in this study revealed that the total number of infiltrating TAMs increased during alectinib treatment despite the absence of substantial alterations in the Gas6 expression (Figure 5A–C). The results support the findings of Loges et al. indicating that tumor‐infiltrating TAMs produce GAS6 and additionally reveal that the number of TAMs increase after alectinib treatment. In a mouse model using the lung cancer cell line H1299, Kanzaki et al. reported that CAFs enhanced GAS6 expression in response to cisplatin administration [36]. The results of this study indicate that CAFs enhance Gas6 expression in response to alectinib treatment (Figure 6A). This transcriptional alteration was characterized by Mmp11 upregulation (Figure 6B). Matrix metalloproteinases (MMPs) catalyze proteolytic activities and are involved in a range of physiological processes, including tissue remodeling. Moreover, they play a role in various pathological conditions, including cancer [40, 41]. MMP11 is mainly supplied by stromal cells, including CAFs and macrophages, and is expressed in numerous tumors. However, its expression in normal tissues is uncommon [42]. Haoran Yang et al. reported that MMP11 is highly expressed in lung adenocarcinoma (LUAD) tissues and that MMP11 deletion considerably inhibits the growth, migration, and invasion of LUAD cells [43]. High MMP11 expression has been reported to be negatively correlated with the efficacy of immunotherapy in EGFR‐mutated lung cancer [44] and is associated with poorer OS in patients with lung cancer [45]. Notably, Mmp11, which promotes malignant traits in cancer, is upregulated in CAFs during alectinib treatment. However, the mechanism underlying this change in gene expression and its association with Gas6 upregulation remain unclear and warrant further investigation. This study provides new insights into the regulation of GAS6 in the TME during alectinib treatment. A detailed understanding of the mechanisms underlying these changes in the TME may, in the future, facilitate the identification of novel therapeutic targets for intervention in the TME.

There are some important limitations to the current study. First, the findings regarding GAS6/AXL‐mediated resistance to ALK‐TKIs and changes in the TME were derived from experiments conducted using two cell lines. It remains unclear whether these findings universally occur in ALK‐rearranged lung cancers. Future studies are needed to examine whether similar changes will be observed in other ALK‐rearranged NSCLC cells in addition to H3122 and JFCR‐028‐3/4 cells. Second, the findings from sc‐RNA‐seq have not been experimentally validated. Since the cell counts detected by sc‐RNA‐seq are affected by multiple factors, the increase in macrophage counts during alectinib treatment needs to be confirmed by immunostaining of tumor sections or other methods. Third, it remains unclear whether MMP11 in fibroblasts functions as a cell marker or a pivotal regulator of GAS6. Additional experiments, such as MMP11 depletion in tumor hosts, are required to address this question.

In summary, the findings of this study indicate that increased TAMs and MMP11‐expressing CAFs during alectinib treatment increase GAS6 production in the TME, thereby enhancing resistance in AXL‐overexpressing ALK‐rearranged NSCLC. Furthermore, gilteritinib was shown to be effective in overcoming this GAS6/AXL mediated resistance.

Author Contributions

Takahiro Utsumi: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, software, validation, visualization, writing – original draft, writing – review and editing. Hayato Mizuta: data curation, formal analysis, investigation, methodology, resources, validation, writing – review and editing. Yosuke Seto: data curation, formal analysis, methodology, software, writing – original draft, writing – review and editing. Ken Uchibori: funding acquisition, resources, supervision, validation, writing – review and editing. Makoto Nishio: funding acquisition, resources, supervision, validation, writing – review and editing. Isamu Okamoto: funding acquisition, supervision, validation, writing – review and editing. Ryohei Katayama: conceptualization, formal analysis, funding acquisition, investigation, methodology, resources, software, supervision, validation, visualization, writing – original draft, writing – review and editing.

Ethics Statement

Approval of the research protocol by an Institutional Review Board: Japanese Foundation for Cancer Research (approval no. 2013‐1093). All animal procedures were performed in accordance with protocols approved by the JFCR Animal Care and Use Committee.

Consent

Written informed consent for all genetic and cell biological analyses was obtained from the patients.

Conflicts of Interest

R. Katayama is an editorial board member of Cancer Science. R. Katayama reports receiving research funding from Chugai Pharma, TOPPAN, and UBE. I. Okamoto reports receiving research funding and lecture fees from Chugai Pharma, as well as lecture fees from Takeda Pharma, and Pfizer. The others declare no conflicts of interest.

Supporting information

Data S1. Detailed Supporting Information and methods.

CAS-116-1034-s004.docx (46.7KB, docx)

Figures S1‐S4.

Figure S1. The patient derived cell line (PDC) established after development of alectinib resistance exhibited a partial EMT phenotype.

Figure S2. AXL/GAS6 signaling activation results in alectinib resistance, which can be overcome with gilteritinib.

Figure S3. The results of RNA sequencing analysis obtained from the in vivo tumor tissues.

Figure S4. The result of single cell RNA sequencing analysis of host mouse cells from vehicle or alectinib treated tumor tissue.

CAS-116-1034-s006.pdf (3MB, pdf)

Table S1. Primer lists used in this study.

CAS-116-1034-s003.xlsx (10.1KB, xlsx)

Table S2. Gene expressions of 58 RTKs in the two PDCs.

CAS-116-1034-s001.xlsx (12.2KB, xlsx)

Table S3. Gene lists of each cluster in scRNA sequencing analysis.

CAS-116-1034-s002.xlsx (238.2KB, xlsx)

Table S4. Gene lists of differentially expressed in cluster 5 and 6.

CAS-116-1034-s005.xlsx (33.7KB, xlsx)

Acknowledgments

We would like to thank Prof. Siro Simizu for the helpful discussion, and thank Ms. Sumie Koike, Dr. Nobuyuki Kondo and Dr. Ai Takemoto from the JFCR for their help with in vitro and in vivo experiments. We would also like to thank the patient whose cells were used in this research, who participated in this study.

Funding: This study was supported in part by JST SPRING grant no. JPMJSP2136 (to T. Utsumi), and by MEXT/JSPS KAKENHI grant nos. JP22K18383, JP24K02333 (to R. Katayama), JP22K16205 (to K. Uchibori), and the grant from the AMED grant nos. JP24ama221210h0003, JP24ck0106795h0002, and JP24ama221231h0001 (to R. Katayama) and the grants from the Chugai Foundation for Innovative Drug Discovery Science, Kobayashi Foundation for Cancer Research, and Mitsubishi Foundation (to R. Katayama), and the grants from the Nippon Foundation and Takeda Science Foundation.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1. Detailed Supporting Information and methods.

CAS-116-1034-s004.docx (46.7KB, docx)

Figures S1‐S4.

Figure S1. The patient derived cell line (PDC) established after development of alectinib resistance exhibited a partial EMT phenotype.

Figure S2. AXL/GAS6 signaling activation results in alectinib resistance, which can be overcome with gilteritinib.

Figure S3. The results of RNA sequencing analysis obtained from the in vivo tumor tissues.

Figure S4. The result of single cell RNA sequencing analysis of host mouse cells from vehicle or alectinib treated tumor tissue.

CAS-116-1034-s006.pdf (3MB, pdf)

Table S1. Primer lists used in this study.

CAS-116-1034-s003.xlsx (10.1KB, xlsx)

Table S2. Gene expressions of 58 RTKs in the two PDCs.

CAS-116-1034-s001.xlsx (12.2KB, xlsx)

Table S3. Gene lists of each cluster in scRNA sequencing analysis.

CAS-116-1034-s002.xlsx (238.2KB, xlsx)

Table S4. Gene lists of differentially expressed in cluster 5 and 6.

CAS-116-1034-s005.xlsx (33.7KB, xlsx)

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