Abstract 138: Establishment of monoclonal cell subpopulations as model systems for functionally exploring resistance mechanisms in EGFR mutant non-small cell cancers
Authors: Blanchard Claire , Avery Kyle , Ward Chelsea , Alsubaie Abduljalil M. , Baldelli Elisa , Pierobon Mariaelena 
Published: June 2023

Abstract
Background: Coexisting subgroups of cells within a tumor are always evolving. Understanding the molecular events that drive this evolution remains a high priority for understanding dynamic response to treatment. However, models that accurately map individual clones’ activity and sensitivity to treatment remain a challenge. To fill this gap, we established monoclonal cell populations (MCP) from a cell line and used this model system to dissect coexisting cellular and molecular events within an individual tumor and modulated response to anti-EGFR treatment in Non-Small Cell Lung Cancers (NSCLC).
Materials/Methods: MCPs were established from the commercially available H1975 EGFR-mutant NSCLC cell line. Parental cells were resuspended at a density of 10 cells per mL and 100 μL of cell suspension was added to each well of a 96-well plate. Eight plates were used, and each well was inspected 24 hours post-seeding by two or more independent scientists. Wells with individual cells were monitored every 3-4 days and followed throughout the expansion process. Once MCPs were established, morphological comparison was performed using the opensource QPath software and clones were classified as circular, branched, or mixed. Selected clones were then treated with 0.7 μM of Osimertinib for 72 hours, in line with the IC50 value of the parental line and treatment responses were compared.
Results: Of the 120 single cells detected, 25 expanded successfully and 16 were treated with Osimertinib. Rates of clonal expansion varied from 19 to 49 days and the most prominent morphology was branched (68.75%), followed by mixed (18.75%) and circular (12.5%). No direct correlation was observed between rate of expansion and cell morphology. Response to Osimertinib varied significantly across the clones compared to the parental line from which they were established. Cell viability after 72 hours of treatment with Osimertinib was similar between the parental line and 5 of the 25 clones. For the remaining clones, cell viability was significantly lower in the monoclonal cell subpopulations compared to the parental line. Specifically, cell viability decreased by 40% compared to the parental cell in 5 clones. In the most sensitive clones, cell viability was reduced by 63% and 75% compared to the parental.
Conclusion: 25 monoclonal cell subpopulations were successfully established and expanded from the H1975 cell line. MCPs showed significant variations in their growth rate and sensitivity to Osimertinib suggesting that this approach may help identify coexisting mechanisms of response to treatment within an individual tumor. Defining molecular profiles of coexisting MCPs may lead to the identification of biomarkers that may assist physicians in predicting response to treatment or design combination regimens that concomitantly target subclones within the tumor microecology.
Citation Format: Claire Blanchard, Kyle Avery, Chelsea Ward, Abduljalil M. Alsubaie, Elisa Baldelli, Mariaelena Pierobon. Establishment of monoclonal cell subpopulations as model systems for functionally exploring resistance mechanisms in EGFR mutant non-small cell cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 138.