Abstract The effect of cell culture conditions on the proliferation and response to EGFR inhibitors of 4 patient-derived NSCLC lines (PDC) was determined. Variables included monolayer vs spheroid with only tumor cells or with HUVEC and MSC added, RPMI vs DMEM/F12 with or without EGF and other additives, growth time (10 days) and drug exposure time up to 4 days. We explored the growth of 4 NSCLC PDC lines grown in RPMI1640 or in advanced DMEM/F12 media supplemented with 10% fetal bovine serum (R10 or D10) or with 5% FBS, hydrocortisone, rhEGF, adenine, penicillin-streptomycin and glutamine (complete media; RC or DC). Cells were cultured as monolayer or mixed with stromal cells HUVEC and MSC to recapitulate a tumor microenvironment. The lines form multicellular spheroids under low adhesion culture conditions. We investigated the growth inhibitory effect of EGFR inhibitors, erlotinib and osimertinib, in 4 patient-derived (one with an EGFR mutation) and 3 established NSCLC lines with known EGFR mutations. Erlotinib and osimertinib were tested in 9-point, half log concentration response starting at 30µM and 10µM, respectively. 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 and mixed 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. In general, the NSCLC lines grew better in advanced DMEM/F12 media than in RPMI 1640 media with the largest effect seen in complex spheroid growth at 10-12 days. The Matrigel, hydrocortisone, rhEGF, adenine, and glutamine additives did not affect cell growth. The effect of media was more pronounced with some cell lines than others. For all 4 NSCLC lines, 3D growth in 96-well ULA culture peaked on day 8 and declined on days 10-12. Three of 4 lines cultured in monolayer or mixed monolayer were unresponsive to erlotinib (>30 μM) and osimertinib (>10 μM). The responsive line harbors a EGFR (L858R) mutation. Simple & complex spheroids formed by LG0567-F671-PDC in DMEM/F12 complete media 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 >30 and 13 μM, respectively. LG0567-F671-PDC was unresponsive to osimeritinib under all culture conditions because this PDC cell line does not harbor EGFR mutation Patient-derived NSCLC and NSCLC cell lines response to anticancer drugs varied with culture conditions. The differential response to EGFR inhibitors in 3D simple, 3D complex spheroids, versus 2D culture, suggests that the tumor microenvironment and attachment factors may play a role in EGFR inhibitor activity and needs to be explored further using imaging techniques. Citation Format: Gurmeet Kaur, James H. Doroshow, Beverly A. Teicher. Effect of 2D vs. 3D, exposure time, media and additives on growth and EGFR inhibitor response in patient-derived NSCLC lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2170.