Abstract Introduction High risk non muscle invasive bladder cancer is commonly treated with intravesical BCG therapy, yet many patients fail treatment or develop recurrent disease. Effective anti tumor immunity in bladder cancer depends on dendritic cell activation and IL-12 production, which promotes T cell priming and effector function. We developed CANDI, a modular nanoparticle immunotherapy platform designed to deliver small molecule innate immune agonists directly to tumor antigen-presenting cells and enhance IL-12-driven immunity. Using optical screening, we identified a combination of innate stimulants that robustly induce IL-12. Unexpectedly, inclusion of the JAK inhibitor ruxolitinib further enhanced IL-12 production when co-delivered with innate agonists. Methods Bone marrow-derived dendritic cells from IL-12 reporter mice were used to quantify cytokine production following CANDI treatment. Therapeutic efficacy and immune responses were evaluated in orthotopic and metastatic MB49 bladder cancer models using intravesical and intravenous delivery. Tumor burden, dendritic cell migration, and T cell responses were assessed by imaging and flow cytometry. Results Triple combination CANDI significantly increased IL-12 production and dendritic cell activation compared to dual formulations. In tumor-bearing mice, CANDI reduced tumor burden, enhanced dendritic cell trafficking to tumor-draining lymph nodes, and improved antigen presentation and T cell priming. Comparable immune activation and tumor control were observed following intravesical and systemic delivery. In metastatic disease, CANDI reduced lung tumor burden and improved survival. Therapeutic efficacy was largely dependent on CD4 T cells, which amplified local IL-12 production by tumor resident dendritic cells. Conclusion Triple combination CANDI is a potent dendritic cell activating nano immunotherapy that drives durable anti tumor immunity and provides a rational strategy to overcome limitations of prior innate immune agonist therapies. Funding Source NIH, DOD, BICAN Topic Categories Vaccines and Immunotherapy (VAC)

The proliferation of human cervical cancer (Hela) cells was investigated on a series of nanostructured polymer latex surfaces. The physico-chemical properties of the surfaces, composed of mixtures of polystyrene and acrylonitrile butadiene styrene dispersions, were precisely controlled in the nanoscale range by adjusting the mixing ratio of the components and thermal treatment. In addition, the proliferation response of HeLa cells was compared to that of human dermal fibroblast (HDF) cells. A low dispersive surface energy and peak or valley dominance (Spk/Svk) were observed to increase the proliferation yield of the Hela cells. The HDF cells were less influenced by the surface chemistry and showed improved proliferation on surfaces without dominant peak or valley features (Spk and Svk). The observed changes in Hela cell behaviour underscored the critical role of material surface properties in influencing cellular responses, with more significant accumulation of nuclear patterning of filamentous actin (F-actin) on stiffer and smoother surfaces (e.g., borosilicate glass) due to higher mechanical stress. A more dynamic reorganisation of the cytoskeleton was observed for cells grown on polymer surfaces with moderate roughness and surface energy. These results emphasise the importance of characterising and tuning surface properties to accommodate the specific behaviours of different cell types.

Epithelial-mesenchymal transition (EMT) is a key biological process in physiological and pathological conditions, spanning development, wound healing, and cancer. Vimentin, a key cytoskeletal intermediate filament (IF) protein, is an established intracellular determinant of EMT. Recently, extracellular vimentin has also emerged with important functions, and we demonstrated that vimentin from fibroblast-derived extracellular vesicles (EVs) promotes wound healing. Building on these findings, we explored whether extracellular vimentin regulates EMT. We employed fibroblast-derived EVs to assess their EMT-driving capacity. Using coculture models and EV treatments from WT and vimentin-KO fibroblasts, we observed that fibroblasts induce an EMT phenotype in epithelial cells, marked by elevated mesenchymal markers and reduced epithelial markers. EVs from vimentin-deficient fibroblasts showed a decreased EMT-inducing capacity and failed to stimulate cell cover closure, underscoring vimentin's critical role in orchestrating these processes. Coculturing epithelial cells with WT fibroblasts mirrored these outcomes, while vimentin-deficient fibroblasts produced similarly poor EMT induction. Proteomic profiling revealed that WT EVs contained an enriched set of EMT-associated proteins, including those involved in cytoskeletal organization, cell adhesion, and EMT-regulating signaling pathways. Notably, these proteins, such as fibronectin and N-cadherin, were significantly diminished in vimentin-deficient EVs. Moreover, we identified over 600 additional proteins uniquely present in WT-derived EVs, with enrichment in key biological processes like wound healing and cell migration. These findings demonstrate that vimentin-positive EVs drive EMT by transmitting a specific protein cargo that supports EMT-related cellular changes. The vimentin-positive EV proteome will help understand EMT mechanisms and develop targeted therapies for pathological conditions related to abnormal EMT.

Despite extensive progress in cancer therapeutic research, translating promising anticancer compounds into clinical treatments often fails due to suboptimal pharmacokinetic and safety profiles. These shortcomings underscore the critical need for comprehensive pharmacokinetic (PK) analyses in the early stages of drug development. Among the compounds that have shown promising anticancer effects in multiple preclinical studies are anisomelic acid (AA) and ovatodiolide (OVT) - two diterpenoids from plant Anisomeles malabarica. However, their pharmacokinetic and toxicity profile remain poorly characterized. To explore their potential as chemotherapy agents, we first evaluated their key in vitro pharmacokinetic (PK) parameters, followed by an acute oral toxicity assessment and complementary in vivo PK analyses. In vitro experiments showed that both AA and OVT exhibited near-complete solubility in phosphate buffer, high stability, and strong permeability across MDR1-MDCK cell monolayer, and were not substrates of multidrug resistance protein (MDR1). However, OVT underwent rapid metabolism in liver microsomes in the presence of NADPH, whereas AA showed comparatively greater stability under the same conditions. Subsequent in vivo pharmacokinetic (PK) analyses in mice also demonstrated rapid clearance and low systemic bioavailability for both compounds following intravenous (IV) or transdermal (TD) administration. Metabolite identification revealed extensive conjugation to cysteine, and no acute toxicity or mortality was observed at high oral doses. Collectively, these data underscore the distinct metabolic and clearance patterns that limit systemic bioavailability but highlight the favorable safety of AA and OVT. Moreover, while topical administration may offer therapeutic advantages for localized conditions, additional formulation strategies will be crucial to overcome limited bioavailability for systemic use of AA or OVT.

In this issue of Developmental Cell, Huang et al. assess the role of vimentin intermediate filaments during extracellular matrix (ECM) degradation by macrophages. Vimentin stabilizes podosome clusters via CD11b in M2 macrophages, thereby reducing tumor collagen fiber and enhancing lung adenocarcinoma cancer invasion.

Human papillomavirus (HPV) is a major driver of cervical and other epithelial cancers, with the viral oncoprotein E6 playing a central role in tumorigenesis by promoting degradation of the tumor suppressor p53. While prophylactic vaccines prevent infection, there remains a critical need for therapeutic strategies that eliminate established HPV-positive cells. Here, we identify anisomelic acid (AA), a natural diterpenoid, as a novel pharmacological principle that selectively induces the degradation of HPV16 E6. Using cellular thermal shift assay, we demonstrate that AA directly interacts with E6, likely triggering a conformational change that promotes its ubiquitination. Proteomic analysis of the E6 interactome in AA-treated cells revealed consistent enrichment of E3 ubiquitin ligases, including E6AP, UBR4, CDC20, and TRIP12, as well as proteasomal subunits. To our knowledge, this represents the first comprehensive proteomics framework of the HPV16 E6 interactome under small-molecule treatment conditions. These findings support a model in which AA facilitates proteasome-mediated elimination of E6, and the dataset itself provides a timely and valuable resource for HPV biology and therapeutic development.

Immunotherapy represented by programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) monoclonal antibodies has led tumor treatment into a new era. However, the low overall response rate and high incidence of drug resistance largely damage the clinical benefits of existing immune checkpoint therapies. Recent studies correlate the response to PD-1/PD-L1 blockade with PD-L1 expression levels in tumor cells. Hence, identifying molecular targets and pathways controlling PD-L1 protein expression and stability in tumor cells is a major priority. In this study, we performed a Stress and Proteostasis CRISPR interference screening to identify PD-L1 positive modulators. Here, we identified TRAF6 as a critical regulator of PD-L1 in melanoma cells. As a non-conventional E3 ubiquitin ligase, TRAF6 is inclined to catalyze the synthesis and linkage of lysine-63 (K63) ubiquitin which is related to the stabilization of substrate proteins. Our results showed that suppression of TRAF6 expression down-regulates PD-L1 expression on the membrane surface of melanoma cells. We then used in vitro and in vivo assays to investigate the biological function and mechanism of TRAF6 and its downstream YAP1/TFCP2 signaling in melanoma. TRAF6 stabilizes YAP1 by K63 poly-ubiquitination modification, subsequently promoting the formation of YAP1/TFCP2 transcriptional complex and PD-L1 transcription. Inhibition of TRAF6 by Bortezomib enhanced cytolytic activity of CD8+ T cells by reduction of endogenous PD-L1. Notably, Bortezomib enhances anti-tumor immunity to an extent comparable to anti-PD-1 therapies with no obvious toxicity. Our findings reveal the potential of inhibiting TRAF6 to stimulate internal anti-tumor immunological effect for TRAF6-PD-L1 overexpressing cancers.

Pancreatic ductal adenocarcinoma (PDAC) is a solid organ malignancy with a high mortality rate. Statistics indicate that its incidence has been increasing as well as the associated deaths. Most patients with PDAC show poor response to therapies making the clinical management of this cancer difficult. Stromal cells in the tumor microenvironment (TME) contribute to the development of resistance to therapy in PDAC cancer cells. Cancer-associated fibroblasts (CAFs), the most prevalent stromal cells in the TME, promote a desmoplastic response, produce extracellular matrix proteins and cytokines, and directly influence the biological behavior of cancer cells. These multifaceted effects make it difficult to eradicate tumor cells from the body. As a result, CAF-targeting synergistic therapeutic strategies have gained increasing attention in recent years. However, due to the substantial heterogeneity in CAF origin, definition, and function, as well as high plasticity, majority of the available CAF-targeting therapeutic approaches are not effective, and in some cases, they exacerbate disease progression. This review primarily elucidates on the effect of CAFs on therapeutic efficiency of various treatment modalities, including chemotherapy, radiotherapy, immunotherapy, and targeted therapy. Strategies for CAF targeting therapies are also discussed.

Intermediate filaments (IFs) comprise a large family of versatile cytoskeletal proteins, divided into six subtypes with tissue-specific expression patterns. IFs have a wide repertoire of cellular functions, including providing structural support to cells, as well as active roles in mechanical support and signaling pathways. Consequently, defects in IFs are associated with more than 100 diseases. In this Cell Science at a Glance article, we discuss the established classes of IFs and their general features, their functions beyond structural support, and recent advances in the field. We also highlight their involvement in disease and potential use as clinical markers of pathological conditions. Finally, we provide our view on current knowledge gaps and the future directions of the IF field.

Vimentin, a type III intermediate filament, reorganizes into what is termed the ‘vimentin cage’ in response to various pathogenic infections. This cage-like structure provides an envelope to key components of the pathogen's life cycle. In viral infections, the vimentin cage primarily serves as a scaffold and organizer for the replication factory, promoting viral replication. However, it also occasionally contributes to antiviral functions. For bacterial infections, the cage mainly supports bacterial proliferation in most observed cases. These consistent structural alterations in vimentin, induced by a range of viruses and bacteria, highlight the vimentin cage's crucial role. Pathogen-specific factors add complexity to this interaction. In this review, we provide a thorough overview of the functions and mechanisms of the vimentin cage and speculate on vimentin's potential as a novel target for anti-pathogen strategies.

As a member of the large family of intermediate filaments (IFs), vimentin has emerged as a highly dynamic and versatile cytoskeletal protein involved in many key processes of wound healing. It is well established that vimentin is involved in epithelial-mesenchymal transition (EMT) during wound healing and metastasis, during which epithelial cells acquire more dynamic and motile characteristics. Moreover, vimentin participates in multiple cellular activities supporting growth, proliferation, migration, cell survival, and stress resilience. Here, we explore the role of vimentin at each phase of wound healing, with focus on how it integrates different signaling pathways and protects cells in the fluctuating and challenging environments that characterize a healing tissue.

In the context of three-dimensional (3D) cell cultureand tissueengineering, 3D printing is a powerful tool for customizing in vitro3D cell culture models that are critical for understanding the cell-matrixand cell-cell interactions. Cellulose nanofibril (CNF) hydrogelsare emerging in constructing scaffolds able to imitate tissue in amicroenvironment. A direct modification of the methacryloyl (MA) grouponto CNF is an appealing approach to synthesize photocross-linkablebuilding blocks in formulating CNF-based bioinks for light-assisted3D printing; however, it faces the challenge of the low efficiencyof heterogenous surface modification. Here, a multistep approach yieldsCNF methacrylate (CNF-MA) with a decent degree of substitution whilemaintaining a highly dispersible CNF hydrogel, and CNF-MA is furtherformulated and copolymerized with monomeric acrylamide (AA) to forma super transparent hydrogel with tuneable mechanical strength (compressionmodulus, approximately 5-15 kPa). The resulting photocurablehydrogel shows good printability in direct ink writing and good cytocompatibilitywith HeLa and human dermal fibroblast cell lines. Moreover, the hydrogelreswells in water and expands to all directions to restore its originaldimension after being air-dried, with further enhanced mechanicalproperties, for example, Young's modulus of a 1.1% CNF-MA/1%PAA hydrogel after reswelling in water increases to 10.3 kPa from5.5 kPa.

Extracellular vesicles (EVs) are important mediators of intercellular communication involved in local and long-range signalling of cancer metastasis. The onset of invasion is the key step of the metastatic cascade, but the secretion of EVs has remained unexplored at that stage due to technical challenges. In this study, we present a platform to track EVs over the course of invasive development of human prostate cancer cell (PC3) tumoroids utilizing in vivo-mimicking extracellular matrix-based 3D cultures. Using this EV production method, combined with proteomic profiling, we show that PC3 tumoroids secrete EVs with previously undefined protein cargo. Intriguingly, an increase in EV amounts and extensive changes in the EV protein composition were detected upon invasive transition of the tumoroids. The changes in EV protein cargo were counteracted by chemical inhibition of invasion. These results reveal the impact of the tumoroids' invasive status on EV secretion and cargo, and highlight the necessity of in vivo-mimicking conditions for uncovering novel cancer-derived EV components.

Vimentin, an intermediate filament protein typically located in the cytoplasm of mesenchymal cells, can also be secreted as an extracellular protein. The organization of extracellular vimentin strongly determines its functions in physiological and pathological conditions, making it a promising target for future therapeutic interventions. The extracellular form of vimentin has been found to play a role in the interaction between host cells and pathogens. In this review, we first discuss the molecular biophysics of extracellular vimentin, including its structure, secretion, and adhesion properties. We then provide a general overview of the role of extracellular vimentin in mediating pathogen-host interactions, with a focus on its interactions with viruses and bacteria. We also discuss the implications of these findings for the development of new therapeutic strategies for combating infectious diseases.

Vimentin is a cytoskeletal protein important for many cellular processes, including proliferation, migration, invasion, stress resistance, signaling, and many more. The vimentin-deficient mouse has revealed many of these functions as it has numerous severe phenotypes, many of which are found only following a suitable challenge or stress. While these functions are usually related to vimentin as a major intracellular protein, vimentin is also emerging as an extracellular protein, exposed at the cell surface in an oligomeric form or secreted to the extracellular environment in soluble and vesicle-bound forms. Thus, this review explores the roles of the extracellular pool of vimentin (eVIM), identified in both normal and pathological states. It focuses specifically on the recent advances regarding the role of eVIM in wound healing and cancer. Finally, it discusses new technologies and future perspectives for the clinical application of eVIM.

The heat shock (HS) response is crucial for cell survival in harmful environments. Nuclear lamin A/C, encoded by the LMNA gene, contributes towards altered gene expression during HS, but the underlying mechanisms are poorly understood. Here, we show that upon HS, lamin A/C was reversibly phosphorylated at serine 22 in concert with HSF1 activation in human cells, mouse cells and Drosophila melanogaster in vivo. Consequently, the phosphorylation facilitated nucleoplasmic localization of lamin A/C and nuclear sphericity in response to HS. Interestingly, lamin A/C knock-out cells showed deformed nuclei after HS and were rescued by ectopic expression of wild-type lamin A, but not by a phosphomimetic (S22D) lamin A mutant. Furthermore, HS triggered concurrent downregulation of lamina-associated protein 2α (Lap2α, encoded by TMPO) in wild-type lamin A/C-expressing cells, but a similar response was perturbed in lamin A/C knock-out cells and in LMNA mutant patient fibroblasts, which showed impaired cell cycle arrest under HS and compromised survival at recovery. Taken together, our results suggest that the altered phosphorylation stoichiometry of lamin A/C provides an evolutionarily conserved mechanism to regulate lamina structure and serve nuclear adaptation and cell survival during HS.

The impact of small alkali halide additions on the melting behavior and corrosivity of a synthetic sulfate deposit at 500, 550, and 600 degrees C was investigated. Three differently alloyed commercial heat-transfer materials; low -alloyed 10CrMo9-10, stainless AISI 347, and high-alloyed Sanicro 28, were studied. The samples were exposed for 168 h in a tube furnace to a K2SO4 + Na2SO4 mixture containing 0.85 mol% KCl, KBr, or KF. The extent of material degradation was determined by weight loss measurements, while the morphology, thickness, and composition of the formed oxide scale were characterized with SEM-EDS. Additionally, the melting behavior of the mixtures was studied with TG-DTA. It could be concluded that already small amounts of reactive alkali halides in an otherwise inert K2SO4 + Na2SO4 mixture change significantly the corrosion and melting behavior of the mixture.

Fibroblastic migration is of key importance in wound healing. While the intermediate filament (IF) protein vimentin is required for normal wound healing, we examined whether vimentin-mediated regulation of fibroblast migration could be involved. In wound healing assays triggering cell polarization and directed migration, we observed that vimentin-deficient mouse and rat embryonic fibroblasts lost their directional persistence. We show that vimentin maintains directionality by guiding focal adhesions (FAs) in fibroblasts. Detailed analysis showed that vimentin stabilizes FAs and regulates their disassembly rate. The destabilization of Vim-/- FAs was reflected by smaller FAs. Live cell and super-resolution imaging demonstrate that vimentin interacts dynamically with the key molecules of FAs and, importantly, with FAK, which is crucial for the maturation of FAs. These results demonstrate that vimentin IFs control the maturation, stability, dynamics, arrangement, and overall orientation of FAs, with a net effect on FA coordination during migration. ### Competing Interest Statement The authors have declared no competing interest.

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.