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Article

Gilteritinib overcomes second‑generation TKIs resistance in ALK‑rearranged non‑small‑cell lung cancer by inhibiting PD‑L1 and CD8 co‑expression

  • Authors:
    • Hang Guo
    • Yaoji Li
    • Chengyan Jin
    • Xiao Qi
    • Jincheng Wang
    • Baofeng Li
    • Chunguang Wang
  • View Affiliations / Copyright

    Affiliations: Department of Thoracic Surgery, Second Hospital Affiliated to Jilin University, Changchun, Jilin 130000, P.R. China, Department of Pharmacy, Jilin Cancer Hospital, Changchun, Jilin 130000, P.R. China
  • Article Number: 168
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    Published online on: April 28, 2026
       https://doi.org/10.3892/ijmm.2026.5839
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Abstract

The present study investigated how gilteritinib overcomes resistance to second‑generation tyrosine kinase inhibitors in anaplastic lymphoma kinase (ALK)‑rearranged non‑small‑cell lung cancer (NSCLC), providing new theoretical support for NSCLC treatment. The GSE191078 dataset was downloaded from Gene Expression Omnibus database. Cell clustering was performed using the FindClusters function, followed by uniform manifold approximation and projection dimensionality reduction and data filtering. Epithelial and T cells were extracted for single‑cell transcriptome sequencing and pseudotime analysis was conducted using the Monocle 2 algorithm. The inhibitory effects of gilteritinib on H2228/Al cells were evaluated using CCK8 and TUNEL assays. Western blotting, reverse transcription‑quantitative PCR and immunofluorescence were used to examine programmed death‑ligand 1 (PD‑L1) and cluster of differentiation 8 (CD8) expression. A PD‑L1/CD8 co‑culture system was established for rescue experiments and a nude mouse xenograft model was used to assess the anti‑tumor efficacy of gilteritinib. A total of 21,866 cells were obtained and grouped into 12 major cell types. In ALK‑rearranged NSCLC, epithelial cells were associated with regulation of the P53, glycolysis and hypoxia pathways, while pseudotemporal analysis linked T cells to endothelial cell‑related processes and ribosomal functions. In vitro, gilteritinib inhibited H2228/Al cell growth, induced apoptosis and reduced ALK protein levels. Co‑culture and rescue experiments suggested the mechanism involved inhibiting PD‑L1 and CD8 co‑expression, corroborated by animal experiments. Gilteritinib can overcome alectinib resistance and inhibit PD‑L1 and CD8 co‑expression in ALK‑rearranged NSCLC, providing a new therapeutic strategy.
View Figures

Figure 1

Identification of DEGs, pathway
enrichment analysis and characteristics of immune cell infiltration
in ALK-TKI resistant groups. (A) Volcano plot of DEGs in
single-cell sequencing. (B) KEGG pathway enrichment analysis of
upregulated genes in ALK-TKI resistant group vs. control group. (C)
Volcano plot of DEGs in single-transcriptome sequencing. (D) KEGG
pathway enrichment analysis of upregulated genes in ALK-TKI
resistant group vs. control group. (E) Box plot showing the
proportion of immune cell subsets. (F) Lollipop plot illustrating
the correlation between CD274 expression and immune cell subsets.
(G) Box plot showing the expression levels of CD274 and PDCD1.
*P<0.05, **P<0.01. DEGs, differential expressed genes; ALK,
anaplastic lymphoma kinase; TKIs, tyrosine kinase inhibitors; KEGG,
Kyoto Encyclopedia of Genes and Genomes; CD, cluster of
differentiation; PDCD1, Programmed cell death protein 1; NK,
natural killer; DT, drug tolerant.

Figure 2

Gilteritinib inhibits ALK-rearranged
NSCLC cells in vitro. (A) Relative inhibitory effects of
alectinib and gilteritinib on H2228 cells at various concentration
gradients; (B) Cell viability of alectinib and gilteritinib on
H2288/Al cells; (C) TUNEL staining (magnification, ×40; scale bar,
200 µm) and quantitative analysis of cell apoptosis. A and G
indicate alectinib and gilteritinib respectively. *P<0.05,
***P<0.001. ALK, anaplastic lymphoma kinase; NSCLC, non-small
cell lung cancer.

Figure 3

Effect of gilteritinib on PD-L1 and
CD8 expression and ALK rearrangement in H2288/Al and CD8 T cell
co-culture system. (A) Reverse transcription-quantitative PCR of
the expression level on PD-L1; (B) Protein expressions of PD-L1 in
H2288/Al and CD8 T cells after treating with alectinib and
gilteritinib; (C) Western blotting analysis and (D) IF staining of
T-cell polarization in co-culture system (CD8: green; DAPI: blue).
Magnification, ×40; scale bar, 200 µm. (E) IF detection of ALK
rearrangements following alectinib and gilteritinib treatment (ALK:
green; DAPI: blue). ×200 magnification; Scale bar, 50 µm. A and G
indicate alectinib and gilteritinib respectively. *P<0.05,
**P<0.01, ***P<0.001. PD-L1, programmed death-ligand 1; CD,
cluster of differentiation; ALK, anaplastic lymphoma kinase; IF,
immunofluorescence.

Figure 4

The effect of co-expression of PD-L1
and CD8 on the Drug Resistance of ALK-rearranged NSCLC. (A)
H2228/Al cell viability assay performed after transfection for 24
h; (B) Reverse transcription-quantitative PCR of PD-L1 and CD8 mRNA
expression in cells of each group; (C and D) Western blotting
analysis of PD-L1 and CD8 expression in H2228/Al cells; (E) TUNEL
staining (magnification, ×40; scale bar: 200 µm) and (F)
quantification for detection of apoptosis in H2288/Al cells.
*P<0.05, **P<0.01, ***P<0.001. PD-L1, programmed
death-ligand 1; CD, cluster of differentiation; ALK, anaplastic
lymphoma kinase; NSCLC, non-small cell lung cancer; PD-L1,
programmed death-ligand 1; sh, short hairpin.

Figure 5

Rescue assay. (A) H2228/Al cell
viability assay performed after transfection for 24 h; (B) Reverse
transcription-quantitative PCR of PD-L1 and CD8 mRNA expression in
cells of each group; (C and D) Western blotting of PD-L1 and CD8
expression in H2228/Al cells; (E) TUNEL staining (magnification,
×40; scale bar: 200 µm) and (F) quantification for detection of
apoptosis in H2288/Al cells. *P<0.05, **P<0.01,
***P<0.001. PD-L1, programmed death-ligand 1; CD, cluster of
differentiation.

Figure 6

Effect of gilteritinib on PD-L1 and
CD8 expression levels in tumor tissues. (A) Image of tumor removed
from nude mice and tumor growth curve, the × axis represents time
(d), the y axis represents the volume of the tumor
(mm3). Reverse transcription-quantitative PCR for
determination of (B) PD-L1 and (C) CD8 expression level in each
group; (D and E) Quantification of immunofluorescence results; (F)
Immunofluorescence staining of PD-L1 and CD8 (magnification, ×200;
scale bar, 50 µm; PD-L1: red; CD8: green; DAPI: blue). ns, not
significant, *P<0.05, **P<0.01, ***P<0.001. PD-L1,
programmed death-ligand 1; CD, cluster of differentiation.

Figure 7

Validation of gilteritinib regarding
PD-L1 and CD8 expression levels as well as ALK rearrangement in
tumor tissues. (A) Western blot analysis and (B) the relative
expression level of PD-L1 and CD8 protein; (C) Immunofluorescence
staining (magnification, ×200; scale bar, 50 µm) and (D)
quantification of ALK (ALK: green; DAPI: blue). ns, not
significant, *P<0.05, **P<0.01, ***P<0.001. PD-L1,
programmed death-ligand 1; CD, cluster of differentiation; ALK,
anaplastic lymphoma kinase.
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Copy and paste a formatted citation
Spandidos Publications style
Guo H, Li Y, Jin C, Qi X, Wang J, Li B and Wang C: Gilteritinib overcomes second‑generation TKIs resistance in ALK‑rearranged non‑small‑cell lung cancer by inhibiting PD‑L1 and CD8 co‑expression. Int J Mol Med 57: 168, 2026.
APA
Guo, H., Li, Y., Jin, C., Qi, X., Wang, J., Li, B., & Wang, C. (2026). Gilteritinib overcomes second‑generation TKIs resistance in ALK‑rearranged non‑small‑cell lung cancer by inhibiting PD‑L1 and CD8 co‑expression. International Journal of Molecular Medicine, 57, 168. https://doi.org/10.3892/ijmm.2026.5839
MLA
Guo, H., Li, Y., Jin, C., Qi, X., Wang, J., Li, B., Wang, C."Gilteritinib overcomes second‑generation TKIs resistance in ALK‑rearranged non‑small‑cell lung cancer by inhibiting PD‑L1 and CD8 co‑expression". International Journal of Molecular Medicine 57.6 (2026): 168.
Chicago
Guo, H., Li, Y., Jin, C., Qi, X., Wang, J., Li, B., Wang, C."Gilteritinib overcomes second‑generation TKIs resistance in ALK‑rearranged non‑small‑cell lung cancer by inhibiting PD‑L1 and CD8 co‑expression". International Journal of Molecular Medicine 57, no. 6 (2026): 168. https://doi.org/10.3892/ijmm.2026.5839
Copy and paste a formatted citation
x
Spandidos Publications style
Guo H, Li Y, Jin C, Qi X, Wang J, Li B and Wang C: Gilteritinib overcomes second‑generation TKIs resistance in ALK‑rearranged non‑small‑cell lung cancer by inhibiting PD‑L1 and CD8 co‑expression. Int J Mol Med 57: 168, 2026.
APA
Guo, H., Li, Y., Jin, C., Qi, X., Wang, J., Li, B., & Wang, C. (2026). Gilteritinib overcomes second‑generation TKIs resistance in ALK‑rearranged non‑small‑cell lung cancer by inhibiting PD‑L1 and CD8 co‑expression. International Journal of Molecular Medicine, 57, 168. https://doi.org/10.3892/ijmm.2026.5839
MLA
Guo, H., Li, Y., Jin, C., Qi, X., Wang, J., Li, B., Wang, C."Gilteritinib overcomes second‑generation TKIs resistance in ALK‑rearranged non‑small‑cell lung cancer by inhibiting PD‑L1 and CD8 co‑expression". International Journal of Molecular Medicine 57.6 (2026): 168.
Chicago
Guo, H., Li, Y., Jin, C., Qi, X., Wang, J., Li, B., Wang, C."Gilteritinib overcomes second‑generation TKIs resistance in ALK‑rearranged non‑small‑cell lung cancer by inhibiting PD‑L1 and CD8 co‑expression". International Journal of Molecular Medicine 57, no. 6 (2026): 168. https://doi.org/10.3892/ijmm.2026.5839
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