Abstract A161: Comparison of EGFR inhibitor response of patient-derived NSCLC lines grown as monolayers, simple spheroids, and mixed cell complex spheroids
Authors: Kaur Gurmeet , Connelly John W. , Rapisarda Annamaria , Doroshow James H. , Teicher Beverly A. 
Published: August 2018

Abstract
Background: Human cancer cell lines are important tools in cancer research and drug discovery. NCI has established cell lines from patient-derived xenografts. These lines form multicellular spheroids under low adhesion culture conditions. In the tumor microenvironment, stromal (fibroblast, endothelial cells) and cancer cells engage in crosstalk mediated by soluble factors and cell-cell contact; therefore, we generated complex spheroids by mixing tumor cells, endothelial, and mesenchymal stem cells. We investigated the growth inhibitory activity of EGFR inhibitors, erlotinib and osimertinib, in eight patient-derived NSCLC lines. Materials and Methods: Erlotinib and osimertinib were tested in 9-point concentration response starting at 30uM and 5uM, respectively, and diluting by half-logs. Tumor cells were cultured in 96-well plates as monolayers, in 96-well U-bottom ultra-low adhesion (ULA) plates as 3D simple spheroids, and tumor cells were cocultured with human bone marrow-derived mesenchymal stem cells (hMSC) and human endothelial cells (HUVEC) in 96-well ULA plates as 3D complex spheroids. At 72h post plating, the cells were exposed to the test drugs for 72h and 96h. Cell viability was measured using luminescence by 2D CellTiter-Glo® in 2D monolayer cultures and 3D CellTiter-Glo® in 3D simple and complex spheroids. Cell viability was determined relative to a vehicle-treated control and IC50s were calculated from concentration response curves. To determine the localization of HUVEC and hMSC in complex spheroids, GFP-labeled HUVEC and REP-labeled hMSC were used. Images were captured at several time points using a Leica DMi8 microscope. Results: The response of 4 lung adenocarcinoma lines and 1 lung neuroendocrine line with 3 sublines exposed to the EGFR inhibitors erlotinib and osimertinib was assessed. All lines cultured as monolayer were unresponsive to erlotinib (&gt;30 µM) and osimertinib (&gt;5 µM). Simple spheroids formed by TLG0904F1496 and TLG0703F948 tumor cells; complex spheroids formed by TLG0703F948, TLG0904F1496 and its three clones were sensitive to erlotinib with IC50s below the clinical Cmax, indicating the importance of the 3D culture format. When tested separately as monolayers, hMSC and HUVEC were resistant to erlotinib with an IC50s of &gt;30 and 13 µM, respectively. We observed varied patterns of HUVEC and hMSC localization in the complex spheroids. The TLG0904F1496 and three clones formed spheroids with a tight cluster of HUVEC and hMSC in the center while the other four lines have HUVEC and hMSC dispersed throughout the spheroid. Conclusion: Patient-derived NSCLC line response to anticancer drugs varied with culture conditions. Osimertinib showed activity in the T790 mutant TLG0703F948 line in 3D complex spheroids only. The differential response to EGFR inhibitors in 3D simple and 3D complex spheroids, versus 2D culture, suggests that the tumor microenvironment may play a role in EGFR inhibitor activity and needs to be explored further using imaging techniques.
Citation Format: Gurmeet Kaur, John W. Connelly, Annamaria Rapisarda, James H. Doroshow, Beverly A. Teicher. Comparison of EGFR inhibitor response of patient-derived NSCLC lines grown as monolayers, simple spheroids, and mixed cell complex spheroids [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A161.