Abstract 3835: High-purity patient-derived cells: An advanced cell culture model for precision cancer medicine
Authors: Kim Seok-Young , Kim Hwan , Kim Sung-Moo , Kim Hye Ryun , Yun Mi Ran , Lee Ji Min , Choi Hun Mi , Cho Byoung Chul 
Published: July 2017

Purpose: Patient-derived cells (PDCs) established from malignant effusions can represent an excellent tool for the study of non-small cell lung cancer (NSCLC) with oncogenic driver mutations. However, because of cellular paucity and low tumor purity, PDCs have a limited utility for in-depth molecular analysis. In this study, we developed a high-purity patient-derived cell (HP-PDC) method for primary culture of malignant effusions.

Experimental design: HP-PDCs were established by a multistep tumor cell isolation protocol as follows. 1) Initial cultures of malignant effusions were observed under a light microscope to identify spheroid-forming cells. 2) Tumor purity of PDCs was analyzed by Immunofluorescence (IF) and flow cytometry. 3) Mutation status of EGFR or ALK in PDCs was screened by direct or PCR-based sequencing and RT-PCR and then was compared with driver mutations detected in matching patient tumor samples. 4) HP-PDCs were tested for drug efficacies by a cell viability assay, a colony-forming assay, and western blot. 5) HP-PDCs were screened with a panel of 84 kinase inhibitors to identify effective drugs.

Results: A consecutive series of 45 malignant effusion samples was collected from 36 patients with advanced NSCLC. Spheroid-forming cells were detected in 19 samples (42.2%) which were cultured in AR5 media to establish PDCs. IF analysis revealed that TTF-1 was upregulated in 9 PDCs. These 9 PDCs with high TTF-1 expression also showed EpCAM-positive cell population in flow cytometry with high purity of over 70%. Using Sanger sequencing, EGFR exon 19 deletion was detected 3 cases, EGFR T790M with L858R in 2 cases, and ALK rearrangements in 4 cases. Two of PDCs established from patients, who carried EGFR mutations and progressed on gefitinib, showed resistance to gefitinib in vitro. One of gefitinib-resistant PDCs was sensitive to AZD9291 with IC50 value of 47 nM. Three of PDCs established from patients, who carried ALK fusion genes and progressed on ALK inhibitors (crizotinib, alectinib, and ceritinib respectively), also recapitulated patient’s response to the ALK inhibitors. In an alectinib-resistant PDC, crizotinib and ceritinib exhibited potency with IC50 values of 321 nM and 215 nM, respectively. Colony-forming assay and western blot results were correlated with the cell viability assay data. Time spent for establishment of HP-PDCs ranged from 26 days to 132 days.

Conclusions: Nine HP-PDCs established by multistep tumor cell isolation protocol had high tumor purity, and recapitulated patient’s genomic profile and drug responses. HP-PDC is a powerful preclinical model which predicts responses to targeted drugs in a short period of time and may provide an excellent platform for personalized therapies in patients with advanced NSCLC.

Citation Format: Seok-Young Kim, Hwan Kim, Sung-Moo Kim, Hye Ryun Kim, Mi Ran Yun, Ji Min Lee, Hun Mi Choi, Byoung Chul Cho. High-purity patient-derived cells: An advanced cell culture model for precision cancer medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2017-3835