Deuterium Concentration as a Dual Regulator: Depletion and Enrichment Elicit Divergent Transcriptional Responses in A549 Lung Adenocarcinoma Cells

ABSRACT Deuterium abundance has been proposed as a modulator of cellular metabolism, yet its influence on cancer associated transcriptional networks remains incompletely characterized. We analyzed A549 lung adenocarcinoma cells cultured across four deuterium concentrations (40, 80, 150, and 300 ppm) using NanoString nCounter pro-filing. Expression data were processed through multistep filtering, symbolic trajectory encoding, density based spatial clustering (DBSCAN) to identify extreme transcrip-tional responders, and Gaussian mixture modeling (GMM 6) to resolve coordinated gene expression modules. DBSCAN identified 11 sentinel outlier genes under deuter-ium depletion, including reduced expression of multidrug resistance–associated ABCB1 (−42% at 80 ppm), proliferative signaling component FGFR4 (−19%), and tran-scriptional amplifier MYCN (−24%). In contrast, enrichment at 300 ppm produced a broad increase in oncogenic transcription (mean +44%), with marked elevation of in-flammation related (IL6, TGFBR2) and invasion associated (MMP9) genes. GMM 6 clustering of the remaining core network resolved six functional modules, indicating that depletion preferentially reduces expression of genes associated with plastici-ty related programs (Cluster 5: TGFB1, S100A4), while basal survival associated genes (Cluster 6: BIRC5, RET) remain comparatively stable. Together, these results indicate that deuterium concentration acts as a bidirectional modulator of transcriptional pro-grams in the A549 model, with enrichment broadly elevating oncogenic gene expres-sion and moderate depletion associated with selective downregulation of genes linked to resistance, signaling, and invasive behavior.