Differential Expression of LncRNA NEAT1 in 3D Tumoroids Compared to 2D Cultures Highlights Its Role in Glioblastoma Progression.

NEAT1 (Nuclear-Enriched Abundant Transcript1) is a long non-coding RNA (lncRNA) that critically regulates tumorigenesis, with growing recognition of its potential as a therapeutic target. However, most functional in vitro studies of lncRNAs rely on 2D cell culture systems, which lack the architectural and physiological complexity of tumors. Here, we demonstrate that 3D tumor architecture reshapes lncRNA-driven oncogenic programs. In 3D tumoroids, microenvironmental features such as mechanical cues, cell-cell interactions, and metabolic gradients modulate NEAT1 expression, and function. Using GBM as a proof-of-concept, we show that these context-dependent changes influence stemness, invasion, and EMT pathways. NEAT1 expression was significantly elevated in 3D tumoroids and positively correlated with stemness, invasion, glucose transporter expression, and epithelial-mesenchymal transition (EMT), both at the mRNA and functional levels. siRNA-mediated NEAT1 downregulation in 3D tumoroids to levels comparable to 2D culture, reduced the expression of these cancer-associated markers and suppressed proliferation, migration, and invasion, establishing a causal relationship. To establish broader relevance, we further examined NEAT1 and another oncogenic lncRNA, MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript (1) expression levels across breast, cervical, GBM, liver, and lung cancer models observing consistent expression differences. Collectively, our findings highlight the importance of evaluating the role of lncRNAs in physiologically relevant 3D systems.