Abstract LB176: Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids
Authors: Vella Nathan , Estêvão Diogo , Cruz Tânia , Oliveira Maria José , Fenech Anthony George , Magri Vanessa Petroni 
Published: April 2024

Abstract
A major barrier in lung cancer pre-clinical research is the lack of simple, replicative and reproducible cell culture models. Most studies are still focused on 2D, completely ignoring cell-to-cell interactions, in vivo tissue architecture and the surrounding microenvironment. To address this void in cancer research, we aimed to develop a biomimetic 3D lung cancer spheroid model that can be used as a more reliable tool for pharmacological studies. Therefore, 3D spheroids of A549 (adenocarcinoma), H460 (large cell carcinoma) and H520 (squamous cell carcinoma) non-small cell lung cancer (NSCLC) cells were optimized and consequently submitted to AZD2014 treatment, a dual mTOR inhibitor targeting mTORC1 and mTORC2, members of the PI3K/Akt/mTOR pathway, which is commonly deregulated in lung cancer. Each cell line was seeded at 2000, 5000 or 10000 cells/spheroid in agarose micro-molds for eight days. The 3D NSCLC spheroids were further characterized for their tumor features, namely 3D dynamics and morphology, including diameter, circularity and compactness, viability properties, as well as proliferative and necrotic areas. Based on this characterization, the 2000 cells/spheroid condition was selected for further treatments and spheroid treatment time-points were selected. For each cell line, spheroids were treated with AZD2014, using concentrations based on the IC30 values (sensitization concentration) defined under 2D conditions: 10 μM, 10 μM and 0.2 μM for A549, H460 and H520 spheroids, respectively. Treated and untreated spheroids were followed during six days post-treatment, and their morphological characteristics, cell viability, necrotic core formation and proliferative areas, as well as ATP production, were assessed. Spheroids of all cell lines exhibited a significant inhibition of spheroid growth with AZD2014 treatment, which was maintained over the six days. Moreover, a reduction in spheroid intracellular ATP was observed at days three and six post-treatment. An additional treatment regimen was studied by re-treating spheroids with the same concentration of AZD2014 after 48hrs from the first treatment. Spheroids exposed to this dual treatment regimen exhibited a significant inhibition of spheroid growth, while the respective controls exhibited intensified growth. While AZD2014 did not induce significant reductions in 3D cell viability, a clear inhibition of spheroid growth was observed. Having a more intricate cellular organization, this study alleviates the increased complexity of pharmacological responses in 3D spheroids when compared to 2D models. Further studies are being carried out to define the enhanced biomimetic ability of these NSCLC spheroids and the underlying mechanisms involved when targeting the PI3K/Akt/mTOR pathway using AZD2014 as mono-therapy or in combination with two novel anti-sense oligonucleotides (ASO).
Citation Format: Nathan Vella, Diogo Estêvão, Tânia Cruz, Maria José Oliveira, Anthony George Fenech, Vanessa Petroni Magri. Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB176.