Xenograft tumors derived from malignant pleural effusion of the patients with non-small-cell lung cancer as models to explore drug resistance

摘要

Background Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor ( EGFR ) mutations or anaplastic lymphoma kinase ( ALK ) fusions show dramatic responses to specific tyrosine kinase inhibitors (TKIs); however, after 10–12?months, secondary mutations arise that confer resistance. We generated a murine xenograft model using patient-derived NSCLC cells isolated from the pleural fluid of two patients with NSCLC to investigate the mechanisms of resistance against the ALK- and EGFR-targeted TKIs crizotinib and osimertinib, respectively. Methods Genotypes of patient biopsies and xenograft tumors were determined by whole exome sequencing (WES), and patients and xenograft-bearing mice received targeted treatment (crizotinib or osimertinib) accordingly. Xenograft mice were also treated for prolonged periods to identify whether the development of drug resistance and/or treatment responses were associated with tumor size. Finally, the pathology of patients biopsies and xenograft tumors were compared histologically. Results The histological characteristics and chemotherapy responses of xenograft tumors were similar to the actual patients. WES showed that the genotypes of the xenograft and patient tumors were similar (an echinoderm microtubule-associated protein-like 4- ALK ( EML4 – ALK) gene fusion (patient/xenograft: CTC15035_EML4–ALK) and EGFR L858R and T790M mutations (patient/xenograft: CTC15063_{EGFR L858R, T790M})). After continuous crizotinib or osimertinib treatment, WES data suggested that acquired ALK E1210K mutation conferred crizotinib resistance in the CTC15035_EML4–ALK xenograft, while decreased frequencies of EGFR L858R and T790M mutations plus the appearance of v-RAF murine sarcoma viral oncogene homolog B ( BRAF ) G7V mutations and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha ( PIK3C2A ) A86fs frame shift mutations led to osimertinib resistance in the CTC15063_{EGFR L858R, T790M} xenografts. Conclusions We successfully developed a new method of generating drug resistance xenograft models from liquid biopsies using microfluidic technology, which might be a useful tool to investigate the mechanisms of drug resistance in NSCLC.