How Nutrient Availability Rewires Mitochondria and Cytoskeleton in 3D Lung Cancer Models

Lung cancer cells are master adapters. Within a growing tumour, some cells bathe in a plentiful supply of glucose, amino acids and growth factors, while their neighbours just a few dozen micrometres away sit in a starved, acidic wasteland where oxygen and nutrients are scarce. A new systems-level study published in the Journal of Experimental & Clinical Cancer Research shows that this patchwork of nutrient availability does far more than simply slow tumour growth in poor conditions. Instead, it pushes non-small cell lung cancer (NSCLC) cells into two fundamentally different biological states, each defined by a coordinated reorganisation of mitochondria, the cytoskeleton and the molecular machinery of migration and invasion.

The research, led by Claudia De Vitis and Giulia Ricci with Rita Mancini at Sapienza University of Rome, together with a large multi-institution Italian collaboration, tackled a problem that has long frustrated cancer biologists: most of what we know about tumour metabolism comes from cells grown flat on plastic dishes in a sea of rich culture medium. Those conditions bear little resemblance to the three-dimensional, nutrient-graded architecture of a real tumour. To bridge that gap, the team grew lung adenocarcinoma cells as three-dimensional spheroids, both in an established NSCLC cell line, NCI-H460, and in a patient-derived model known as PUC36, which was established from the malignant pleural effusion of a lung adenocarcinoma patient with appropriate ethics approval and informed consent. Crucially, they cultured these spheroids under two defined conditions: a nutrient-rich medium and a nutrient-restricted regime using RPMI medium supplemented with only two percent foetal bovine serum, which they refer to as FBSlow.

The first striking result concerns the mitochondria. Using transmission and scanning electron microscopy, the researchers resolved the internal ultrastructure of spheroid cells at high resolution and found that nutrient supply dictated mitochondrial shape and internal architecture. Cells in nutrient-rich spheroids displayed elongated mitochondria with preserved cristae, the folded inner membranes where the enzymes of oxidative phosphorylation are packed. Consistent with this, transcriptomic and proteomic profiling revealed enhanced expression of fusion-associated programmes involving proteins such as Mitofusin-2 and OPA1, the molecular machinery that joins mitochondria together and maintains their networked, healthy configuration. Functional assays measuring oxygen consumption confirmed that these cells ran a more vigorous oxidative metabolism with greater respiratory flexibility, meaning they could adjust their respiration rate dynamically when challenged with metabolic stressors.

Under nutrient restriction, the picture changed dramatically. Spheroid cells cultured in the FBSlow condition showed altered mitochondrial morphology and a marked reduction in respiratory flexibility. Reactome pathway enrichment analysis of the multi-omics data pointed to shifts in mitochondrial protein import and mitochondrial transport processes, while proteomic string network analysis revealed extensive mitochondrial remodelling across the two nutrient states. Total ATP production measured with Seahorse metabolic flux technology was reduced in the nutrient-restricted spheroids compared with their nutrient-rich counterparts. The authors interpret this as a stress-adaptive mitochondrial remodelling characterised by reduced oxidative phosphorylation and increased glycolytic reliance, a metabolic pivot familiar from the Warburg tradition of cancer research but here tied explicitly to the three-dimensional architecture of the tumour model.

What elevates this study beyond a conventional metabolic comparison is its demonstration that these metabolic states are physically coupled to the cell’s structural skeleton. The cytoskeleton, a dynamic scaffold of actin filaments and microtubules, governs how a cell holds its shape, grips its surroundings and crawls through tissue. The researchers found that nutrient-rich spheroids displayed dynamic F-actin remodelling, with abundant stress fibres, filopodia and lamellipodia, the protrusive structures that cells extend to probe and invade their environment. Nutrient-restricted spheroids, by contrast, maintained a reinforced cortical actin network, a dense band of filaments just beneath the cell membrane that stiffens the cell periphery, and showed accumulation of the protein zyxin at focal adhesion sites. Zyxin, together with partners such as tensin 3, integrin alpha 6 and alpha-actinin 1, anchors cells to the surrounding matrix, and its accumulation is consistent with a more constrained, mechanically reinforced structural phenotype.

These two cytoskeletal states were accompanied by distinct signalling profiles. Nutrient-rich spheroids showed increased AKT signalling, demonstrated by elevated phosphorylation of AKT at both Ser473 and Thr308, alongside mesenchymal-like transcriptional profiles, including markers of epithelial-mesenchymal plasticity such as vimentin and N-cadherin with reduced E-cadherin. Epithelial-mesenchymal plasticity, or EMP, is the process by which epithelial cancer cells loosen their cell-cell junctions and acquire the deformability and motility of mesenchymal cells, a change widely associated with metastatic potential. The nutrient-rich state therefore combined active oxidative metabolism, AKT-driven growth signalling and a mesenchymal, motile molecular identity. Nutrient-restricted cells instead retained a more compact architecture with stronger cell-matrix adhesion and evidence of extracellular matrix interaction programmes, including changes in laminin chain expression and metalloproteinase gene expression patterns.

The functional consequences were perhaps the most surprising. When the researchers tested how spheroids behaved after being deposited onto adherent surfaces, they observed a nutrient-dependent uncoupling between spreading and invasion, two behaviours often assumed to go hand in hand. Nutrient-restricted spheroids adhered efficiently and spread radially across the surface, expanding outward in a controlled, sheet-like manner. Nutrient-rich spheroids, despite their motile molecular signature, adhered and spread poorly, yet they exhibited significantly enhanced invasive potential, penetrating into surrounding matrices rather than simply flattening onto them. In other words, abundance of nutrients did not make cells better colonisers of a surface, but it did make them better infiltrators of tissue, which is the behaviour most relevant to metastasis.

Multi-omics integration reinforced this interpretation. By combining RNA sequencing data with quantitative proteomics and applying single-sample gene set enrichment analysis against the Molecular Signature Database, the team found coordinated regulation of extracellular matrix remodelling and mechanotransduction networks, the signalling systems that convert mechanical forces at the cell surface into biochemical instructions inside the cell. This suggests that nutrient availability does not tune metabolism and structure independently; rather, it coordinates mitochondrial function, cytoskeletal organisation and mechanical signalling into integrated adaptive programmes. The graphical summary presented by the authors captures this duality: nutrient-restricted spheroids rely on stress-adaptive mitochondrial remodelling, cortical actin reinforcement and zyxin-mediated adhesion to favour compact architecture and surface spreading, while nutrient-rich spheroids deploy MFN2- and OPA1-related fusion programmes, enhanced oxidative metabolism and AKT activation to support dynamic actin remodelling and elevated cytoskeletal plasticity that favours invasion.

The clinical implications are significant, even if they remain at the framework stage. Tumour metabolic heterogeneity is a well-recognised obstacle in NSCLC, where genetically similar cells within the same lesion can respond very differently to therapy. This study provides a mechanistic account of how the metabolic microenvironment alone, independent of mutation, can select for distinct phenotypic states with different invasive behaviours. It also highlights a methodological lesson: two-dimensional culture in nutrient-rich medium would have hidden the nutrient-restricted state entirely and misrepresented the behaviour of cells in nutrient-poor tumour regions. By validating their findings in both an established cell line and a patient-derived model, the authors strengthened the case that these nutrient-dependent mitochondrial-cytoskeletal states are a genuine feature of lung adenocarcinoma biology rather than a laboratory artefact. Future therapeutic strategies aimed at metabolic vulnerabilities in NSCLC may need to account for the fact that starving a tumour of nutrients does not simply weaken it; it may lock cancer cells into a reinforced, adhesive state that behaves in entirely unexpected ways.

Subject of Research: Nutrient-dependent coordination of mitochondrial function and cytoskeletal organisation in 3D non-small cell lung cancer models

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

Article References: Nutrient availability shapes tumour adaptation: a systems-level view in 3D lung adenocarcinoma models. (n.d.). https://doi.org/10.1186/s13046-026-03818-z

Image Credits: AI Generated

DOI: 10.1186/s13046-026-03818-z

Keywords: lung adenocarcinoma, NSCLC, 3D spheroids, cancer metabolism, mitochondrial dynamics, cytoskeletal remodelling, nutrient availability, epithelial-mesenchymal plasticity, AKT signalling, tumour invasion, multi-omics, tumour microenvironment