Abstract 5028: Mapping metabolic networks and drug resistance in 3D spheroids using stable isotope-resolved metabolomics (SIRM)

Introduction: 2D cells lack cell-cell and cell-extracellular matrix interactions, critical for regulating cell functions. 3D cells in matrigel, hydrogels, hanging drops, and micropatterned plates can overcome these drawbacks but suffer from long spheroid formation times with variable efficiency. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles-magnetized cells for spheroid formation. M3DB spheroids can emulate tissue microenvironments and exhibit higher resistance to toxic agents than their 2D counterparts. It is, however, unclear how M3DB spheroids influence toxic responses in cells via metabolic perturbations. We employed Stable Isotope-Resolved Metabolomics (SIRM) with 13 C 6 -glucose to map metabolic networks in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells ± anti-cancer Na 2 SeO 3 . Experimental: A549 and PANC1 cells were grown in 2D culture in DMEM to ca. 70% confluence for loading with magnetic nanoparticles (Nanoshuttle, N3D Biosciences), detached, and placed in cell repellent plates for spheroid formation. Spheroids were then treated in [U- 13 C]-glucose ± Na 2 SeO 3 for 24 h. Metabolites were extracted in cold 70% methanol and analyzed by IC-FTMS and NMR 1,2 to determine the 13 C isotopologue and isotopomer distributions for reconstructing metabolic networks. Findings: We found that spheroids remained metabolically active in 10-day cultures. 13 C label incorporation into glycolytic, Krebs cycle, and nucleotide metabolites was similar between spheroids and their 2D counterparts. The exceptions were higher 13 C-ribose incorporation into uracil nucleotides in 2D than M3DB cultures of A549 cells and activation of gluconeogenesis in PANC1 M3DB spheroids. Selenite inhibited these pathways less in spheroids than 2D counterparts for both cell lines, consistent with the growth changes. Thus, the increased resistance of cancer spheroids to selenite may be mediated by these metabolic pathways central to growth and survival. Conclusions: 3D spheroids offer convenient models with more relevant 3-D architectures for preclinical studies on metabolic reprogramming and drug response of human cancer cells and cancer-stromal cell co-cultures. Spheroid formation in the M3DB system is fast, high throughput without matrix interferences for metabolic analyses. Supported by NCI P01CA163223-01A1, P30 CA177558 and NIDDK 1U24DK097215-01A1 1. Sun RC, Fan TW-M, Deng P, et al. Liquid diet delivery of stable isotopes into PDX mice for deep metabolic pathway tracing. Nat. Commun. 2017;9:1646. 2. Fan TW-M, Warmoes MO, Sun Q, et al. Distinctly perturbed metabolic networks underlie differential tumor tissue damages induced by immune modulator β-glucan in a two-case ex vivo non-small cell lung cancer study. CSH Molec Case Studies 2016;2:a000893. Citation Format: Teresa W. Fan, Salim S. El-Amouri, Jessica K. Macedo, Qiushi Sun, Andrew N. Lane. Mapping metabolic networks and drug resistance in 3D spheroids using stable isotope-resolved metabolomics (SIRM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5028.