The method builds on Cell Painting, a high-content imaging technique used in drug discovery to capture cellular changes across multiple cellular structures. It labels major components such as nuclei, mitochondria and the internal scaffolding with stains that can be imaged under a microscope. For this study, the researchers first grew bowel cancer cells as small three-dimensional spheres in standard laboratory plates. They then made the minispheres transparent with a solution of sugar and glycerol, allowing the microscope to image cells located deeper inside the clusters. Thousands of measurements of cell shape and structure could then be collected at single-cell resolution while retaining the three-dimensional environment in which the drugs were tested.

That combination addresses a central limitation of existing screening approaches. Cells can behave differently when they grow in three dimensions, yet many screening methods either examine flat cultures or collapse a complete three-dimensional sample into a single summary image. The adapted Cell Painting workflow preserves the detail of individual cells without giving up the throughput needed to test many compounds at once. In the reported comparison, 5-fluorouracil, a chemotherapy, and olaparib, a targeted cancer drug, looked almost alike in conventional flat cultures. Their profiles separated in minispheres, but only when the investigators inspected the cells individually; whole-sphere views did not show the difference.

Follow-up experiments produced a counterintuitive result. Although 5-fluorouracil caused less DNA damage than olaparib, it was more effective at preventing the cancer cells from dividing. Measurements of gene activity indicated that the chemotherapy activated the p53 pathway more strongly. The p53 pathway is a central stress-response system, and the study therefore associated the drugs’ different effects in three dimensions with distinct cellular responses, even though their effects in two-dimensional cultures appeared similar. The experiments were conducted in two cancer cell lines under laboratory conditions, so the results do not establish how either drug acts in patients.

The approach was also tested beyond the laboratory where it was developed through collaboration with colleagues at AstraZeneca, which the researchers described as a useful test of whether the method could be adopted by other laboratories. Images, data and code from the study were made openly available. The authors now hope to extend the imaging strategy to more complex three-dimensional models, including organoids grown from patient tissue. The work is presented as a peer-reviewed imaging-analysis study rather than a clinical trial, and its immediate contribution is methodological: it shows that making three-dimensional samples transparent can expose drug-dependent responses at the individual-cell level that would otherwise remain hidden in flat cultures or averaged tissue-scale measurements.

Subject of Research: Single-cell Cell Painting analysis of drug responses in three-dimensional colorectal cancer minispheres

Article Title: Transparent minispheres reveal that some look-alike cancer drugs part ways in 3D

Article References: Transparent minispheres reveal that some look-alike cancer drugs part ways in 3D. Original publication (Bibliographic details unavailable.)

Image Credits: AI Generated

DOI: Not provided

Keywords: Cell Painting, 3D spheroids, drug screening, 5-fluorouracil, olaparib, p53 pathway, cancer cells, high-content imaging, morphological profiling, Transparent, minispheres, reveal

The study, led by Abdallah Fallah, Alain Zgheib, Bogdan Alexandru Danalache, Nicoletta Eliopoulos and Borhane Annabi at the Université du Québec à Montréal and McGill University, used focused PCR arrays to compare U87 glioblastoma cells grown as conventional two-dimensional monolayers or as three-dimensional spheroids. The researchers then perturbed SORT1 in two ways: genetically, using SORT1-targeted siRNA (siSORT1), and pharmacologically, using AF38469, a Vps10p-domain antagonist that blocks the receptor’s ligand-binding arm. A non-targeting scrambled siRNA served as the genetic control.

The results reveal that when U87 cells are grown as 3D spheroids, they activate a coordinated transcriptional program that is largely absent in 2D monolayers. This program includes upregulation of angiogenic markers such as VEGFA and HIF1A, adipogenic and lipogenic regulators including PPARG, DDIT3, ADIPOQ, CEBPD and FOXO1, and a battery of glioblastoma stemness and drug-resistance markers including PROM1/CD133, SOX2, NANOG, CD44, ABCG2, ABCB5, TAZ and KITLG. Together, these changes indicate that 3D growth triggers a lipid-remodeling program with cancer stem cell features that are directly relevant to chemoresistance.

When the researchers silenced SORT1 or blocked it with AF38469, both interventions reduced spheroid size and repressed the adipogenic module, supporting a role for SORT1 in regulating adipogenesis and lipid-synthesis programs in glioblastoma spheroids. However, the two perturbations did not behave identically across the full transcriptional landscape. The cancer stem cell module proved far more sensitive to genetic SORT1 silencing, with 18 of 27 genes responding, than to pharmacological AF38469 treatment, which affected only 10 of 27 genes.

Among the transcripts that emerged as siSORT1-dependent but AF38469-insensitive were CXCL8, CD38, ZEB2, SOX2, NANOG and CD44. This bifurcation is significant because it suggests that SORT1 functions segregate into two arms: a ligand-dependent arm that AF38469 can target, and a ligand-independent, scaffold-like arm that requires SORT1 expression itself. The pharmacological inhibitor reproduces the genetic silencing effect nearly perfectly on the lipid and metabolic module but fails to do so on the developmental backbone of the cancer stem cell program.

Principal component analysis confirmed this dual-module organization. When siSORT1 and AF38469 were applied together, they jointly displaced the adipogenesis profile, whereas only siSORT1 clearly separated the cancer stem cell profile. This systems-level observation supports the idea that the bifurcation operates on entire transcriptional modules rather than on individual genes. The lipid and metabolism machinery plus drug-resistance elements form a co-regulated, ligand-dependent arm, while stemness, plasticity and the developmental backbone form a scaffold-dependent persistence arm.

The SORT1-dependent transcriptional state is organised into six functionally coherent modules, providing what the researchers describe as a modular blueprint for combinatorial therapeutic targeting. An AF38469 phenocopy index, a quantitative measure of how well the pharmacological inhibitor mimics genetic SORT1 silencing, provides strong support for this bifurcation model. The index shows that AF38469 reproduces the siSORT1 effect on the lipid and metabolic module but fails on the developmental backbone.

These findings are consistent with a model in which SORT1 behaves as a state-persistence node, associating adipogenic and lipogenic reprogramming with cancer stem cell-associated and drug-resistance-associated transcriptional programs. The researchers caution that this model remains to be tested with direct functional assays in 3D U87 spheroids, and the current study is primarily transcriptional in scope. Nevertheless, the modular organization offers a framework for designing combination strategies that could circumvent the acquired SORT1-regulated cancer stem cell and drug-resistance phenotype.

The broader implications are considerable. By creating hydrophobic compartments that sequester lipophilic chemotherapeutics, increased adipogenic reprogramming and lipid synthesis correlate with poor treatment response and increased drug tolerance in glioblastoma. SORT1 has been implicated in cancer stem cell biology and chemoresistance, and this study now provides a systems-level dissection of how the receptor links lipid remodeling to a chemoresistance-associated molecular signature. If the two-arm model holds up under direct functional testing, it could open new avenues for therapeutic intervention, combining AF38469-like ligand-dependent antagonists with strategies that target the scaffold-dependent arm of SORT1 biology.

The choice of a three-dimensional spheroid model deserves particular attention when weighing these results. Standard two-dimensional monolayer culture has long been criticized for stripping glioblastoma cells of the hypoxic, nutrient-gradient and cell–cell contact conditions that define the tumour microenvironment in patients. Hanging-drop spheroid systems restore some of these gradients, and it is precisely under these conditions that the adipogenic and stemness programs described here emerge. The fact that markers such as CD133 and SOX2 rise in spheroids relative to monolayers echoes a broader literature showing that 3D architecture alone, without any genetic manipulation, can push glioma cells toward a stem-like, therapy-tolerant state. This makes the spheroid platform a useful intermediate between simplified monolayers and animal models for probing drug-resistance biology.

The lipid dimension of the study connects to a growing appreciation of metabolic reprogramming in aggressive cancers. Lipid droplets act as hydrophobic sinks that can partition weakly lipophilic drugs away from their intracellular targets, effectively lowering the free drug concentration reaching the nucleus or microtubules. Enzymes of fatty acid synthesis and adipogenic transcription factors such as PPARG and CEBPD have each been independently linked to poor prognosis in high-grade gliomas. What the present work adds is a plausible coordinator: a single membrane receptor whose perturbation collapses much of this lipid program, suggesting that lipid remodeling in glioblastoma spheroids is not a diffuse set of parallel changes but a regulated module with an identifiable upstream node.

Sortilin itself has an unusual profile among trafficking receptors. It shuttles between the trans-Golgi network and endosomes, sorting cargo proteins involved in metabolism, inflammation and growth factor signaling. Its involvement in lipoprotein handling made it an attractive candidate when the authors sought a molecular bridge between lipid biology and cancer stem cell maintenance. The observation that a ligand-binding antagonist and a genetic knockdown diverge in their transcriptional consequences is consistent with a receptor that does more than transmit signals through ligand occupancy; it may also act as a physical scaffold, organizing protein complexes or cargo trafficking routes that persist as long as the protein is expressed, regardless of whether its ligand-binding pocket is occupied.

The specific transcripts that resist pharmacological blockade are informative. CXCL8, the gene encoding interleukin-8, is a chemokine associated with inflammatory recruitment, angiogenesis and stem cell maintenance in multiple tumour types. ZEB2 is a transcriptional repressor best known in epithelial–mesenchymal transition, but it also participates in developmental gene-regulatory networks in neural lineages. CD38, SOX2, NANOG and CD44 round out a set of markers tied to self-renewal and immune interaction. That these genes respond to loss of SORT1 expression but not to ligand-pocket antagonism implies that the scaffold-dependent arm of the receptor touches developmental and inflammatory programs that the ligand-dependent arm does not.

The drug-resistance markers in the spheroid signature also merit comment. ABCG2 and ABCB5 encode ATP-binding cassette transporters that actively pump structurally diverse chemotherapeutics out of cells, and both have been detected on glioblastoma stem-like populations. TAZ, a co-activator in the Hippo pathway, links mechanical cues from the 3D environment to transcriptional programs supporting proliferation and survival. The co-regulation of transporter genes with adipogenic regulators in a single module raises the possibility that lipid accumulation and drug efflux are not merely coincident but co-selected features of the same cellular state, which would explain why lipid-rich tumour cells so often prove chemoresistant.

Several caveats frame the interpretation. The work relies on a single cell line, U87-MG, which, although widely used, does not capture the molecular heterogeneity of patient glioblastomas, and the analysis is transcriptional rather than functional: no direct assays of spheroid viability under temozolomide challenge, stem cell frequency or lipid droplet burden are reported. Supplementary data on fibronectin distribution between cytosolic and nuclear fractions following SORT1 perturbation hint at downstream mechanistic threads, but these remain to be pulled. Validation across patient-derived glioblastoma stem cell cultures, organoids and in vivo models would be the natural next step.

If the two-arm model is confirmed, the therapeutic logic is straightforward but demanding. Blocking the ligand-dependent arm with AF38469-like molecules could deflate the lipid and metabolic machinery that shelters tumour cells from chemotherapy, while the scaffold-dependent arm would require entirely different approaches, such as degrading the receptor protein itself or disrupting its intracellular trafficking partners. The modular blueprint offered by this study, with its quantitative phenocopy index, gives medicinal chemists and translational researchers a measurable target profile for such combination strategies, and the open-access availability of the underlying datasets should facilitate independent evaluation of this provocative model.

Subject of Research: Sortilin-associated transcriptional programs in 3D glioblastoma spheroids

Article Title: A SORT1-associated transcriptional signature links adipogenic plasticity, stemness and drug-resistance programs in 3D glioblastoma spheroids: dissection of ligand-dependent and ligand-independent arms

Article References: Fallah, A., Zgheib, A., Danalache, B. A., Eliopoulos, N., & Annabi, B. (2026). A SORT1-associated transcriptional signature links adipogenic plasticity, stemness and drug-resistance programs in 3D glioblastoma spheroids: dissection of ligand-dependent and ligand-independent arms. BMC Cancer. https://doi.org/10.1186/s12885-026-16856-9

Image Credits: AI Generated

DOI: 10.1186/s12885-026-16856-9

Keywords: glioblastoma, SORT1, sortilin, cancer stem cells, 3D spheroids, chemoresistance, adipogenesis, AF38469, drug resistance, transcriptional modules, SORT1-associated, transcriptional

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: De Vitis, C., Salerni, L., Telese, S., Cavazzuti, L., Federici, G., Mirabilii, S., Maiello, R. G., Berardini, M., Roca, M. S., Messina, E., Dezi, C., Ascenzi, F., Bruschini, S., Bassi, M., Di Gennaro, E., Ricciardi, M. R., Catizone, A., Ricci, A., Sacconi, A., … Mancini, R. (2026). Nutrient availability shapes tumour adaptation: a systems-level view in 3D lung adenocarcinoma models. Journal of Experimental & Clinical Cancer Research. 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

The research, led by Federica Banche-Niclot, Rosalia Ferraro, Francesca Taraballi, and Sergio Caserta across institutions in the United States and Italy, addresses a persistent gap in translational cancer research. Mechanical alterations within the tumor microenvironment, including stiffening of the extracellular matrix, altered tissue viscoelasticity, and increased cellular deformability, are not passive byproducts of disease. Instead, these biomechanical cues actively reshape cellular behavior, influencing how cancer cells invade, migrate, survive under stress, and respond to chemotherapy. Yet despite this recognized importance, mechanical phenotype remains underutilized in diagnostic and therapeutic workflows, and few platforms exist that allow controlled, quantitative exploration of these mechanical dynamics.

Conventional tools for studying tissue mechanics have significant limitations. Atomic force microscopy, indentation, micro tweezers, and compression assays have all contributed valuable insights into cellular elasticity, but they often oversimplify the inherently viscoelastic nature of biological systems. These techniques are sensitive to tool geometry and substrate properties, producing high variability and limited comparability, especially when moving from isolated cells to tissue-level constructs. The new study instead places rotational rheology at the center of its strategy. By measuring the elastic storage modulus G-prime and the viscous loss modulus G-double-prime, rheology captures both energy storage and dissipation, the key parameters governing how tumors deform, resist, or transmit mechanical stress. These moduli can be converted into conventional metrics such as Young’s modulus under defined assumptions, enabling comparisons across biological models.

To build a physiologically relevant in vitro system, the team cultured two distinct cell lines as three-dimensional spheroids using the liquid overlay technique. Cancer spheroids were formed from MDA-MB-231 human breast adenocarcinoma cells, a line derived from a patient with metastatic disease, while non-tumoral spheroids were formed from primary human mesenchymal stem cells isolated from infrapatellar fat pad tissue under institutionally approved protocols. Spheroids were grown in U-bottom 96-well plates coated with a nonstick polyHEMA film, which promotes cell-to-cell aggregation. Viability was tracked over 72 hours using an ATP-based metabolic assay alongside live/dead fluorescent staining. The mesenchymal spheroids developed a necrotic core by 72 hours, so they were mechanically tested at 24 hours, whereas the cancer spheroids reached a compact, nearly spherical morphology with sustained viability at 72 hours and were tested at that timepoint.

A critical design challenge was how to hold the spheroids in place during rheological measurement without contaminating the mechanical signal. The researchers embedded the spheroids in a purified methacrylated Type I bovine collagen hydrogel at 3 milligrams per milliliter, cast into disks matching the 25-millimeter rheometer plate. Spheroid numbers were calculated to achieve roughly 1 percent surface coverage of the supporting disk, requiring 15 cancer spheroids or 85 mesenchymal spheroids per sample. The bare collagen gel proved to be approximately three orders of magnitude softer than the spheroid-laden constructs, with a storage modulus of only about 78 pascals at an angular frequency near 1 radian per second, confirming that the support matrix did not mask the mechanics of the cells themselves.

The rheological results revealed a striking mechanical contrast. At an angular frequency of about 1 radian per second, the storage modulus reached 9.70 plus or minus 1.83 times 10 to the fourth pascals for constructs containing healthy spheroids and 2.45 plus or minus 1.13 times 10 to the fifth pascals for those containing cancer spheroids, a 2.5-fold difference in stiffness. The loss tangent, the ratio of viscous to elastic response, stayed nearly constant at around 0.30 for healthy constructs and 0.15 for tumoral ones, indicating that the cancer spheroids behaved in an even more solid-like fashion. Converting the storage moduli into Young’s modulus values assuming incompressible behavior yielded approximately 2.91 times 10 to the fifth pascals for healthy constructs and 7.36 times 10 to the fifth pascals for tumoral constructs. This stiffening is consistent with the extracellular matrix remodeling, increased collagen crosslinking, and heightened contractile forces that characterize aggressive cancer phenotypes and promote migration and invasion.

To validate the in vitro platform against real biology, the team benchmarked it with ex vivo tissues: healthy mammary gland fat pads from mice and tumor masses generated by injecting MDA-MB-231 cells into mice. Tissues were rehydrated in phosphate-buffered saline and standardized to 5-millimeter cylinders with a biopsy punch. Rheological testing showed the same directional pattern as the spheroid models, with tumoral tissues approximately ten times stiffer than healthy counterparts, exhibiting storage moduli of 2.38 plus or minus 1.04 times 10 to the fifth pascals versus 2.45 plus or minus 0.93 times 10 to the fourth pascals. Both tissue types displayed solid-like behavior, with the storage modulus consistently exceeding the loss modulus across the tested frequency range of 0.01 to 100 radians per second.

One of the study’s most technically important findings emerged from comparing rheology with compression testing. Compression measurements using a Hertz model analysis of the stress-strain curve, restricted to strains below 10 percent to remain in the linear elastic regime, gave Young’s modulus values of 3.68 plus or minus 0.38 times 10 to the fourth pascals for healthy tissue and 5.22 plus or minus 0.62 times 10 to the fourth pascals for tumoral tissue, making tumors about 1.42 times stiffer. These absolute values differed from the rheological estimates, but the discrepancy was resolved when the applied normal force was taken into account. Healthy tissues exhibited force-softening, with moduli decreasing by at least an order of magnitude as normal force increased, while tumoral tissues showed force-stiffening, with moduli rising under higher load. When rheological values were compared within the same normal force window reached during compression, roughly 0.1 to 0.15 newtons, the two methods agreed closely. The authors emphasize that this nonlinear, force-dependent behavior can introduce serious artifacts into biomechanical characterization if not standardized, and that mechanical comparisons must be made within consistent force regimes.

The translational implications are considerable. Increased tumor stiffness is known to hinder drug diffusion, impair immune cell infiltration, and activate mechanotransduction pathways that promote chemoresistance. By reproducing this stiffness landscape in vitro and aligning it with ex vivo profiles, the platform enables a more predictive evaluation of mechanically responsive therapies before animal or clinical testing. The authors also point toward mechanical fingerprinting as a potential physical biomarker strategy, particularly if integrated with imaging techniques or functional assays. The standardized hydrogel scaffolds, controlled spheroid assembly, and quantitative mechanical outputs make the approach suitable not only for academic investigation but also for industrial drug screening and biomaterials testing pipelines.

The researchers are careful to note the limitations and the road ahead. Bulk rheological metrics provide standardization and throughput, but they cannot fully describe the mechanical microenvironment experienced by individual cells, which varies spatially due to heterogeneity in matrix density and local architecture. Cell-scale techniques such as atomic force microscopy and microrheology capture local stiffness gradients relevant to mechanotransduction but require careful model assumptions and are sensitive to sampling location, so bulk and microscale measurements should be treated as complementary rather than competing descriptors. Future work will expand the platform to larger ex vivo cohorts, incorporate additional cell populations and tumor subtypes, add dynamic cues such as perfusion and immune components, and account for sex as a biological variable, since hormone-dependent signaling can modulate stromal behavior and matrix remodeling. Mycoplasma testing was not performed in this proof-of-concept study, though no morphological abnormalities or altered growth kinetics were observed. Even so, the framework establishes a reproducible mechanical benchmark for breast cancer, laying the groundwork for embedding biomechanical profiling into preclinical workflows, mechanics-informed diagnostics, and patient-specific modeling.

Subject of Research: Viscoelastic biomechanical profiling of healthy and cancerous breast tissue using 3D spheroid models and ex vivo rheology

Article Title: Mechanical fingerprints in breast cancer research: A multimodal experimental approach

Article References: Banche‐Niclot, F., Ferraro, R., Di Palo, V., de Paolis, P., Taraballi, F., & Caserta, S. (2026). Mechanical fingerprints in breast cancer research: A multimodal experimental approach. Bioengineering & Translational Medicine, 11(5), Article e70148. https://doi.org/10.1002/btm2.70148

Image Credits: AI Generated

DOI: 10.1002/btm2.70148

Keywords: breast cancer, tumor biomechanics, rheology, 3D spheroids, viscoelasticity, extracellular matrix, tumor stiffness, Young's modulus, compression testing, drug delivery, mechanotransduction, ex vivo tissue analysis

The review, conducted by a team led by Giacomo Colella, Valentina Piccioni, and Gianmarco Contino of the University of Birmingham, adhered to the PRISMA 2020 reporting guidelines and searched the PubMed database for studies published between 2000 and 2025. The team focused specifically on in vitro and ex vivo models of solid tumors that had been tested in an immuno-oncology context, deliberately excluding animal studies to concentrate on human-relevant laboratory platforms. From this search, the researchers identified 75 studies that met their inclusion criteria, spanning six principal experimental model types and 23 different cancer types, with lung, breast, and colorectal cancers appearing most frequently in the literature.

The six model categories catalogued in the review represent a spectrum of biological fidelity and technical simplicity. Two-dimensional cell line cultures, the workhorse of cancer biology for decades, remain valuable for rapid initial screening of immunotherapeutic compounds. Three-dimensional spheroid cultures introduce a level of spatial organization that better recapitulates the architecture of tumors, including nutrient and oxygen gradients that influence immune cell behavior. Patient-derived organoids, or PDOs, go further by preserving many of the genetic and phenotypic characteristics of the original tumor from which they were grown, offering a personalized window into how an individual patient’s cancer might respond to immunotherapy.

At the ex vivo end of the spectrum, tumor tissue slices preserve the native architecture of the tumor microenvironment, including its stromal cells, extracellular matrix, and endogenous immune infiltrate, although they can typically be maintained in culture only for short windows of time. Tumor-on-a-chip systems, often built from polydimethylsiloxane or cyclic olefin copolymer, add another dimension of realism by enabling controlled perfusion, the establishment of chemical gradients, and the observation of immune cell trafficking under dynamic flow conditions that mimic the vasculature. The review also noted a residual category of other model types, including immune co-culture cell microarrays and platforms derived from circulating tumor cells or induced pluripotent stem cells.

On the immunotherapy side, the studies surveyed by the review interrogated a range of treatment modalities. Immune checkpoint inhibitors, particularly antibodies targeting the programmed death-1 receptor and its ligand PD-L1, dominated the landscape, reflecting their clinical prominence. Adoptive cell therapies formed the second major pillar, encompassing chimeric antigen receptor T cells, tumor-infiltrating lymphocytes, and natural killer cell-based approaches. The immune cell types most commonly incorporated into these experimental systems were T lymphocytes, including both tumor-infiltrating lymphocytes and CAR-T cells, followed by natural killer cells, a distribution that mirrors the current emphasis of clinical immuno-oncology.

A central theme of the review is the concept of transferability, meaning the degree to which findings generated in one model system can be generalized to other models, to different cancer types, and ultimately to patients. The authors recorded the culture setup, validation status, and species of origin for each study to enable contextual comparison across platforms. Their analysis suggests that no single model is sufficient on its own: two-dimensional cultures are useful for hypothesis generation and high-throughput screening, but they strip away the spatial and stromal context that governs immune cell function in real tumors. Three-dimensional spheroids and organoids restore much of that context, and organoids in particular retain patient-specific characteristics that make them attractive for precision immuno-oncology, yet they typically lack a complete immune component unless immune cells are deliberately co-cultured.

Tumor tissue slices occupy a distinctive niche because they retain the endogenous immune microenvironment, including tumor-associated macrophages, cancer-associated fibroblasts, regulatory T cells, and myeloid-derived suppressor cells, all of which are increasingly recognized as critical determinants of immunotherapy response and resistance. The limitation is practical rather than conceptual: slice viability declines over days, restricting the duration of experiments and complicating the assessment of therapies that require prolonged immune priming. Tumor-on-a-chip devices, by contrast, can sustain perfused cultures for longer periods and allow researchers to observe how immune cells migrate, extravasate, and infiltrate tumor tissue under physiologically relevant flow, but they demand specialized engineering expertise and remain difficult to standardize across laboratories.

The systematic nature of the review, prospectively registered with PROSPERO under registration number CRD420251153061, lends weight to its conclusions. The authors extracted data across defined domains including model type, immune cell representation, the class of immunotherapy assessed, and measures of transferability, summarizing the results into structured tables intended to serve as a practical reference. The work was supported by a Cancer Research UK Training Fellowship and by the European COST Action CA21135, and the authors acknowledged the Queen Elizabeth Upper Gastrointestinal Cancer Patient Group for its input. The team declared no competing interests, and because the review relied exclusively on publicly available published data, no primary patient samples or ethical approvals were required.

The practical significance of this catalogue lies in its potential to reduce the attrition that has plagued immuno-oncology drug development. Many immunotherapeutic agents that show promise in simplified laboratory systems fail in clinical trials, a pattern that experts attribute in part to the mismatch between conventional preclinical models and the immunologically complex reality of human tumors. By providing a curated, searchable dataset of 75 studies organized by model type, cancer type, immune context, and therapy class, the review gives researchers a tool for selecting the most appropriate platform for a given experimental question, whether that involves screening checkpoint inhibitor combinations in organoids, testing CAR-T cell cytotoxicity in spheroids, or probing immune trafficking in microfluidic chips.

The review also highlights gaps that the field must address. The representation of immune cell types remains narrow, with myeloid populations and dendritic cells underrepresented relative to T cells and natural killer cells, despite their established roles in shaping antitumor immunity. Validation standards vary widely across studies, and the authors note that culture conditions, extracellular matrix substitutes such as basement membrane extract, and air-liquid or gel-liquid interface techniques differ enough to complicate cross-study comparisons. As cancer immunotherapy continues to expand into new modalities and tumor types, the Birmingham team’s systematic inventory provides both a snapshot of current practice and a benchmark against which the next generation of preclinical platforms can be measured, bringing the laboratory one step closer to faithfully predicting which patients will benefit from the immune-based treatments reshaping modern oncology.

Subject of Research: A systematic review of in vitro and ex vivo preclinical models used to evaluate cancer immunotherapy in solid tumors

Article Title: Preclinical models for cancer immunotherapy: a systematic review of ex vivo and in vitro platforms

Article References: Colella, G., Piccioni, V., Swirsky, F., Kunene, V., & Contino, G. (2026). Preclinical models for cancer immunotherapy: a systematic review of ex vivo and in vitro platforms. Cancer Immunology, Immunotherapy. https://doi.org/10.1007/s00262-026-04494-w

Image Credits: AI Generated

DOI: 10.1007/s00262-026-04494-w

Keywords: cancer immunotherapy, patient-derived organoids, immune checkpoint inhibitors, CAR-T cells, tumor tissue slices, tumor-on-a-chip, 3D spheroids, tumor microenvironment, adoptive cell therapy, preclinical models, natural killer cells, systematic review