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Abstract
Background The dynamic interplay between tyrosine kinase inhibitors (TKIs) and the tumor immune microenvironment (TME) plays a crucial role in the therapeutic trajectory of non-small cell lung cancer (NSCLC). Understanding the functional dynamics and resistance mechanisms of TKIs is essential for advancing the treatment of NSCLC.
Methods This study assessed the effects of short-term and long-term TKI treatments on the TME in NSCLC, particularly targeting epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations. We analyzed changes in immune cell composition, cytokine profiles, and key proteins involved in immune evasion, such as laminin subunit γ−2 (LAMC2). We also explored the use of aspirin as an adjunct therapy to modulate the TME and counteract TKI resistance.
Results Short-term TKI treatment enhanced T cell-mediated tumor clearance, reduced immunosuppressive M2 macrophage infiltration, and downregulated LAMC2 expression. Conversely, long-term TKI treatment fostered an immunosuppressive TME, contributing to drug resistance and promoting immune escape. Differential responses were observed among various oncogenic mutations, with ALK-targeted therapies eliciting a stronger antitumor immune response compared with EGFR-targeted therapies. Notably, we found that aspirin has potential in overcoming TKI resistance by modulating the TME and enhancing T cell-mediated tumor clearance.
Conclusions These findings offer new insights into the dynamics of TKI-induced changes in the TME, improving our understanding of NSCLC challenges. The study underscores the critical role of the TME in TKI resistance and suggests that adjunct therapies, like aspirin, may provide new strategies to enhance TKI efficacy and overcome resistance.
- Lung Cancer
- Tumor microenvironment - TME
- Immunosuppression
Data availability statement
No data are available.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor mutations and anaplastic lymphoma kinase fusions have improved non-small cell lung cancer (NSCLC) outcomes but often face limitations due to acquired resistance. Changes in the tumor microenvironment (TME) are suspected to play a role in this resistance, yet the interactions between TKIs and the TME remain poorly understood.
WHAT THIS STUDY ADDS
This study reveals dynamic TME changes with TKI treatment in NSCLC: short-term use boosts antitumor immunity, while long-term use leads to immunosuppression. It identifies aspirin as a potential adjunct therapy to modulate these changes and enhance TKI efficacy. Additionally, it addresses clinical issues like short treatment duration, high regression rates, advantages of ALK-TKIs, and reduced immunotherapy effectiveness after TKI resistance.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
This study provides a deeper understanding of TKI-induced TME dynamics, potentially guiding future research on overcoming resistance in NSCLC. It supports the development of combination therapies with TKIs and aspirin, which may improve treatment efficacy.
Introduction
In the evolving treatment landscape of non-small cell lung cancer (NSCLC), the recent advent of immune checkpoint inhibitors (ICIs) and targeted tyrosine kinase inhibitors (TKIs) has marked a significant paradigm shift.1 2 TKIs have become crucial in NSCLC management by specifically targeting genetic mutations, such as epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusions.3 While TKIs demonstrate notable initial efficacy, their long-term effectiveness is often challenged by the emergence of resistance.4 Unlike the response to TKIs, the overall response rate to ICIs may be lower,5 6 but for patients who respond, ICIs offer durable effects, reducing the likelihood of resistance development and enabling longer-term survival.7 8 Their success predominantly stems from their profound impact on the tumor microenvironment (TME), reactivating the immune system’s ability to identify and eradicate tumor cells, thereby fostering a milieu conducive to sustained immune responses.9–11 A thorough understanding of the TME is increasingly recognized as crucial in optimizing cancer treatments, as the TME plays key roles in the effectiveness and development of resistance to therapies, particularly in NSCLC.12 13 Traditionally, resistance to TKIs in NSCLC has been primarily attributed to tumor cell-intrinsic factors, including genetic mutations and alterations in signaling pathways.14 As a result, it is generally believed that targeted drugs may exert control through inhibition rather than elimination. However, recent research has unveiled the potential roles of TKIs in modulating the TME, suggesting that different oncogenic driver mutations may lead to distinct TME characteristics.3 15 16
The increasing application of neoadjuvant therapies has significantly enhanced our understanding of how both immunotherapy and targeted treatments influence the TME in NSCLC.17 In particular, neoadjuvant immunotherapy, often used in conjunction with chemotherapy, has been associated with higher pathological response rates.18 19 Patients achieving pathological remission typically exhibit better long-term survival outcomes, which may be largely attributed to the profound impact of ICIs on the TME,18 20–22 a phenomenon linked to increased functional T-cell infiltration within the tumor areas.17 These observations support the notion that an immune-activated TME is instrumental in achieving pathological remission and improving prognosis.23 Consequently, the modulation of the TME to amplify immune responses has emerged as a promising strategy to enhance the efficacy of NSCLC treatments.24 In contrast, studies on various neoadjuvant targeted therapies, especially those involving EGFR-TKIs, have demonstrated lower rates of pathological remission.25 This disparity in pathological responses between immunotherapy and targeted therapy highlights a potential factor contributing to the development of resistance in targeted treatments.26 This observation raises critical questions regarding the underlying mechanisms that constrain the long-term effectiveness of targeted therapies and contribute to the development of resistance.
Additionally, certain clinical observations remain puzzling, such as the ability of targeted therapies to significantly inhibit overall tumor growth despite only targeting a small subset of tumor cells with specific mutations. Furthermore, the effectiveness of immunotherapy often appears to be diminished when administered after the development of resistance to targeted therapy.27 ALK-TKIs, in contrast to EGFR-TKIs, tend to result in longer progression-free survival (PFS) and higher rates of pathological remission in neoadjuvant therapy.28 Unraveling the exact mechanisms behind these complex phenomena requires in-depth explorations into the nuances of targeted therapy.
Our research endeavors to unravel the intricacies of how the TME influences the efficacy of targeted treatments. Our findings indicate that the degree of immune suppression within the TME significantly contributes to the development of resistance against TKIs. Furthermore, we have identified novel strategies for its modulation, which hold the promise of delaying the onset of resistance and enhancing the durability of TKI efficacy. Moreover, our research helps to explain many confusing clinical issues in targeted therapy for lung cancer.
Materials and methods
Cell lines and reagents
Human lung cancer cell lines HCC827, H3255, PC-9 and H2228 were purchased from the American Type Culture Collection. The H3122 human lung adenocarcinoma cell line was obtained from Shanghai Bioleaf Biotech (Shanghai, China). PC-9GR (gefitinib resistance due to T790M mutation) was created through chronic exposure of the drug at gradual dose increments. The osimertinib-resistant cell lines PC-9OR, H1975OR, PC-9GROR, HCC827OR and lorlatinib-resistant cell lines H3122LR, and crizotinib-resistant cell lines H2228CR were also established in our laboratory. All of these cell lines were maintained in Roswell Park Memorial Institute 1640 medium (Gibco, California, USA) with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/mL) in an incubator with 5% CO2 atmosphere at 37°C.
Osimertinib (S7297), lorlatinib (S7536), gefitinib (S1025), crizotinib (S1068),and aspirin (S3017) were obtained from Selleck Chemicals. alectinib (T1936) was from TargetMol. Anifrolumab (HY-P99168) was obtained from MedChemExpress.
Cell viability assay
Cell viability was determined using a Cell Counting Kit-8 assay (CCK-8, Bioground, Chongqing, China). Briefly, cells were seeded in a 96-well plates at a density of 3×103 cells per well and cultured overnight. On the next day, the medium was replaced with the indicated doses of drug-containing medium and cultured for another 48 hours. Following the replacement of the treatments with fresh medium, the absorbance in each well was measured at 450 nm on a Sunrise R Microplate Reader (Thermo Fisher Scientific, Germany).
Cell proliferation
Cell proliferation was assessed by Ki67 staining. Briefly, cells were seeded in 6-well plates (3×105) and treated as indicated for 48 hours. Then, the cells were fixed and incubated overnight with Ki67 (#M00254-8, Boster, Wuhan, China). They were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min and observed under a fluorescence microscope.
Western blot analysis
Tumor cells were grown and treated as indicated, and total cell proteins were harvested by scraping and quantitated using a bicinchoninic acid protein assay kit (Bioground, Chongqing, China). The following primary antibodies against Bim (#2933S), B-cell lymphoma extra-large (BCL-XL, #2764S), myeloid leukemia cell 1 (MCL-1, #94296), protein kinase B (Akt, #9272S), phospho (Ser473)-Akt (#4060S), caspase-3 (#9662S), cleaved caspase-3 (#9664S), phospho (Tyr1068)-EGFR (#3777S), EGFR (#4267S), phospho (Ser172)-TANK-binding kinase 1 (TBK1, #5483S), TBK1 (#38066S), stimulator of interferon genes (STING, #13647S), phospho (Ser386)-interferon regulatory factor 3 (IRF3, #37 829S), IRF3 (#4302S), phospho (Ser536)-nuclear factor κb (NF-κb) p65 (#3033S), NF-κb p65 (#4764S), programmed death ligand 1 (PD-L1, #13684S), CD8α (#85336S), granzyme B (GB, #46 890S), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3, #45208S), CD68 (#97778S), CD86 (#91882S), CD163 (#68922S), inducible nitric oxide synthase (iNOS, #20609S), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, #2118) were obtained from Cell Signaling Technology. An antibody against CD206 (PTM-6020) was obtained from PTM-BioLab. An antibody against laminin subunit γ−2 (LAMC2, ab210959) was obtained from Abcam. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG was from Sino Biological (Beijing, China).
Immunohistochemistry staining
Tumor tissues were fixed and embedded with paraffin. Then, 3 mm formalin-fixed, paraffin-embedded slides were prepared for immunohistochemical staining. Briefly, tumor sections were deparaffinized, and tissues were incubated with specific primary antibodies. An anti-rabbit goat IgG labeled with horseradish peroxidase (HRP, ZSGB-Bio, China) was used as the secondary antibody. Staining was performed using the protocol from the DAB display reagent kit.
Generation of activated T cells
Activated T cells were acquired as previously reported.29 Briefly, human peripheral blood T cells were cultured in ImmunoCult-XF T-cell expansion medium with ImmunoCult Human CD3/CD28/CD2 T-cell activator (both from STEMCELL Technologies, Vancouver, California, USA) and interleukin 2 (IL-2, 10 ng/mL; Sino Biological, China) for 1 week according to the manufacturer’s protocol. All experiments were performed in DMEM/F12 with anti-CD3 antibody (100 ng/mL; eBioscience, Thermo Scientific, Massachusetts, USA) and IL-2 (10 ng/mL).
T cell-mediated cancer cell killing assay
Cells were seeded in 6-well plates (3×105) and treated as indicated for 48 hours. T cells and cell debris were removed by washing with phosphate-buffered saline, and cancer cells were subjected to crystal violet staining.
ELISA
Interferon (IFN)-γ and tumor necrosis factor α (TNF-α) ELISA kits were purchased from Solarbio (Beijing, China), and IFN-α and IFN-β ELISA kits were purchased from FineTest (Wuhan, China). The ELISAs were performed according to the manufacturers’ instructions. Values represent the average of three replicates from at least three independent experiments.
Immunofluorescence staining
Immunofluorescence staining of tumor tissue sections was performed as described in a previous study.30 For cyto-immunofluorescence staining, cancer cells were seeded in 6-well plates with pre-placed 20 mm glass slides. The immunofluorescence staining procedures were similar to the tissue staining procedure mentioned above.
Multiplex immunofluorescence detection
Multiplex immunofluorescence (mIF) analysis of tumor tissue sections was performed as described in a previous study.31
Flow cytometry
Cells were stained with antibodies against cell-surface molecules for 20 min at 4°C. The surface staining antibodies included an antibody against CD8 (IM2469, Beckman). For intracellular staining, cells were fixed and permeabilized with forkhead box P3 (Foxp3) fixation/permeabilization solution (IM2469, Thermo Fisher Scientific) for 20 min at 4°C and were then stained with antibodies against intracellular molecules for 20 min at 4°C. The stained cells were analyzed using CytoFLEX fluorescence-activated cell sorting (FACS, Beckman Coulter, Brea, California, USA), and the data were analyzed using FlowJo software V.10 (Treestar, San Carlos, California, USA).
T cells chemotaxis assay
T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Beyotime) at 37°C for 20 min. Horizontal slide chemotaxis chambers (#80326, Ibidi) with or without Matrigel (#356230, Corning Incorporated) were used to investigate the chemotaxis and migration of CFSE-labeled T cells. Matrigel was supplemented with activated or heat-inactivated laminin γ2 (Ln-γ2, #MBS2030234, MyBioSource) or conditioned medium. The number of migratory T cells was imaged with a fluorescence microscope (Leica, German) after 24 hours of culture at 37°C in a humidified chamber containing 5% CO2.
Macrophages induced polarization
THP-1 cells were polarized into M0, M1, and M2 macrophages as previously described. The cells were subjected either to 100 ng/mL phorbol 12-myristate 13-acetate (PMA, Solarbio, China) for 48 hours to obtain M0 macrophages, 20 ng/mL IFN-γ (300–02, PeproTech, USA) plus 100 ng/mL lipopolysaccharide (LPS, L2280, Sigma, USA) for 48 hours to obtain M1 macrophages, or 20 ng/mL IL-4 (200–04, PeproTech, USA) plus 20 ng/mL IL-13 (200–13, PeproTech, USA) for 48 hours to obtain M2 macrophages.
Animal experiments
Animal experiments were approved by the ethics committee on animal experimentation of the Army Medical University. To establish PBMCs-CDX(Peripheral blood mononuclear cells-cell derived xenograft) mouse model (Lin et al, 2018), PC-9 cells (5×105) were injected into the hind flanks of 6-week-old female NOD-SCID mice. The mice were subjected to tumor growth monitoring, and tumor volumes were calculated from caliper measurements using the following formula: (length×width2/2. When tumors reached 100 mm3 in volume, 5×106 human PBMCs were intravenously transplanted. Osimertinib (2 mg/kg) and aspirin (20 mg/kg) were dissolved in drinking water and given to mice orally. Tumor growth was monitored every 3 days. Tumors were harvested, fixed with 4% paraformaldehyde, and embedded in paraffin. The infiltration of CD8+ T cells were stained with immunohistochemistry and the expression of LAMC2, CD68, CD206 was determined by immunofluorescence staining.
Statistical analysis
All datas are expressed as the mean±SEM, and the statistical analyses were performed in GraphPad Prism V.8 for Windows (GraphPad Software, San Diego, California, USA, www.graphpad.com). Differences between the two groups were analyzed by Student’s t-test. Results were considered to be statistically significant at p<0.05.
Result
TKI treatment alters the tumor immune microenvironment
To explore the features of immune cell composition in the TME between EGFR-TKI and ALK-TKI in patients with NSCLC, we first performed multiplex immunochemistry staining to quantitatively analyze immune cells in the TME of pre-TKI and post-TKI treatment tissue samples. In the post-TKI treatment tissue samples, we observed significant increases in the density of CD8+, CD8+GB+, and CD8+ programmed cell death protein 1 (PD-1) + cells within the TME, particularly in ALK fusion-positive samples (figure 1A,B and online supplemental figure S1A), indicating that TKI treatment enhances the antitumor immune response in the TME. In contrast, tissue samples treated with EGFR-TKI exhibited significant increases in CD68+ and CD163+ macrophages that were not observed in ALK TKI-treated pathological samples, indicating a potential enrichment of M2 macrophages (online supplemental figure S1B and figure 1B). Further pathological analysis revealed a tendency toward near-complete pathological remission in ALK fusion-positive tissue samples following targeted therapy (online supplemental figure S1C). These results suggest that TKI treatment can alter the immune cell landscape.
Supplemental material
TKI therapy alters the tumor immune microenvironment. (A) Representative mIF images of pretreatment tumor and resected samples analyzed for immune-related biomarkers. (B) Densities (cells/mm2) of CD8+, CD8+GB+, CD8+PD-1+, CD163+, CD68+, and CD163+CD68+ by mIF quantification in paired pretreatment tumor samples and resected tumors. (C) Cell viability CCK-8 assay for cells treated with TKIs (osimertinib: 10 nM, lorlatinib: 10 nM), activated T cells (1:1 ratio to cancer cells), or the combination. (D) T cell-mediated cancer cell-killing assay. PC-9 and H3122 cells co-cultured with activated T cells for 48 hours with or without TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were subjected to crystal violet staining. Ratio of cancer cells to T cells: 1:1. (E) Ki67 incorporation assay on PC-9 and H3122 cells treated as indicated. Activated T cells (1:1 ratio to cancer cells) or TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into mice via the tail vein on day 7, and osimertinib was administered as indicated. (G) Macroscopic appearance of tumors after drug application for 4 weeks. (H) The tumor weight (g) for each mouse is shown. *p<0.05, **p<0.01. ns, no significance. (I) Immunofluorescence staining with an antibody against CD8 to detect T cells and antibodies against CD68 and CD206 to detect macrophages in TKI-resistant non-small cell lung cancer tissues (11 cases of EGFR-TKI resistance, 5 cases of ALK-TKI resistance). Scale bar: 50 µm. ALK, anaplastic lymphoma kinase; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; ALKi,anaplastic lymphoma kinase inhibitor; EGFRi,epidermal growth factor receptor inhibitor; hu-PBMC,human-Peripheral blood mononuclear cell; mIF, multiplex immunofluorescence; PD-1, programmed cell death protein 1; TKI, tyrosine kinase inhibitor.
To further validate the crucial role of activated T cells in enhancing the tumoricidal effects of TKI, we first defined the concentration of T cells co-cultured with cancer cells (online supplemental figure S1D,E). Subsequently, we observed that EGFR-TKI and ALK-TKI combined with activated T cells not only significantly reduced the viability of cancer cells but also reduced the number of Ki67-positive cells (figure 1C–E and online supplemental figure S1F,G). We then examined the expression levels of several proapoptotic proteins, including Bim and caspase 3, and found that they were increased in a time-dependent manner in the presence of T cells (online supplemental figure S1I). In addition, in the PBMC-CDX humanized mouse model, we observed that treatment with osimertinib alone reduced tumor sizes, while osimertinib treatment in the presence of PBMCs resulted in more significant tumor shrinkages (figure 1F–H). Thus, we further validated that the therapeutic efficacy of TKI treatment was enhanced by adaptive immunity.
To further explore the effect of TKI treatment on T-cell function, we observed that TKI drugs had no effect on the release of pro-inflammatory cytokines (IFN-γ and TNF-α) from T cells by ELISA (online supplemental figure S1H). This result indicated that TKIs may not directly alter the function of T cells. Interestingly, we observed that CD8 expression decreased and M2 macrophages (CD68+ and CD206+ macrophages) increased in the tissues of patients resistant to TKIs, especially in patients resistant to EGFR-TKI. Immunofluorescence staining revealed an immunosuppressive state in TKI-resistant NSCLC tissues (figure 1I). Under the co-culture conditions (macrophages and cancer cells), we also found that M2 macrophages attenuated the antitumor efficacy of EGFR-TKIs, whereas M1 macrophages potentiated the antitumor efficacy of EGFR-TKIs (online supplemental figure S1J).
The above findings suggest that TKIs have the potential to modulate the tumor immune microenvironment, thereby influencing the development of TKI resistance through their impact on immune cell function in the presence of tumor cells.
Short-term TKI treatment activates T-cell functionality
Focusing on the impact of short-term TKI treatment on T-cell function, we particularly examined the activation of the type I IFN signaling pathway at various TKI concentrations. We first observed dose-dependent increases in the phosphorylation of the key proteins TBK1 and IRF3 following EGFR-TKI (osimertinib) treatment, and ALK-TKI (lorlatinib) treatment led to the activation of Phosphorylation-Nuclear factor kappa B(p-NFκB) (figure 2A and online supplemental figure S2A). Correspondingly, increases in the secretion of INF-α and INF-β post-TKI treatment were noted, underscoring the capacity of TKIs to rapidly activate T-cell immune functions in a dose-dependent manner (figure 2B). Further investigations, focusing on the expression levels of the STING protein, revealed that EGFR-mutant cell lines exhibited relatively lower expression levels of STING pathway proteins compared with ALK fusion-positive cell lines (online supplemental figure S2B), while TKI treatment did not significantly alter their expression (online supplemental figure S2C). These results suggest that STING may not be the dominant player in the immune activation process induced by TKIs. Furthermore, we noted that short-term treatment with osimertinib resulted in significant dose-dependent and time-dependent upregulations of the type I IFN signaling pathway. Notably, this phenomenon first increased and then decreased in HCC827 cells (figure 2C,D). A contrasting downregulation of the pathway was observed in drug-resistant cell lines treated with a first-generation TKI, such as gefitinib (online supplemental figure S2D). Then, we found that administration of the type I IFN blocker anifrolumab weakened the cell-killing effect induced by the combination treatment of TKIs and T cells (figure 2E and online supplemental figure S2E), indicating that type I IFN may function as the mediator underlying the T cell-mediated antitumor effect induced by TKIs. Additionally, our research further revealed that short-term TKI treatment led to the suppression of PD-L1 expression (figure 2F and online supplemental figure S2F). Further, we observed increases in INF-γ and TNF-α levels in T cells co-cultured with ALK fusion-positive cell lines post-TKI treatment, while decreases were commonly noted in T cells co-cultured with EGFR-mutant cell lines (figure 2G,H). Moreover, as the medication time increased, the expression levels of CD8 and GB in EGFR-TKI-treated T cells co-cultured with tumor cells first increased and then decreased, while the expression levels of ALK-TKI-treated proteins decreased and then increased (figure 2I). These results suggest that short-term TKI treatment has a complex impact on enhancing the immune response against tumors, and EGFR-TKI and ALK-TKI have distinct effects on the TME.
Short-term treatment with TKI activates T-cell function. (A) Levels of phosphorylated AKT (p-AKT), p-TBK1, and p-IRF3 in cancer cells treated with different concentrations of TKI for 12 hours, analyzed using western blotting. (B) ELISA analysis of IFN-α and IFN-β levels in cancer cells following treatment with different concentrations of TKI (0, 1, or 5 µM) for 24 hours. (C) Levels of the indicated proteins in cancer cells treated with different concentrations of TKI for 48 hours, analyzed using western blotting. (D) Levels of the indicated proteins in tumor cells treated with 1 µM TKI for different periods of time, analyzed by western blotting. (E) Cell viability CCK-8 assay for cells treated with TKIs (osimertinib: 10 nM, lorlatinib: 10 nM), activated T cells (1:1 ratio to cancer cells), anifrolumab (10 µg/mL), or the combination. (F) Levels of PD-L1 in cancer cells treated with different concentrations of TKI for 12 hours, analyzed by western blotting. (G) Schematic workflow for the co-culture of cancer cells and T cells in Transwells. (H) ELISA analysis of the TNF-α and IFN-γ protein expression levels in T cells treated under the indicated conditions for 24 hours. (I) Levels of CD8 and GB in T cells treated under the indicated conditions, analyzed by western blotting. EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GB, granzyme B; IFN, interferon; IRF3, interferon regulatory factor 3; PD-L1, programmed death ligand 1; TBK1, TANK-binding kinase 1; TNF, tumor necrosis factor; TKI, tyrosine kinase inhibitor.
Long-term TKI treatment induces resistance in NSCLC and fosters an immunosuppressive microenvironment
We then asked how TKI treatment impacted the immune microenvironment in NSCLC over the long-term. By comparing sensitive cell lines and their counterpart TKI-resistant cell lines, we observed that resistant cell lines demonstrated notable resistance to T cell-mediated cytotoxicity compared with their parental cell lines (figure 3A,B and online supplemental figure S3A,B). Further molecular-level analyses revealed significant changes in the expression of immune checkpoint molecules compared with the sensitive cells, and western blot analysis showed that the expression of TIM3, an immune checkpoint molecule, in T cells co-cultured with osimertinib-resistant cells increased, while the expression levels of GB and CD8, key proteins related to T-cell activity, decreased; furthermore, the levels of secreting proinflammatory cytokines (eg, INF-γ) also decreased. Following long-term treatment with osimertinib or in osimertinib-resistant cells, the expression levels of TIM3 and PD-1 were increased, while GB and CD8 expression levels were decreased, as was the secretion of INF-γ. The expression levels of TIM3, PD-1, and CD8 and INF-γ secretion were decreased in TKI-resistant ALK fusion-positive cells. After lorlatinib treatment, the expression of TIM3 in T cells decreased, and the expression levels of PD-1, GB, and CD8 showed decreasing trends, as did the secretion of IFN-γ (figure 3C–E and online supplemental figure S3C). These results revealed that resistant cells may evade T cell-mediated immune responses by modulating surface molecules and cytokine secretion.
Long-term treatment with TKI inactivates T-cell function. (A) Cell viability CCK-8 assay for cells treated with T cells (at the indicated ratios to cancer cells). (B) T cell-mediated cancer cell-killing assay. PC-9OR and H3122LR cells co-cultured with activated T cells for 48 hours were subjected to crystal violet staining. Ratio of cancer cells to T cells: 1:3. (C) Schematic workflow for the co-culture of resistant cells and T cells in transwells. (D) Levels of the indicated proteins in T cells treated with different TKIs (osimertinib: 1 µM, lorlatinib: 1 µM). (E) ELISA analysis of IFN-γ protein expression in T cells treated with TKIs under the indicated conditions for 24 hours (osimertinib: 1 µM, lorlatinib: 1 µM). (F) PD-L1 protein expression levels in parent cells and resistant cells, analyzed by western blotting. (G) Western blotting and immunofluorescence staining (H) of PD-L1 protein expression in parent cells and resistant cells following treatment with TKIs (osimertinib: 1 µM, lorlatinib: 1 µM). (I) Schematic workflow of the co-culture of resistant cell medium and macrophages. (J) Levels of CD206 and CD86 in the indicated groups, analyzed by western blotting. (K) Immunofluorescence staining with antibodies against CD68 and CD206 to detect macrophages in PC-9 xenografts following treatment with osimertinib. (L) Schematic workflow for the co-culture of macrophages, resistant cells, and T cells by direct contact. (M) T cell-mediated cancer cell-killing assay. PC-9 and H3122 cells co-cultured under the indicated conditions for 48 hours were subjected to crystal violet staining. Ratio of cancer cells to T cells: 1:3. (N) Cell viability CCK-8 assay for T cells treated under the indicated conditions (ratio of cancer cells to T cells: 1:3). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFN, interferon; PD-L1, programmed death ligand 1; TIM3, T-cell immunoglobulin and mucin domain-containing protein 3; TKI, tyrosine kinase inhibitor.
Moreover, we observed a series of significant immune-related changes in TKI-resistant cell lines, such as a decrease in INF-α levels (online supplemental figure S3D). Western blot analysis further revealed decreases in p-TBK1 and p-IRF3 expression in drug-resistant cell lines (online supplemental figure S3E), reflecting the strategies employed by drug-resistant cells to evade immune surveillance by weakening type I IFN production. Then, we observed that PD-L1 expression decreased after TKI treatment in parental cells, while it increased in EGFR mutation-carrying resistant cells; however, there was no significant change in ALK fusion-positive resistant cells (figure 3F–H and online supplemental figure S3F–H), suggesting that ALK fusion-positive resistant cells might not suppress immune responses through PD-L1-mediated pathways.
Through co-culture with conditioned medium with TKI-resistant cells, we observed an increase in the expression of the M2 macrophage marker CD206 and a decrease in the expression of the M1 macrophage marker CD86 (figure 3I,J). The enrichment of M2 macrophages was further confirmed through histological analysis, indicating that TKI-resistant cells not only altered direct immunosuppressive molecules but also affected immune cells like macrophages in the TME, further enhancing immunosuppression (figure 3K). Additional experiments revealed the significant inhibitory impact exerted by TKI-resistant cells and their associated M2-polarized macrophages on the cytotoxic function of T cells (figure 3L–N), highlighting the critical role of tumor-induced macrophage polarization in the attenuation of T cell-mediated antitumor responses. Taken together, these findings suggest that long-term TKI treatment might contribute to dampening the direct regulation of T-cell immune efficacy in TKI-resistant cells and indirect regulation by inducing the polarization of M2 macrophages.
TKI treatment enhances T-cell infiltration by downregulating LAMC2 expression
We then explored how short-term TKI therapy enhances T-cell infiltration within tumors. Through the analysis of mIF data from pre-TKI and post-TKI treatment tissue samples, we observed a significant increase in immune cell infiltration in the tumor area following short-term TKI treatment (figure 4A). Then, a significant shrinkage of transplanted tumors (PC-9 and H3122 cells) and an increase in CD8+ T cells were observed after TKI treatment (figure 4B–D and online supplemental figure S4A), emphasizing the important role of short-term TKI treatment in activating the TME.
TKI treatment promotes T-cell infiltration by inhibiting LAMC2 protein expression. (A) Density (cells/mm2) of CD8+, CD8+GB+, CD68+, and CD68+CD163+ cells in tumors and stroma, quantified by multiplex immunofluorescence in paired pretreatment tumor samples and resected tumors. (B) PC-9 cells and H3122 cells were injected into Hu-HSC mice (n=3 mice per group) on day 0, and osimertinib or crizotinib was administered as indicated. (C) Tumor growth curves of cell-derived xenograft mouse models treated as indicated. Tumor volume is shown as the mean±SEM (n=3). The tumor weight (g) of each mouse is shown. (D) Immunohistochemistry analysis of CD8 expression in tumor sections from the different groups. Representative images are shown. (E) LAMC2 expression levels in the indicated cells, analyzed by western blotting. (F) ELISA analysis of LAMC2 levels in PC-9 cells and H3122 cells following treatment for 24 hours. (G) Schematic diagram of a T-cell chemotaxis assay directly regulated by the addition of medium from PC-9 and H3122 cells. (H) Representative images of infiltrating T cells stained with CFSE dye. (I) Immunofluorescence staining with antibodies against CD8 and LAMC2 in tissues harboring EGFR mutations and ALK fusions. (J) LAMC2 expression levels in cancer cells treated with different concentrations of TKIs for 12 hours, analyzed by western blotting. (K) Representative images of infiltrating T cells stained with CFSE dye, with TKI inhibition directly regulated by the recombinant protein Ln-γ2. (L) Immunofluorescence staining with antibodies against CD8 and LAMC2 in non-small cell lung cancer tissues harboring EGFR mutations and ALK fusions following treatment with TKIs. ALK, anaplastic lymphoma kinase; CFSE, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMC2, laminin subunit γ−2; PBS, phosphate-buffered saline; TKI, tyrosine kinase inhibitor.
Previous studies have shown that LAMC2 expression plays a key role in regulating the ability of T cells to penetrate and attack tumors.32 We first analyzed LAMC2 expression in a Gene Expression Profiling Interactive Analysis (GEPIA) data set (http://gepia.cancer-pku.cn/) and found LAMC2 to be highly expressed in patients with lung cancer (online supplemental figure S4B). Then, the differential LAMC2 expression patterns observed in western blot and ELISA analyses revealed that tumor cells positive for ALK fusions exhibited significantly lower LAMC2 expression and secretion compared with cells with EGFR mutation, which may explain why better clinical outcomes were achieved with ALK-TKI (figure 4E,F). T-cell infiltration experiments further confirmed that EGFR mutant cells more strongly impeded the infiltration of immune cells compared with ALK fusion cells (figure 4G,H). As observed in tissue immunofluorescence analysis, LAMC2 expression in EGFR mutant tumor tissue was higher than that in ALK fusion tissue (figure 4I), suggesting that LAMC2 expression in EGFR mutant tumors leads to reduced T-cell infiltration, thereby limiting the sensitivity of tumors to immune responses.
Our study found that LAMC2 expression was significantly inhibited by short-term treatment with various TKIs (online supplemental figure S4C). We also observed a dose-dependent decrease in LAMC2 protein expression after TKI treatment (figure 4J and online supplemental figure S4D–E). Immunohistochemical analysis of mouse transplanted tumor tissues further confirmed that TKI treatment could inhibit the expression of LAMC2 protein (online supplemental figure S4F). Moreover, T-cell infiltration experiments demonstrated that short-term TKI treatment inhibited the expression of LAMC2, thus promoting T-cell infiltration (figure 4K). In mouse models, immunofluorescence labeling revealed a significant correlation between the decrease in LAMC2 protein expression in TKI-treated tumor tissues and the increase in T-cell infiltration (online supplemental figure S4G). Pathological tissue immunofluorescence analysis of human tumor samples confirmed that TKI treatment reduced LAMC2 protein expression in tumor tissues, accompanied by a significant increase in T-cell infiltration (figure 4L).
These results collectively indicate that TKI treatment effectively alters the TME by regulating the expression of LAMC2 protein, enhancing T-cell infiltration and activity.
Long-term TKI treatment induces LAMC2 overexpression and inhibits T-cell infiltration
To explore the effects of long-term TKI treatment on the TME, we detected the expression of LAMC2 at different time points (2, 4, and 6 weeks) during osimertinib treatment in PC-9 xenografted tumors and observed that LAMC2 expression gradually increased in a time-dependent manner; in contrast, it was suppressed in the early stages of short-term TKI treatment (figure 5A), consistent with the results in published RNA sequencing data (GSE193258) (online supplemental figure S5A). Subsequent pathological tissue immunofluorescence analysis revealed significant overexpression of LAMC2 protein following long-term TKI treatment and resistance, accompanied by a significant decrease in T-cell infiltration (figure 5B), indicating a shift in the TME toward an immunosuppressive state. To delve deeper into the role of the LAMC2 protein in tumor immune evasion, we observed increased expression of LAMC2 in TKI-resistant cells (figure 5C–E and online supplemental figure S5B); in addition, LAMC2 overexpression could not decrease the sensitivity to TKI in the PC-9 and H3122 cell lines. These results bolstered the hypothesis that prolonged TKI treatment may induce alterations in the TME, particularly affecting immune cell infiltration and functionality. By employing ELISA and T-cell infiltration assays, we established a link between increased LAMC2 secretion in TKI-resistant cells and a corresponding decrease in T-cell infiltration capabilities (figure 5F,G). These observations underscore the pivotal role of the LAMC2 protein in modulating the tumor immune environment. We also observed that inhibiting LAMC2 protein expression in resistant cells effectively restored the T cell-mediated tumor-killing functions (figure 5H–J, online supplemental figure S5E,F). These findings are crucial for understanding the long-term effects of TKI treatment and their impact on the tumor immune environment, offering a new perspective on the profound influence of long-term TKI treatment on the TME.
Long-term treatment with TKIs inhibits T-cell infiltration by increasing LAMC2 expression. (A) Immunofluorescence staining with an antibody against LAMC2 in PC-9 xenografts treated with osimertinib. (B) Immunofluorescence staining with antibodies against CD8 LAMC2 in TKI-resistant non-small cell lung cancer tissues. (C) LAMC2 expression in parent cells and resistant cells, analyzed by western blotting. (D) Immunofluorescence staining and western blotting (E) of LAMC2 expression in parent cells and resistant cells treated with TKIs (osimertinib: 1 µM, lorlatinib: 1 µM). (F) ELISA analysis of LAMC2 levels in parent cells and resistant cells following treatment for 24 hours. (G) Schematic diagram of a T-cell chemotaxis assay directly regulated by the addition of media from parent cells and resistant cells. Representative images of infiltrating T cells stained with CFSE dye. (H) LAMC2 expression levels in cells following transfection with LAMC2 siRNAs. (I) Cell viability CCK-8 assay for T cells and cancer cells co-cultured at different ratios. (J) T cell-mediated cancer cell-killing assay. Cells co-cultured in the indicated groups for 48 hours were subjected to crystal violet staining. Ratio of cancer cells to T cells: 1:1. CFSE, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMC2, laminin subunit γ−2; TKI, tyrosine kinase inhibitor.
Aspirin intervention alters the tumor immune environment, overcoming TKI resistance
To further explore effective treatment options for overcoming TKI resistance by modulating the TME, we initially screened a range of potential drugs, including vinblastine, AZD0156, L-5-hydroxytryptophan, bis(benzonitrile) dichloroplatinum (II), albendazole, metformin, and aspirin. Among these, aspirin stood out due to its significant impact on the expression levels of key proteins in drug-resistant cells, namely PD-L1 and LAMC2 (online supplemental figure S6A), highlighting its potential as an immunomodulatory agent in cancer treatment. Our in vivo studies demonstrated that the combination of aspirin with osimertinib significantly overcame resistance to osimertinib in tumor models (figure 6A,B). This effect is speculated to be due to aspirin’s ability to enhance the immune response, potentially by decreasing PD-L1 expression. Further research revealed that aspirin significantly enhanced T cell-mediated tumor-killing effects (figure 6C–E and online supplemental figure S6B,C). These findings indicate that aspirin can activate T cells and augment their capacity to target tumor cells effectively.
Aspirin enhances the antitumor immunity response. (A) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into the tail veins of mice on day 7, and osimertinib/osimertinib plus aspirin was administered as indicated. (B) Macroscopic appearance of tumors after drug application for 4 weeks. The tumor weight (g) of each mouse is shown. *p<0.05, ***p<0.001. (C) Cell viability CCK-8 assay for cells treated with aspirin (200 µM), activated T cells (1:2 ratio to cancer cells), or the combination for 48 hours. Data are shown as the mean±SEM. *p<0.01. (D) T cell-mediated cancer cell-killing assay. PC-9OR and HCC827OR cells co-cultured in the indicated groups for 48 hours were subjected to crystal violet staining. Ratio of cancer cells to T cells: 2:1. (E) Ki67 incorporation assay on PC-9OR and HCC827OR cells treated as indicated. Activated T cells (1:2 ratio to cancer cells) or aspirin (200 µM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) Flow cytometry analysis of the exhaustion- and activation-related molecule Foxp3 in activated T cells co-cultured with the indicated cells (ratio of cancer cells to T cells: 1:1) for 48 hours with or without aspirin (200 µM). (G) PD-L1 levels in total protein extracts from indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (H) LAMC2 levels in total protein extracts from the indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (I) Immunofluorescence staining with an antibody against LAMC2 in PC-9 xenografts treated with osimertinib or osimertinib plus aspirin. (J) Schematic diagram of a T-cell chemotaxis assay directly regulated by the treatment of PC-9OR cells with aspirin (200 µM). Representative images of infiltrating T cells stained with CFSE dye. (K) Representative images of the immunofluorescence staining of CD68 (red), CD206 (green), and DAPI (blue) in mouse tumor tissue sections. (L) Immunofluorescence staining with antibodies against CD8 (red) and LAMC2 (green) in non-small cell lung cancer tissues treated with osimertinib or osimertinib plus aspirin. Scale bar: 50 µm.hu-PBMC,human-Peripheral blood mononuclear cell; S.C,Subcutaneous; CFSE, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; Foxp3, forkhead box P3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMC2, laminin subunit γ−2; PBS, phosphate-buffered saline; PD-L1, programmed death ligand 1.
We then assessed aspirin’s influence on molecules associated with T-cell activation and exhaustion, particularly focusing on Foxp3 (figure 6F). The results verified that aspirin could ameliorate T-cell exhaustion. In addition, aspirin upregulated GB levels, thereby bolstering their tumoricidal capabilities and indicating aspirin’s role in activating cytotoxic T cells (online supplemental figure S6D). When co-cultured with T cells, aspirin-induced apoptosis, further underscoring its contribution to enhancing the tumor-killing efficacy of T cells (online supplemental figure S6E). In TKI-resistant cells, aspirin was observed to reduce the expression of PD-L1 (figure 6G and online supplemental figure S6F), a critical factor in preventing the inhibition of T-cell function and enhancing their assault on cancer cells. Finally, our study revealed that aspirin inhibited the expression of LAMC2 in TKI-resistant cells and transplanted tumors (figure 6H,I and online supplemental figure S6G–H). T-cell infiltration experiments confirmed that aspirin treatment inhibited LAMC2 expression, thus promoting T-cell infiltration (figure 6J), and decreased the number of M2 macrophages in transplanted tumors (figure 6K).
We collected postoperative specimens from two patients who received neoadjuvant therapy with osimertinib—one patient received osimertinib treatment alone, while the other patient received concomitant aspirin (100 mg once daily) treatment due to a cardiovascular disease comorbidity. The postoperative specimens were stained to detect CD8+ T cells and LAMC2 protein by immunohistochemical analysis, and the results showed that LAMC2 protein expression decreased after osimertinib treatment, while CD8+ T cells increased; LAMC2 protein expression decreased further after treatment with aspirin combined with osimertinib, while CD8+ T cells increased (figure 6L).
In summary, the above results demonstrated that aspirin not only altered the behaviors of macrophages and T cells but also directly impacted the biological characteristics of tumor cells, indicating its potential role in overcoming TKI resistance by modulating the TME.
Discussion
Within the dynamic landscape of NSCLC therapeutics, the advent of TKIs has represented a paradigm shift toward precision oncology.33 Despite the initial therapeutic triumphs of TKIs, the emergence of drug resistance poses a formidable challenge, eluding simple resolution.34 The evolving mutation spectrum, intrinsic heterogeneity, and complex interactions between tumors and immune elements pose significant challenges for achieving durable responses in patients with NSCLC.35 36 The current state of managing TKI resistance is problematic, and while new generations of targeted drugs are in clinical trials,37 they only solve the issue for a small portion of resistant patients.38 A more practical solution may lie in enhancing our understanding of resistance mechanisms and achieving prolonged use of existing targeted drugs without the onset of resistance. Our previous research has already demonstrated that an immunologically active TME is the reason for the pathological remission and better prognosis obtained with immunotherapy.31 This phenomenon has also drawn our attention to whether the tumor immune microenvironment affects the action and development of resistance to targeted therapy drugs. While partial progress has been made in unraveling the complex interplay between TKIs and the TME,39 40 revealing dynamic changes in immune cell populations and PD-L1 expression post-treatment,41 42 research in this field is still in its nascent stages, with much left to explore. Current studies present a labyrinthine portrait: some studies have observed immune activation following TKI treatment,43 such as findings that EGFR-positive lung cancer may become “hot” tumors with increased immune infiltration post-TKI resistance, thereby benefiting from immunotherapy, while others report increased PD-L1 expression and the development of an immune-suppressive TME.44 These disparate observations underscore the multifaceted dynamics of TME behavior in the context of TKI therapy and accentuate the imperative for a more profound comprehension of these interactions. Despite these insights, the detailed mechanisms through which TKIs modulate the TME, particularly in the context of resistance, remain to be fully elucidated.
In this study, we elucidated the dynamic interactions between TKI treatment, TME changes, and the development of resistance in NSCLC. Our research initially emphasized the short-term effects of TKIs, which include enhancing T cell-mediated tumor clearance and suppressing the expression of the glycoprotein LAMC2,45–47 which is associated with tumor immune evasion. These short-term effects suggest a potential therapeutic window during which TKI application may optimally harness the immune system’s attack on tumors. However, the long-term effects of TKI treatment are more complex. With continued treatment, we observed the development of resistance and the formation of an immunosuppressive TME, suggesting that tumor cells adapt and resist the sustained pressure of TKIs by altering their microenvironment. Our data provide new insights into how TKI treatment improves the TME through three parallel mechanisms: first, by modulating T cell-mediated tumor clearance; second, by regulating the infiltration of pro-inflammatory and immunosuppressive M2 macrophages; and third, by adjusting LAMC2 expression, which is closely associated with tumor immune evasion. The combined action of these mechanisms highlights the potential for TKIs to induce positive immune responses in the short-term while also suggesting the potential for adaptive immune suppression with long-term treatment (figure 7). Therefore, some previously confusing clinical issues can be well explained.
Schematic of TKI-induced changes in the tumor immune microenvironment in EGFR-mutant and ALK-fusion NSCLC in sensitive and resistant states. In EGFR-mutant NSCLC, TKI treatment in the sensitive state results in decreased LAMC2 and PD-L1 expression, increased type I IFN levels, and enhanced M2 macrophage infiltration. In the resistant state, these tumors exhibit increased LAMC2 and PD-L1 expression, decreased type I IFN levels, and elevated infiltration of immunosuppressive M2 macrophages, leading to a shift towards an immunosuppressive environment. In ALK-fusion NSCLC, sensitive state TKI treatment also leads to decreased LAMC2 and PD-L1 expression and increased type I IFN levels. In the resistant state, ALK-fusion tumors show increased LAMC2 expression, and decreased type I IFN levels, though to a lesser extent than in EGFR-mutant resistant tumors, indicating a similar transition to an immunosuppressive environment. ALK, anaplastic lymphoma kinase; EGFR, epidermal growth factor receptor; IFN, interferon; LAMC2, laminin subunit γ−2; NSCLC, non-small cell lung cancer; PD-L1, programmed death ligand 1; TKI, tyrosine kinase inhibitor.
In tumors with driver genes, how do non-driver gene tumor cells, which lack specific targets, undergo elimination? The question of why targeted treatments can lead to substantial tumor regression even when only a small proportion of target cells are sensitive to the drugs? Central to this phenomenon is the “bystander effect” observed in the context of targeted therapy.48 49 Targeted treatments have the potential to induce tumor regression not only by affecting cells directly responsive to the drugs but also by altering the surrounding cells and the TME.50 Furthermore, such treatments can activate the immune system to mount a robust response against the tumor.51 Our exploration of the interactions and implications of TKIs with immune-related molecules, such as PD-1, in NSCLC suggests that TKI treatment suppresses PD-L1 expression and increases CD8 and GB levels, thereby enhancing T cell-mediated antitumor immune responses. This enhancement enables the immune system to eradicate tumor cells irrespective of the presence or absence of driver mutations. However, the development of resistance to TKIs presents a significant challenge, as resistant cells can counteract T-cell attacks by modulating immune checkpoint molecules, reducing the expression of key cellular activity proteins, and altering the secretion of pivotal cellular factors. The emergence of M2 macrophages, downregulation of the type I IFN signaling pathway, and variations in PD-L1 expression contribute to an increasingly immunosuppressive environment. These findings underscore the complexity and dynamic nature of tumor responses to targeted therapies. By understanding these intricate mechanisms and their implications, we can better appreciate the nuances of tumor biology and the potential for targeted therapies to effect significant changes, even in cases where only a minority of cells are initially sensitive to the treatment.
It has been well-established that immunotherapy demonstrates significant long-term benefits in NSCLC, mainly due to its unique mechanism of action.52 53 By activating the immune system and inducing immune-mediated tumor regression, immunotherapy reduces the percentage of surviving tumor cells, thus providing better pathological relief for patients with NSCLC.54 Patients achieving pathological remission exhibit improved long-term survival outcomes.55 However, targeted therapies, especially EGFR-TKIs, show only a lower rate of pathological remission.56 57 Prolonged treatment with TKIs induces significant changes in the TME, including increases in the expression of immune inhibitory factors, such as PD-L1, suppression of the type I IFN signaling pathway, reduced immune cell activity, and the initiation of immune escape mechanisms. These changes render the TME less conducive for immune cells to effectively recognize and attack tumor cells, ultimately leading to resistance. Furthermore, our research indicates that the TME faces immune suppression following TKI resistance. This immune-suppressive state may limit the efficacy of immunotherapy drugs, making it challenging to trigger an effective antitumor immune response. This limitation explains the poor outcomes observed with the use of ICIs and even combined chemotherapy following TKI resistance. Understanding this issue will provide more effective clinical strategies to improve the major pathological response of targeted neoadjuvant therapy and overcome TKI resistance in NSCLC.
ALK fusion-positive tumors in NSCLC exhibit relatively favorable characteristics, especially compared with EGFR-positive tumors.58 59 First-line treatment with EGFR-targeted drugs usually leads to resistance after a certain period, while ALK-targeted drugs, such as lorlatinib, show a higher PFS, providing a more optimistic treatment outlook for patients with NSCLC.60 In this study, we explored the differential effects of EGFR-positive and ALK fusion-positive mutations in NSCLC treatment and provided a detailed comparison in terms of the immune microenvironment. Regarding immune cell infiltration after treatment, ALK fusion-positive samples showed a more significant increase in CD8+ lymphocyte density, especially in CD8+GB+ and CD8+PD-1+ subsets. This difference suggests that ALK-TKI induces a more robust antitumor immune response. Furthermore, in pathological analysis, ALK fusion-positive samples tend to achieve complete pathological remission after treatment, indicating that the immune microenvironment changes induced by ALK-TKI may be more significant, favoring the tumor’s response to treatment. Additionally, long-term TKI treatment induces distinct immune inhibitory states in NSCLC tumors harboring ALK or EGFR mutations. Specifically, an increase in M2 macrophages was observed in EGFR-mutant cells, associated with an immunosuppressive environment, while ALK-mutant cells showed a contrasting trend, with less promotion of M2 macrophage accumulation. Our research reveals the distinct regulatory effects of ALK and EGFR on the immune microenvironment in NSCLC treatment. ALK mutations may induce a more robust antitumor immune response.
Aspirin is a widely used non-steroidal anti-inflammatory drug.61 Recent research has uncovered its potential in activating the tumor immune microenvironment, extending beyond its traditional anti-inflammatory and analgesic effects.62 63 Aspirin exhibits a broad-spectrum anticancer effect by inhibiting inflammation, modulating immune function, and inducing cancer cell death through endoplasmic reticulum(ER) stress, metabolic disruption, and oncogene downregulation.64–66 Furthermore, studies have demonstrated that aspirin, when used alone or in combination with ICIs such as anti-PD-1 and anti-cytotoxic T-lymphocyte-associated protein 4 antibodies, enhances antitumor activity.67 This effect is notably reliant on CD8+ T cells, highlighting aspirin’s significant role in immunotherapy. In our pursuit of enhancing TKI efficacy in NSCLC treatment, we focused on identifying “TKI Synergists” that could improve treatment outcomes. Our systematic selection process for these agents drew from a variety of compounds known to enhance the TME, including vinblastine,68 AZD0156,69 L-5-HTP,70 benzonitrile,71 albendazole,72 metformin,73–75 and notably, aspirin.76 77 Among these candidates, aspirin emerged as a premier TKI synergist, demonstrating unparalleled capacity to modulate crucial resistance-associated proteins—PD-L1, STING, and LAMC2—and influence M2 macrophage markers, thus providing a mechanistic underpinning for its observed clinical prowess in enhancing TKI outcomes.78 Notably, its ability to reduce T-cell exhaustion and augment cytotoxic T-cell activity offers promising avenues to counteract resistance mechanisms in NSCLC. This comprehensive analysis substantiates the clinical synergy between aspirin and TKIs,79 offering a well-grounded explanation for the improved outcomes seen with this combination. Aspirin may not be the best choice, searching for better TME-modified drugs may be a new strategy to prolong the efficacy of targeted therapy and address drug resistance in NSCLC.
In conclusion, our research unveils, for the first time, the close relationship between targeted therapy resistance and the tumor immune microenvironment, offering a clear direction for delaying resistance in the future. We discovered that by improving the tumor immune microenvironment, especially by enhancing T-cell function and promoting T-cell infiltration, targeted therapy resistance can be effectively overcome. This groundbreaking discovery will reshape our understanding of the relationship between targeted therapy and tumor immunity, guiding new directions in cancer treatment.
Data availability statement
No data are available.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by the Ethics Committees of the Daping Hospital, Army Medical University (no. 2021207 and 2022173). Participants gave informed consent to participate in the study before taking part.
References
Supplementary materials
Supplementary Data
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Footnotes
CL, ZG, DW and JZ contributed equally.
Contributors RH and YH conceptualized and designed the study. CLu, ZG, DW, JZ, and RH performed development of methodology. CLu, ZG, DW, JZ, CHu, DH, CHe, YL, CLin, TP, YD, FS, WJ, and GY were involved in data acquisition (including acquiring data and managing patients and facilities). CLu and DW performed data analysis and interpretation (eg, statistical analysis, biostatistics, and computational analysis). CLu, ZG, DW, RH, and YH were involved in writing, reviewing, and revising the manuscript. CLu, DW, and RH provided administrative, technical, and material support (ie, reporting or organizing data and constructing databases). CLu, RH, and YH were involved in study supervision. All authors have read and approved this article. RH and YH responsible for the overall content as the guarantor.
Funding This research was supported by grants from National Natural Science Foundation of China (Grant Nos. 82172623 and 81972189), Chongqing Technology Innovation and Application Development Special Key Project (CSTB2023TIAD-STX0003), Clinical Medical Technology Innovation Ability Training Program (Grant No. 2019CXLCA003), Chongqing Science and Technology Commission (Grant Nos. cstc2021jcyj-msxmX0014), and Chongqing Graduate Scientific Research Innovation Project (Grant No. CYB22277).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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