To Örebro University

Sepsis remains a leading global health challenge, characterized by high mortality and a persistent lack of disease-modifying therapies. Despite decades of investment, therapeutic progress has been constrained by reductionist strategies that target isolated pathogenic components. This perspective argues that these failures reflect a fundamental mischaracterization of sepsis-not as a disorder of discrete pathways, but as the collapse of complex biological systems in which normally coordinated processes become desynchronized. We identify the intermediate filament protein vimentin as a determinant of system fate governing the transition from adaptive host defense to pathological breakdown. Acting as a context-dependent network integrator and signal amplifier, vimentin coordinates antagonistic cellular programs by integrating biochemical and biophysical cues across immune, vascular, and metabolic systems. Under physiological stress, this coordination enables the orderly activation and resolution of inflammatory and suppressive responses required for pathogen control and restoration of homeostasis. In sepsis, persistent or excessive insults drive vimentin-mediated overactivation, uncoupling these programs and propagating systems-level instability that culminates in organ dysfunction. By integrating mechanistic, preclinical, and emerging clinical evidence, this perspective proposes vimentin modulation as a clinically translatable, systems-oriented strategy aimed at realigning host response networks to address the dynamic, opposing pathologies of sepsis that have eluded current therapies.

While normal fibroblasts suppress tumor growth, during cancer initiation and progression, this capacity can be lost and even switched to tumor-promoting, for reasons that are not understood. In this study, we aimed to determine differences between patient-derived cancer-associated fibroblasts and fibroblasts from healthy breast tissue to identify if and how these changes stimulate Triple-negative breast cancer (TNBC). Two-dimensional and three-dimensional mono and co-cultures of TNBC cells with fibroblasts from healthy breast or TNBC were analyzed for cell contractility, migration, distribution, proliferation, and hyaluronan production by traction force microscopy, live cell imaging, flow cytometry, Western blot, and ELISA. In 3D spheroid co-culture, CAFs migrated into the tumor mass, mixing with tumor cells, whereas normal fibroblasts remained separate. In 2D, CAFs showed increased cell migration and contractile force, and, in both 2D and 3D co-culture, CAFs increased the proliferation of TNBC cells. CAFs showed increased production of hyaluronan, as compared to normal fibroblasts, and loss of hyaluronan synthase 2 reduced CAF-induced stimulation of TNBC proliferation. These findings suggest that increased production of hyaluronan by TNBC CAFs enhances their capacity to mix with and induce the proliferation of cancer cells, and that the production of hyaluronan by CAFs can be a future therapeutic target against TNBC.

Background: Tumors of the pleura, such as metastatic lung cancer and mesothelioma, are amongst the most lethal and therapy-resistant tumors. The first manifestation of the disease is often pleural effusion, the first available material for diagnosis. The five-year survival rate is exceptionally low, around 10-20%, and only a small proportion of patients harbor mutations that allow targeted treatments. Almost all patients develop resistance to treatment, which is often palliative. There is therefore an urgent need to refine the selection of drugs and patients for personalized treatment.

Methods: We isolated and cultured cells from pleural effusions in 3D cell aggregates and compared their drug sensitivity ex vivo to the clinical response to the same chemotherapeutic agents, combined with targeted sequencing and network analysis.

Results: The ex vivo drug response showed a positive correlation with the treatment response and survival of patients in the clinic, with a stronger link to overall survival than to progression-free survival. Cryopreserved cells showed a similar response to freshly collected cells from the clinic.

Conclusions: The findings advance the field of ex vivo screening and present an opportunity to combine strategies for functional precision medicine with comprehensive characterization of disease for improved treatment and future management of lung cancer.

Background/objectives: Non-small cell lung cancer and pleural mesothelioma are among the most lethal and therapy-resistant tumors in humans. These tumors are diagnosed late, frequently present pleural effusion and develop drug resistance, and the treatment is often inefficient and largely alliative. Therefore, there is an urgent need to refine the selection of drugs and patients for personalized treatment.

Methods: To progress the field, we performed a systematic literature review in line with the PRISMA guidelines followed by a narrative synthesis approach to identify themes.

Results: The literature to date shows, in general, a positive correlation between the drug-sensitivity of patient-derived cells ex vivo and the clinical outcome. However, only a handful of these studies show a numerical correlation. This, along with the vast diversity of correlated techniques and parameters makes it difficult to directly compare the findings. To build a common knowledge base for future studies, we therefore offer a comprehensive summary of the literature, identify gaps, and suggest future avenues for research.

Conclusions: We present unified recommendations for the collection, preparation, and ex vivo sensitivity testing of samples for the future development of personalized medicine.

Cells sense mechanical cues and convert them into biochemical responses to regulate biological processes such as embryonic development, aging, cellular homeostasis, and disease progression. In this study, we introduce a large-scale, systematic approach to identify proteins with mechanosensitive nuclear localization, highlighting their potential roles in mechanotransduction. Among the proteins identified, we focus here on the splicing factor PTBP1. We demonstrate that its nuclear abundance is regulated by mechanical cues such as cell density, size, and extracellular matrix (ECM) stiffness and that PTBP1 medicates the mechanosensitive alternative splicing of the endocytic adapter protein Numb. Furthermore, we show that PTBP1 and Numb alternative splicing is critical for ECM stiffness-induced epithelial cell spreading and proliferation as well as for mesenchymal stem cell differentiation into osteoblasts on a stiff matrix. Our results underscore the emerging role of alternative splicing in mechanotransduction and provide novel mechanistic insights into how matrix stiffness modulates cellular mechanoresponses.

Metastasis driven by cancer cell migration is the leading cause of cancer-related deaths. It involves significant changes in the organization of the cytoskeleton, which includes the actin microfilaments and the vimentin intermediate filaments. Understanding how these filament change cells from normal to invasive offers insights that can be used to improve cancer diagnosis and therapy. We have developed a computational, transparent, large-scale and imaging-based pipeline, that can distinguish between normal human cells and their isogenically matched, oncogenically transformed, invasive and metastasizing counterparts, based on the spatial organization of actin and vimentin filaments in the cell cytoplasm. Due to the intricacy of these subcellular structures, manual annotation is not trivial to automate. We used established deep learning methods and our new multi-attention channel architecture. To ensure a high level of interpretability of the network, which is crucial for the application area, we developed an interpretable global explainable approach correlating the weighted geometric mean of the total cell images and their local GradCam scores. The methods offer detailed, objective and measurable understanding of how different components of the cytoskeleton contribute to metastasis, insights that can be used for future development of novel diagnostic tools, such as a nanometer level, vimentin filament-based biomarker for digital pathology, and for new treatments that significantly can increase patient survival.

Background: The epithelial-to-mesenchymal transition (EMT) is a common feature in early cancer invasion. Increased vimentin is a canonical marker of the EMT; however, the role of vimentin in EMT remains unknown.

Methods: To clarify this, we induced EMT in lung cancer cells with TGF-beta 1, followed by treatment with the vimentin-targeting drug ALD-R491, live-cell imaging, and quantitative proteomics.

Results: We identified 838 proteins in the intermediate filament fraction of cells. TGF-beta 1 treatment increased the proportion of vimentin in this fraction and the levels of 24 proteins. Variants of fibronectin showed the most pronounced increase (137-fold), followed by regulators of the cytoskeleton, cell motility, and division, such as the mRNA-splicing protein SON. TGF-beta 1 increased cell spreading and cell migration speed, and changed a positive correlation between cell migration speed and persistence to negative. ALD-R491 reversed these mesenchymal phenotypes to epithelial and the binding of RNA-binding proteins, including SON.

Conclusions: These findings present many new interactors of intermediate filaments, describe how EMT and vimentin filament dynamics influence the intermediate filament interactome, and present ALD-R491 as a possible EMT-inhibitor. The observations support the hypothesis that the dynamic turnover of vimentin filaments and their interacting proteins govern mesenchymal cell migration, EMT, cell invasion, and cancer metastasis.

The capacity of cells to adhere to, exert forces upon and migrate through their surrounding environment governs tissue regeneration and cancer metastasis. The role of the physical contractile forces that cells exert in this process, and the underlying molecular mechanisms are not fully understood. We, therefore, aimed to clarify if the extracellular forces that cells exert on their environment and/or the intracellular forces that deform the cell nucleus, and the link between these forces, are defective in transformed and invasive fibroblasts, and to indicate the underlying molecular mechanism of control. Confocal, Epifluorescence and Traction force microscopy, followed by computational analysis, showed an increased maximum contractile force that cells apply on their environment and a decreased intracellular force on the cell nucleus in the invasive fibroblasts, as compared to normal control cells. Loss of HDAC6 activity by tubacin-treatment and siRNA-mediated HDAC6 knockdown also reversed the reduced size and more circular shape and defective migration of the transformed and invasive cells to normal. However, only tubacin-mediated, and not siRNA knockdown reversed the increased force of the invasive cells on their surrounding environment to normal, with no effects on nuclear forces. We observed that the forces on the environment and the nucleus were weakly positively correlated, with the exception of HDAC6 siRNA-treated cells, in which the correlation was weakly negative. The transformed and invasive fibroblasts showed an increased number and smaller cell-matrix adhesions than control, and neither tubacin-treatment, nor HDAC6 knockdown reversed this phenotype to normal, but instead increased it further. This highlights the possibility that the control of contractile force requires separate functions of HDAC6, than the control of cell adhesions, spreading and shape. These data are consistent with the possibility that defective force-transduction from the extracellular environment to the nucleus contributes to metastasis, via a mechanism that depends upon HDAC6. To our knowledge, our findings present the first correlation between the cellular forces that deforms the surrounding environment and the nucleus in fibroblasts, and it expands our understanding of how cells generate contractile forces that contribute to cell invasion and metastasis.

Metastasizing cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumors in the clinic. However, the role of vimentin in cell motility, and if the assembly of non-filamentous variants of vimentin into filaments regulates cell migration remains unclear. We observed that the vimentin-targeting drug ALD-R491 increased the stability of vimentin filaments, by reducing filament assembly and/or disassembly. ALD-R491-treatment also resulted in more bundled and disorganized filaments and an increased pool of non-filamentous vimentin. This was accompanied by a reduction in size of cell-matrix adhesions and increased cellular contractile forces. Moreover, during cell migration, cells showed erratic formation of lamellipodia at the cell periphery, loss of coordinated cell movement, reduced cell migration speed, directionality and an elongated cell shape with long thin extensions at the rear that often detached. Taken together, these results indicate that the stability of vimentin filaments and the soluble pool of vimentin regulate the speed and directionality of cell migration and the capacity of cells to migrate in a mechanically cohesive manner. These observations suggest that the stability of vimentin filaments governs the adhesive, physical and migratory properties of cells, and expands our understanding of vimentin functions in health and disease, including cancer metastasis.

Dendritic cells use amoeboid migration to pass through narrow passages in the extracellular matrix and confined tissue in search for pathogens and to reach the lymph nodes and alert the immune system. Amoeboid migration is a migration mode that, instead of relying on cell adhesion, is based on mechanical resilience and friction. To better understand the role of intermediate filaments in ameboid migration, we studied the effects of vimentin on the migration of dendritic cells. We show that the lymph node homing of vimentin-deficient cells is reduced in our in vivo experiments in mice. Lack of vimentin also reduces the cell stiffness, the number of migrating cells, and the migration speed in vitro in both 1D and 2D confined environments. Moreover, we find that lack of vimentin weakens the correlation between directional persistence and migration speed. Thus, vimentin-expressing dendritic cells move faster in straighter lines. Our numerical simulations of persistent random search in confined geometries verify that the reduced migration speed and the weaker correlation between the speed and direction of motion result in longer search times to find regularly located targets. Together, these observations show that vimentin enhances the ameboid migration of dendritic cells, which is relevant for the efficiency of their random search for pathogens.