AUTOMATED HIGH-THROUGHPUT FLUX ANALYSIS OF NON-SMALL CELL LUNG CARCINOMA CELLS GROWN IN VITRO IN TWO AND THREE DIMENSIONS

Importance of the Topic

Metabolic flux analysis using stable isotope tracers is essential for understanding how cancer cells rewire their energy metabolism. Traditional 2D monolayer cultures often fail to capture the complexity of the in vivo microenvironment. In contrast, 3D spheroid models provide a more physiologically relevant context, enabling more accurate insights into cellular glycolysis and TCA cycle activity.

Objectives and Study Overview

This study aimed to develop and demonstrate an automated, high-throughput workflow for comparing metabolic fluxes in non-small cell lung carcinoma H1299 cells grown as 2D monolayers versus 3D spheroids. By applying [U-13C6]glucose labeling and advanced informatics, the work sought to quantify differences in glycolytic and tricarboxylic acid (TCA) pathway fluxes between the two culture formats.

Methodology and Instrumentation

Cell Culture and Labeling
H1299 cells were cultured in Dulbecco’s Modified Eagle’s Medium for monolayers, while spheroids were generated on pHEMA-coated dishes. Cells were incubated with [U-13C6]glucose, harvested, and subjected to heat shock lysis followed by methanol/chloroform extraction and protein precipitation.

LC-MS Analysis
The extracts were analyzed on a Waters ACQUITY UPLC I-Class system with a BEH C18 column (2.1×50 mm, 1.7 μm), operating a gradient from 5% to 98% acetonitrile with 0.1% formic acid. Detection was performed on a Xevo G2-XS QTof in negative ESI mode, collecting MSE data across 50–1200 m/z at 30,000 FWHM.

Data Processing Pipeline
Automated workflows in Symphony transferred raw MassLynx files to a server, converted them to mzXML via MSConvert, and performed peak detection in El-MAVEN. The processed data were uploaded to PollyPhi Relative LC-MS for natural abundance correction, isotopologue quantitation, and pathway-level visualization.

Used Instrumentation

• Waters ACQUITY UPLC I-Class system with BEH C18 column (2.1×50 mm, 1.7 μm)
• Xevo G2-XS QTof mass spectrometer (ESI negative mode)
• Software: MassLynx, Symphony, MSConvert, El-MAVEN, PollyPhi Relative LC-MS

Results and Discussion

Spheroid cultures displayed significantly higher fractional enrichment of the 13C3 isotopologue of lactate compared to monolayers, indicating an upregulation of glycolysis in the 3D format. Enhanced 13C2 labeling in TCA intermediates such as malate and glutamate demonstrated increased flux through pyruvate dehydrogenase and the TCA cycle in spheroids. Automated processing of over 100 LC-MS files enabled rapid, reproducible quantitation and visualization of metabolic differences imposed by cell geometry.

Practical Applications and Benefits

• Improved in vitro models for drug screening and metabolic studies, offering greater physiological relevance.
• High-throughput automated workflows reduce manual errors and accelerate data turnaround.
• Integrated isotopic correction and pathway mapping facilitate clear interpretation for QA/QC and research efforts.

Future Trends and Opportunities

Emerging technologies in microfluidic 3D culture platforms, coupled with AI-driven data analytics and cloud-based processing, will further enhance the throughput and resolution of metabolic flux studies. Integration of multi-omics data and real-time feedback loops promises to deepen our understanding of complex cellular metabolism under physiologically relevant conditions.

Conclusion

This work establishes a robust, automated pipeline for high-throughput metabolic flux analysis, revealing marked differences in glycolytic and TCA cycle activities between 2D and 3D lung carcinoma cultures. The approach underscores the necessity of 3D models to accurately reflect in vivo metabolic phenotypes and accelerates translational research efforts.

References

1. Li et al. Survival advantages of multicellular spheroids vs. monolayers of HepG2 cells in vitro. Oncol Rep. 2008;20(6):1465–1471.
2. Wallace et al. A Model for Spheroid versus Monolayer Response of SK-N-SH Neuroblastoma Cells to Treatment with 15-Deoxy-PGJ2. Comput Math Methods Med. 2016;2016:3628124.
3. Fernandez et al. The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer. Cell Death Differ. 2018;25(7):1239–1258.