[Establishment of pediatric neuroblastoma cell line and its resistance mechanism to lorlatinib].
AI interpretation is pending for this paper.
Open original publication →What the AI sees
Not AI summarized yet.
Research significance
Pending deeper interpretation.
Source abstract
Objective: To investigate the mechanisms underlying neuroblastoma resistance to the ALK inhibitor lorlatinib. Methods: Primary tissue was obtained from a pediatric patient diagnosed with neuroblastoma who underwent surgery at Beijing Children's Hospital in November 2023.A novel neuroblastoma cell line, PUMC-NB1, was established via in vitro culture and continuous passaging. The growth of PUMC-NB1 cells was tested using cell counting. Tumourigenicity was assessed via subcutaneous transplantation in NOD-SCID mice, and the histopathological concordance between the transplanted tumors and the original tumour was confirmed by haematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis. Neuroblastoma cell lines resistant to lorlatinib, PUMC-NB1-R, and SH-SY5Y-R, were generated from the PUMC-NB1 cell line and the ALK-mutated SH-SY5Y cell line, respectively. The sensitivity of these cells to ALK inhibitors and conventional chemotherapeutic agents was evaluated using the cell counting kit-8 assay. Molecular characteristics were investigated using whole-exome sequencing and transcriptomic sequencing. mRNA expression of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) was validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein expression of ALK, EGFR, and related signalling molecules was detected by Western blot. Results: The population doubling time of PUMC-NB1 cells was 92.35 hours, and 100% tumor formation was observed in NOD-SCID mice. Transplanted tumors showed positive IHC staining for Ki-67 (15%+), chromogranin A (CgA), tyrosine hydroxylase (TH), S-100, PHOX2B, and synaptophysin (Syn). Whole-exome sequencing revealed an ALKF1174Imutation in the PUMC-NB1 cells. The 50% inhibitory concentration of lorlatinib for PUMC-NB1 was (0.04±0.01) μmol/L. Among the differentially expressed molecules between the parental and resistant cells was EGFR by transcriptomic sequencing, RT-qPCR, and Western blot. Western blotting further revealed that lorlatinib treatment inhibited phosphorylation of the ALK downstream effectors ERK1/2 and Akt in SH-SY5Y cells, whereas it significantly enhanced EGFR phosphorylation in SH-SY5Y-R cells. Combination index analysis demonstrated that co-treatment with the EGFR inhibitor afatinib and lorlatinib synergistically inhibited the growth of lorlatinib-resistant PUMC-NB1-R and SH-SY5Y-R cells (combination index＜1). Conclusions: High expression and activation of EGFR, leading to bypass activation of the ALK downstream pathway, is a key molecular mechanism of lorlatinib resistance in neuroblastoma. This suggests that combination therapy could be used clinically to prevent and treat resistance.