Nutrient availability shapes tumour adaptation: a systems-level view in 3D lung adenocarcinoma models

Background Cancer progression in non-small cell lung cancer (NSCLC) depends on the tumour cells’ ability to adapt to metabolic and mechanical limitations in heterogeneous microenvironments. How nutrient availability coordinates mitochondrial function with cytoskeletal organisation and epithelial-mesenchymal plasticity in spatially structured systems remains undefined. Methods Three-dimensional (3D) spheroids NSCLC models were established using both an established cell line and a patient-derived system. Cells were cultured under defined nutrient-rich and nutrient-restricted conditions to mimic heterogeneous tumour microenvironments. Ultrastructural analyses were performed using high-resolution imaging to assess mitochondrial morphology and cristae organisation. Multi-omics profiling, including transcriptomic and proteomic analyses, was integrated with functional metabolic assays to evaluate mitochondrial activity and respiratory flexibility. Cytoskeletal organisation and signalling pathways were investigated through imaging, molecular analyses, and pathway-specific assays, with a focus on actin dynamics, epithelial-mesenchymal plasticity, and AKT signalling. Functional assays were conducted to assess adhesion, spreading, migration and invasive capacity in 3D spheroid systems. Results Nutrient availability defined distinct mitochondrial-cytoskeletal states in 3D spheroid NSCLC models. Under nutrient-rich conditions, cells exhibited elongated mitochondria with preserved cristae architecture, increased fusion-associated programmes, and enhanced oxidative metabolism. Conversely, nutrient-restricted conditions were associated with altered mitochondrial morphology and reduced respiratory flexibility. These metabolic states were coupled with distinct cytoskeletal architectures. Nutrient-rich spheroids displayed dynamic F-actin remodelling, mesenchymal-like transcriptional profiles, and increased AKT signalling, consistent with a more invasive phenotype. In contrast, nutrient-restricted spheroids maintained cortical actin reinforcement, zyxin accumulation, consistent with a more constrained structural phenotype. Multi-omics integration supported coordinated regulation of extracellular matrix remodelling and mechanotransduction networks. Conclusions Functionally, these states revealed a nutrient-dependent uncoupling between spreading and invasion. Nutrient-restricted spheroids demonstrated efficient adhesion and radial expansion, whereas nutrient-rich spheroids showed scarce adhesion/spreading capability but exhibited enhanced invasive potential. Overall, these findings define nutrient-dependent mitochondrial-cytoskeletal states linking metabolic context to structural plasticity and distinct behaviour programmes in 3D systems, providing a clinically relevant framework to understand how metabolic heterogeneity may influence tumour progression and invasion in NSCLC.