Analysis of ERBB ligand-induced resistance mechanism to crizotinib by primary culture of lung adenocarcinoma with EML4-ALK fusion gene

J Thorac Oncol. 2015 Mar;10(3):527-30. doi: 10.1097/JTO.0000000000000381.


Introduction: Using cell line-based assays, the secretion of erythroblastic leukemia viral oncogene homologue (ERBB) ligands has been reported to contribute to resistance against crizotinib in lung cancer with the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase fusion gene. However, it is difficult to predict the role of the ligands in each patient. Here, we report an analysis of the mechanism of resistance behind crizotinib resistance using a primary culture of cancer cells from pleural effusion of an anaplastic lymphoma kinase-positive lung cancer patient who was clinically resistant to crizotinib.

Methods: Primary cancer cells were prepared as cancer tissue-originated spheroids (CTOSs) according to previously described methods. CTOSs were maintained in StemPro medium, and a sensitivity assay was performed under growth factor-free conditions, or under stimulation with epidermal growth factor (EGF) or neuregulin-1/heregulin. The effect of treatment with crizotinib alone or a combination of crizotinib and erlotinib was examined.

Results: Cancer cells (LB53) were established to be CTOSs from a patient who was clinically resistant to crizotinib. The CTOSs were sensitive to crizotinib under growth factor-free conditions in vitro, whereas resistant under stimulation with EGF or neuregulin-1. These ligands rescued the inhibition of intracellular signaling by crizotinib. Pleural effusion from the patient also activated EGF receptor signaling to the similar extent of EGF stimulation. The resistance to crizotinib by EGF was reversed by blocking EGF receptor signaling by erlotinib in vitro.

Conclusion: Stimulation by ERBB ligands is suggested to be responsible for resistance to crizotinib in this patient. The CTOS method may enable analysis of resistance mechanism for targeted therapy in individual patients.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / drug therapy*
  • Adenocarcinoma / genetics
  • Adenocarcinoma / pathology
  • Crizotinib
  • Drug Resistance, Neoplasm / genetics*
  • ErbB Receptors / antagonists & inhibitors*
  • ErbB Receptors / genetics
  • Female
  • Humans
  • Lung Neoplasms / drug therapy*
  • Lung Neoplasms / genetics
  • Lung Neoplasms / pathology
  • Middle Aged
  • Neuregulin-1 / metabolism
  • Oncogene Proteins, Fusion / genetics*
  • Prognosis
  • Protein Kinase Inhibitors / pharmacology
  • Pyrazoles / pharmacology*
  • Pyridines / pharmacology*
  • Spheroids, Cellular / drug effects
  • Spheroids, Cellular / metabolism
  • Spheroids, Cellular / pathology*
  • Tumor Cells, Cultured


  • EML4-ALK fusion protein, human
  • NRG1 protein, human
  • Neuregulin-1
  • Oncogene Proteins, Fusion
  • Protein Kinase Inhibitors
  • Pyrazoles
  • Pyridines
  • Crizotinib
  • EGFR protein, human
  • ErbB Receptors