Radiotherapy (RT) is a central treatment for prostate cancer (PCa), relying on the induction of DNA double-strand breaks (DSBs). Tumor ability to repair these breaks limits RT efficacy, making DSB repair inhibitors potential radiosensitizers. However, many of these inhibitors lack tumor specificity and harm normal cells. Therefore, tumor-specific radiosensitization strategies are critically needed for PCa. Approximately 50% of PCa cases harbor the TMPRSS2-ERG gene fusion, leading to overexpression of the ERG transcription factor (ERG+). In this study, we demonstrate that ERG+ tumors shift DSB repair toward the poly(ADP-ribose) polymerase 1-dependent end-joining (PARP1-EJ) pathway. Proteomic and Western blot analyses revealed elevated PARP1, XRCC1, and LIG3 levels in ERG+ cells. Notably, PARP inhibition with olaparib increased residual γH2AX/53BP1 foci postirradiation in ERG+ cells, indicating enhanced radiosensitization. In tissue slice cultures (TSCs) from 53 tumors of patients with high-risk PCa, olaparib selectively increased γH2AX/53BP1 foci selectively in ERG+ samples. ERG+ patient-derived organoids also showed significantly delayed growth when treated with olaparib plus RT, compared with either treatment alone. Interestingly, ERG-negative cells within ERG+ TSCs were similarly radiosensitized by olaparib, likely through bystander effect, with residual 53BP1 foci levels comparable to those in ERG+ cells. This was confirmed by medium exchange experiments. These findings suggest that ERG expression promotes dependency on the PARP1-EJ pathway, rendering ERG+ PCa more susceptible to PARP inhibition. This supports combining PARP inhibitors with RT for tumor-selective radiosensitization in ERG+ patients.

Abstract Chronic pyelonephritis is characterized by persistent bacterial infection of the kidney and a dysregulated immune response that promotes disease progression. Macrophages are central regulators of antibacterial immunity in the urinary tract. However, the mechanisms underlying their functional reprogramming in chronic infection remain poorly understood. Here, we identify lactate as a key metabolic determinant of macrophage polarization in human pyelonephritis. Proteomic analysis of human kidney tissue revealed extensive metabolic remodelling, including upregulation of enzymes involved in glycolysis. Consistent with this, lactate levels were significantly elevated in urine and plasma of pyelonephritis patients. Concomitantly, we observed a pronounced accumulation of CD163 macrophages in infected kidneys, representing a distinct macrophage subset with immunomodulatory function. Correlation-based network analysis revealed a strong association between CD163 and lactate dehydrogenase A, supporting a functional association between lactate metabolism and macrophage polarization. Mechanistically, exposure of murine bone marrow-derived macrophages to lactate induced intracellular protein lactylation and promoted polarization toward a CD163 phenotype, defining a metabolically imprinted macrophage state distinct from classical activation paradigms. Proteomic profiling demonstrated extensive remodelling of macrophage protein expression in response to lactate with significant alteration of mediators of phagocytosis, Toll-like receptor signalling, and interferon response. Together, these findings identify lactate as a potent metabolic driver of macrophage reprogramming and establish a foundation for investigating lactylation-dependent immune dysfunction in chronic pyelonephritis.

BACKGROUND:Two PARP inhibitors (PARPis), olaparib and talazoparib, have been approved in combination with androgen receptor pathway inhibitors (ARPIs) for metastatic castration-resistant prostate cancer (mCRPC) in Europe, regardless of homologous-recombination repair (HRR) status. However, the mechanism in HRR-negative patients remains unclear. METHODS:We assessed PARP inhibitors (PARPis; olaparib, talazoparib) alone and with ARPIs (abiraterone, enzalutamide, apalutamide) in prostate cancer cell lines, metastatic patient-derived organoids (mPDOs), and LN-metastatic tissue slice cultures. HRDetect and multi-omics analyses were used to investigate response mechanisms. RESULTS:PARPi-ARPI combinations significantly reduced survival in LNCaP, DU145, and 22RV1 cells and produced greater cytotoxicity than either agent alone in all models. In three metastatic mPDOs, olaparib alone had no effect, whereas talazoparib reduced survival in one mPDO and ARPIs reduced survival in two. In all tested models, PARPi-ARPI combinations produced greater cytotoxicity than either agent alone. None of the mPDOs showed BRCAness by HRDetect. ARPIs downregulated RAD51 and induced a BRCAness-like state, with reduced RAD51 foci formation at DNA double-strand break sites and impaired repair capacity, thereby sensitizing cells to PARPis. In LN-metastatic tissue slice cultures, combination therapy reduced double-strand break repair capacity in 67% of samples. Proteomic analysis showed that olaparib suppressed prometastatic pathways, including epithelial-mesenchymal transition and angiogenesis. CONCLUSION:ARPIs induce a BRCAness-like phenotype through RAD51 downregulation and enhance PARPi sensitivity in metastatic prostate cancer models, supporting combined ARPI-PARPi therapy.

Monoallelic variants in catalytic immunoproteasome subunits have recently been linked to proteasome-associated autoinflammatory syndromes with immunodeficiency (PRAAS-ID), yet their molecular mechanisms and clinical spectra are not fully defined. In this study, seven individuals from five unrelated families carrying five distinct monoallelic PSMB8 variants were identified. Individuals presented with neonatal-onset immunodeficiency characterized by recurrent infections, B cell lymphopenia, and hypogammaglobulinemia requiring immunoglobulin replacement. Inflammatory manifestations of variable severity included enteropathy, hepatitis, myositis, and inflammatory lung disease. Additional findings included leukocyte vacuolization in blood and bone marrow. Pathogenic variants in immunoproteasome subunits were analyzed to identify structural features associated with dominant-negative behavior. Immunoproteasome assembly and activity were investigated using complexome profiling, immunoblotting, and in-gel activity assays in proband-derived fibroblasts and transfected HEK293T cells, with downstream effects assessed by proteomic and RT-qPCR analyses. Mutant PSMB8 subunits were inefficiently incorporated into immunoproteasome complexes, leading to impaired assembly, including reduced fully assembled complexes and accumulation of assembly intermediates. This defect was accompanied by activation of the integrated stress response alongside impaired immune signaling. Monoallelic pathogenic variants in PSMB8, PSMB9, and PSMB10 associated with PRAAS-ID affected residues that are highly conserved and biophysically similar between the three immunoproteasome catalytic subunits. These shared structural features may help identify additional variants with similar disruptive effects on immunoproteasome assembly. Together, our data show that monoallelic PSMB8 variants disrupt immunoproteasome assembly, resulting in clinically variable disease with immunodeficiency and systemic inflammation. Our findings support immunoproteasome assembly disruption as a unifying dominant-negative mechanism underlying PRAAS-ID.

Abstract Fic enzymes mediate diverse post-translational modifications, including adenosine monophosphate (AMP) transfer and removal, referred to as AMPylation and deAMPylation, respectively. We identified the prokaryotic translation elongation factor Tu (EF-Tu) as an AMPylation target of the Fic enzyme SoFic. SoFic can constitutively reverse EF-Tu modification via deAMPylation whereas AMPylation depends on SoFic homodimerization. The complex crystal structure between SoFic and EF-Tu confirms a conserved target binding mode across evolutionary distant Fic enzymes. AMPylation disrupts EF-Tu’s regulatory switch-I region, causing translational inhibition. SoFic furthermore binds to its promotor DNA, suggesting a dual function as transcriptional and translational regulator in bacterial cells. Together, our structural and biochemical data provide valuable insights into the functional and regulatory diversity of Fic enzymes.

Introduction:Colorectal cancer (CRC) is common and associated with poor survival, and early detection is pivotal to improving patient outcomes. Therefore, identifying reliable biomarkers reflecting early-stage tumor development is of major clinical importance. This study aimed to identify salivary proteins with potential as non-invasive biomarkers associated with early-stage CRC. Methods:A total of 104 saliva samples were collected from individuals undergoing CRC screening, including patients diagnosed with CRC stage I and non-cancer controls (NCC), at Taleghani Hospital, Tehran, Iran, in this case-control study. A quantitative label-free proteomics approach was applied to determine global proteome differences between CRC patients and NCCs and to identify proteins with altered abundance. Proteins were extracted from saliva samples, digested with trypsin, and peptides were analyzed using LC-MS/MS on an Orbitrap Fusion mass spectrometer. Results:A total of 2,456 salivary proteins were identified, of which 181 showed significantly different abundance between CRC patients and NCCs. Unsupervised clustering demonstrated partial separation of groups with influence from demographic variables. Members of the small proline-rich protein (SPRR) family emerged as consistent core markers distinguishing CRC from NCCs, while additional immune- and metabolism-associated proteins contributed to group differentiation. Demographic subgroup analyses, including age-, sex-, and smoking-stratified comparisons, revealed subgroup-associated differences in protein abundance patterns; however, several CRC-associated alterations remained detectable across subgroups, including middle-aged individuals. Gene set enrichment analysis indicated suppression of cytoskeletal and epithelial structural pathways alongside enrichment of protease regulatory pathways. Ingenuity Pathway Analysis identified an interconnected immune-metabolic network characterized by inflammatory signaling and oxidative stress-related processes. These alterations indicate immune-related and metabolic signaling changes associated with CRC. Discussion:This study supports salivary proteomics as a non-invasive approach for early CRC detection, and the observed proteomic signatures may reflect systemic tumor-immune interactions associated with CRC. Data are available via ProteomeXchange with identifier PXD078610.

Abstract Antimicrobial peptides (AMPs) are key effectors of host defence, however, their functional deployment across renal tissue and urine in pyelonephritis (PN) remains incompletely understood. Here, we integrate kidney and urine proteomics with urinary peptidomics and computational prediction to define AMP organisation and function. Proteomic analysis indicated coordinated induction of multiple AMPs in infected kidneys. These patterns were recapitulated in the urinary proteome, where AMP abundance correlated with leukocyte counts. Multiplex immunofluorescence microscopy localised these AMPs to myeloid cells, identifying them as central effector sources. Importantly, analysis of the urine peptidome revealed multiple encrypted AMPs (EPs), which arise from proteolytic processing of precursor proteins. To systematically assess their relevance for host defence, we applied an ensemble of machine learning-based predictors to prioritise candidates with activity in the urinary environment. This approach identified several potential EPs, among which the S100A12-derived peptide Calcitermin was confirmed in patient urine. Furthermore, it exerts antibacterial activity against uropathogenic E. coli (UPEC) and modulates myeloid cell responses. Together, these findings define a coordinated and compartmentalised AMP defence programme in human PN that extends beyond increased peptide expression, highlighting EPs as functionally relevant effectors with therapeutic potential.

Despite substantial clinical benefit from immune checkpoint inhibitors (ICI), advanced melanoma remains challenging due to frequent treatment resistance. Resistance may be intrinsic (primary) or emerge over time (secondary). Biomarkers predicting distinct resistance phenotypes before therapy are lacking. As key mediators of cellular communication, extracellular vesicles (EVs) represent promising biomarkers. This study aimed to identify baseline EV proteome-derived pathways and biomarkers associated with overall, primary, and secondary resistance to ICI in advanced melanoma and to derive biomarker signatures predictive of progression-free survival (PFS). EVs were isolated from pretreatment plasma samples of 46 patients with advanced melanoma using size exclusion chromatography and ultracentrifugation. Proteomic profiling was performed by liquid chromatography-mass spectrometry using DIA-NN. Pathway enrichment and network analyses were conducted using Reactome, Metascape, Cytoscape, and DAVID. Resistance-associated proteins were integrated into composite biomarker signatures and evaluated for association with PFS. Overall resistance was characterized by enrichment of platelet- and complement-associated pathways. Primary resistance was associated with enhanced Fc gamma receptor (FCGR) signaling and downregulation of KSRP-associated post-transcriptional regulatory processes. In contrast, secondary resistance was preceded by distinct baseline EV proteomic patterns involving complement activation and reduced hemostasis- and platelet-related pathways. EV-derived biomarker signatures for overall, primary, and secondary resistance independently discriminated patients according to PFS. Baseline plasma EV proteomics reveals distinct systemic biological programs associated with different resistance phenotypes to ICI in advanced melanoma. EV-derived biomarker signatures enable stratification by PFS and warrant validation in larger, multicentric cohorts.

Neutrophils, critical components of innate immunity, undergo significant morphological changes during phagocytosis. In this study, we demonstrate that neutrophils exposed to shear stress generate lipid nanotubes (NTs) with an unique composition. Compared to the proteome of whole neutrophils, NTs notably lack cytoskeletal elements and the complement-inhibiting transmembrane protein CD46. Consequently, these NTs are recognized by the complement system and selectively opsonized the complement component C3b. Biophysical characterization of NTs confirmed that their integrity relies on lipid-lipid interactions and that they pinch off from neutrophils to form NT-derived vesicles (NTDVs). We detected C3b+ NTDVs in plasma from patients and animal models experiencing diverse inflammatory conditions, including metastatic melanoma, vasculitis, and polytrauma. Further functional experiments indicate that resting neutrophils phagocytose complement-opsonized NTDVs, leading to cell activation, including the production of reactive oxygen species (ROS). In conclusion, our data suggest a significant role for neutrophil-derived NTs in a wide range of inflammatory diseases and reveal a previously unknown mode of cell-cell communication.

Abstract Neuronal differentiation is a dynamic, multi-layered process that transforms progenitor cells into functionally integrated neurons, yet the coordinated molecular programs underlying this transition remain incompletely understood. Here, we define the developmental trajectory of murine upper-layer cortical neurons using an integrated multi-omics approach combining transcriptomics and proteomics across key stages of neurogenesis. We find that neuronal identity emerges gradually through coordinated transitions from RNA processing and splicing programs toward synaptic and metabolic maturation. Integration of matched transcriptomic and proteomic datasets reveals a compact set of concordant molecular modules that define this trajectory, highlighting post-transcriptional regulation as a central driver of neuronal maturation. We further show that maternal immune activation (MIA), a model of prenatal inflammation, deranges this developmental program. MIA induces sustained upregulation of Wnt signalling pathways alongside a downregulation of synaptic regulators, without detectable global alterations in DNA methylation. These molecular changes are accompanied by defects in neuronal positioning during cortical development, linking altered molecular trajectories to functional outcomes. Together, our findings establish a temporal molecular framework of neuronal differentiation and demonstrate that prenatal environmental perturbations reshape cortical development primarily through post-transcriptional and signalling-based mechanisms.

Abstract Merkel cell carcinoma (MCC) is a highly aggressive skin cancer, with approximately 80% of cases driven by Merkel cell polyomavirus (MCPyV). Although extracellular vesicles (EVs) are increasingly recognized as mediators of intercellular communication within the tumor microenvironment, their molecular cargo in MCPyV-positive MCC has not been comprehensively characterized. Here, we performed a multi-omics characterization of EVs released by two MCPyV-positive MCC cell lines. EVs were isolated by differential ultracentrifugation and characterized by nanoparticle tracking analysis, imaging flow cytometry, cryo-electron microscopy, and immunoblotting, demonstrating a heterogeneous population of small and large EVs. Proteomic and transcriptomic analyses revealed that MCC-derived EVs possess distinct protein, mRNA, and miRNA cargo compared with their parental cells, with enrichment of molecules associated with gene expression, RNA processing, intracellular signaling, and vesicle-mediated transport. Despite differences in the molecular composition of EVs derived from WaGa and MKL-1 cells, functional enrichment analyses revealed highly similar biological pathways. To investigate whether the viral oncoprotein small T antigen (sT) contributes to EV cargo composition, EVs from inducible sT knockdown cells were analyzed. Loss of sT was associated with modest changes in the EV proteome and mRNA cargo, whereas the overall EV-associated miRNA profile remained largely unchanged. Collectively, these findings provide the first comprehensive molecular characterization of EVs released by MCPyV-positive MCC cells and establish a foundation for investigating the contribution of EV-mediated communication to MCC biology and tumor–microenvironment interactions.

Two-dimensional gastric organoid-derived mucosoids provide a physiologically relevant model for studying epithelial differentiation and function. We demonstrated that withdrawal of WNT3a (WNT) and TGFβ inhibitor promotes differentiation and compartmentalization in human gastric mucosoids without impairing functionality. This results in a phenotype resembling the in vivo antral glands. Compared with WNT/TGFβi + conditions, WNT/TGFβi - cultures exhibited increased cell height, enhanced barrier function, and reduced cell density, accompanied by reduced stemness markers and increased tight junction and secreted factors. Phenotypically, WNT/TGFβi⁻ cultures resembled the antral gland tip, showing increased MUC5AC expression, whereas WNT/TGFβi⁺ condition maintained gland base–like features with higher nuclear density. We applied the adenylyl cyclase activator Forskolin (FSK), which enhanced expression of mucus and hormone-related marker without additional WNT supplementation. Differential proteomic confirmed that withdrawal of WNT and TGFβ inhibitor shift cells from stemness toward differentiated epithelial states with enhanced tight junctions and secretory features. FSK promoted a more parietal cell-like state, dependent on the absence of WNT and TGFβ inhibition. Proteomic analysis revealed that the mucus layer has a protein composition distinct from matched cellular lysates, enriched in extracellular matrix, immune, antimicrobial, coagulation, and GPCR-related proteins. This mucus signature was conserved across organoid lines despite marked donor-to-donor variability. WNT, TGFβ, and cAMP signalling further shaped the composition of the mucus proteome while preserving patient-specific protein signatures. We show that the withdrawal of WNT and TGFβ inhibition alone is sufficient to drive broad epithelial differentiation of cells and the enrichment of multiple differentiated subpopulations, without the need for supplementation of additional growth factors. At the same time, standard culture protocols can be used to preserve the proliferative zone, enabling study of both compartments of the gland. Together, these findings establish gastric mucosoids as a versatile model to investigate epithelial differentiation, functional compartmentalization, and secretion with enhanced in vitro differentiation.

Background Neuroaxonal and synaptic loss are hallmarks of multiple sclerosis (MS), the most common autoimmune disorder of the central nervous system. However, it remains unclear at which disease stages synaptic pathology occurs. We hypothesised that synaptic proteins in plasma and cerebrospinal fluid (CSF) reflect synaptic injury in MS. Methods To identify synaptic proteins lost during neuroinflammation, we performed proteomic analysis of synaptoneurosomes from mice with experimental autoimmune encephalomyelitis (EAE), the model of MS. The findings were validated by histology in the cortex of EAE mice and postmortem MS tissue. Next, we developed an ELISA with knockout-validated antibodies for the presynaptic protein Bassoon (BSN) and quantified BSN in the cortex, spinal cords and plasma of EAE mice and in the CSF (total n = 30) and serum (total n = 146) of an observational cohort study with people with MS (pwMS) and controls. We further compared longitudinal trajectories of serum BSN (sBSN) and serum neurofilament light chain (sNfL) in a cohort of people with primary progressive MS (PPMS) (n = 26) using linear mixed-effects models. Findings The synaptoneurosome screen revealed reduced levels of several presynaptic proteins, including BSN, in the cortex of EAE mice. The loss of synaptic BSN was validated in EAE and human postmortem tissues of pwMS. Notably, BSN simultaneously accumulated in neuronal soma during EAE and MS, suggesting that BSN may serve as a suitable biomarker for monitoring disease pathology. Our ELISA showed a gradual loss of BSN in the cortex of EAE mice and consistently, plasma BSN levels were elevated in two independent acute and chronic EAE cohorts. In pwMS, BSN was detectable in all CSF samples and in 81% of the serum samples. CSF BSN levels were higher in both relapsing and PPMS compared with controls, whereas sBSN was elevated in secondary progressive MS (SPMS) and PPMS. In the longitudinal PPMS cohort, sBSN and sNfL remained unchanged over an average follow-up of 37 months and did not correlate with each other. Interpretation In conclusion, the presynaptic protein BSN can be quantified in plasma and CSF to assess synaptic pathologies. BSN elevation was already detectable at the earliest disease stages and persisted in progressive MS, underscoring continuous neurodegeneration in MS. Measuring synaptic proteins may complement established biomarkers of neuronal injury to enhance our understanding of neurodegeneration in MS. Funding This work was funded by the Hamburg Innovation Call for Transfer (C4T959 to M.A.F.), Deutschen Multiple Sklerose Gesellschaft (V6.2 to M.A.F.). This work is supported by the Deutsche Forschungsgemeinschaft (FOR 5705, 523862973 to M.A.F., S.C.R., J.B.E.; 247354600, 247377969, 426788273, 518551069, 516868494 to H.S.).

Abstract Infection with enterohemorrhagic E. coli (EHEC) causes severe changes in the brain leading to angiopathy, encephalopathy and microglial activation. In this study, we investigated the role of tumour necrosis factor alpha (TNF-α) for microglial activation and brain pathology using a preclinical mouse model of EHEC infection. LC–MS/MS proteomics of mice injected with a combination of Shiga toxin (Stx) and lipopolysaccharide (LPS) revealed extensive alterations of the brain proteome, in particular enrichment of pathways involved in complement activation and coagulation cascades. Inhibition of TNF-α by the drug Etanercept strongly mitigated these changes, particularly within the complement pathway, suggesting TNF-α-dependent vasodilation and endothelial injury. Analysis of microglial populations using a novel human-in-the-loop deep learning algorithm for the segmentation of microscopic imaging data indicated specific morphological changes, which were reduced to healthy condition after inhibition of TNF-α. Moreover, the Stx/LPS-mediated angiopathy was significantly attenuated by inhibition of TNF-α. Overall, our findings elucidate the critical role of TNF-α in EHEC-induced brain pathology and highlight a potential therapeutic target for mitigating neuroinflammation, microglial activation and injury associated with EHEC infection. Graphical Abstract

Neutrophil swarming has emerged as a conserved multicellular behaviour observed across tissues and pathological contexts. Yet, the molecular cues and the spatially coordinated cellular circuits that drive the process of neutrophil swarming leading to cluster formation remain poorly understood. Here, we combine spatial proteomics and lipid profiling in a model of urinary tract infection to define the epithelial-immune circuits driving neutrophil cluster formation. We identify thrombospondin-1 (TSP1)-mediated activation of transforming growth factor beta 1 (TGF-β1) as key epithelial signal licensing neutrophil clustering and enhancing bacterial control. Spatial lipid analysis further reveals that TSP1/TGF-β1 signalling locally activates arachidonic acid metabolism in epithelial neutrophils, with 5-lipoxygenase dependent leukotriene synthesis required for swarm formation and infection clearance. These findings uncover a spatially coordinated defence mechanism in which epithelial-derived TSP1/TGF-β1 engages neutrophil lipid metabolism to orchestrate neutrophil swarming behaviour and reinforce antibacterial immunity. ![Graphical abstract][1] Graphical abstract ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, TRR332 (449437943), A2, A3, A5, B4, C5, C6, Z1, FOR5427 (466687329), SP1, SP4, EN984 18-1 (539301313), TRR296 (424957847) P09, INST 20876/486-1 (504501554), 516868494, 518551069, 247354600, 247377969, 426788273 Bundesministerium für Forschung, Technologie und Raumfahrt, 01EW2503 Mildred Scheel Cancer Carrer Center Hamburg [1]: pending:yes

Abstract Ependymomas (EPN) are heterogenous tumours occurring in children and adults. They are believed to arise from the ependymal cells and occur in the three major compartments of the central nervous system (spine (SP), posterior fossa (PF), supratentorial (ST)). EPN are further subdivided into molecular types displaying distinct epigenomic profiles and clinical characteristics. Despite advancements in diagnostics and tumour characterization, the prognosis for EPN remains variable and is largely dependent on the extent of tumour resection. Thus, there is a high need for targeted adjuvant therapy. Focusing on integrated proteomic analyses, the goal of this study was to identify tumour type specific targetable proteins and putative biomarkers to enhance the efficient diagnosis and therapy of EPN. Histomorphology, DNA-methylation-, proteome- and phosphoproteome data was assessed from formalin-fixed paraffin-embedded (FFPE) samples of primary human EPN. Molecular diagnosis was verified based on DNA-methylation data using the brain tumour classifier (V12.8, classifier score > 0.8). Proteome data were generated with label-free quantification and data dependent acquisition of proteins using mass spectrometry. Established epigenomic EPN types were reflected in our main cohort (n = 196 EPN with MPE: n = 41, SP-EPN: n = 31, SP-EPN-MYCN: n = 8, SP-SE: n = 8, ST-SE: n = 12, PFA1: n = 19, PFA2: n = 10, PFB: n = 22, PF-SE: n = 18, EPN-YAP: n = 7, EPN-ZFTA: n = 20). Matched protein samples revealed that EPN types were reflected on the proteome level with subependymomas (SE) of all compartments showing high similarity. SNF clustering integrating both data modalities revealed stable clustering of EPN types in CNS compartments. EPN types displayed distinct protein patterns allowing for detection of putative markers and targetable proteins. Results were further confirmed in a validation cohort (n = 73). Preliminary findings of the phosphoproteome analysis showed high EPN heterogeneity and partial overlap with known methylome subtypes. In-depth integrative analyses are ongoing and will help to identify EPN type specific dysregulated biological pathways, biomarkers and treatment targets.

Circulating tumor cells (CTCs) play an important role in metastasis formation. Aberrant signaling of oncogenic pathways (e.g., PI3K/AKT/mTOR pathway) drives tumor progression. In this work, the susceptibility of the colon cancer CTC-derived cell line CTC-MCC-41 to AKT and mammalian target of rapamycin (mTOR) inhibitors was evaluated. Additionally, the functional role of the expressed AKT isoforms was characterized in this cell line. The efficacy of the AKT inhibitor MK2206, the mTOR inhibitor RAD001, and the combination was examined in CTC-MCC-41 cells in a murine intracardiac xenotransplantation model. Furthermore, stable isoform-specific AKT1 or AKT2 knockdowns (KDs) as well as AKT1/AKT2 double-KD cells were generated. Differentially regulated proteins and phospho-peptides were identified using liquid chromatography coupled mass spectrometry (LC-MS). CTC-MCC-41 cells showed a high susceptibility for dual targeting of AKT and mTOR in vivo, indicating that selective eradication of CTCs by AKT/mTOR inhibitors may be considered a new treatment option in cancer. KD of AKT1 or AKT2 significantly reduced the proliferation of CTC-MCC-41 cells. AKT KDs share commonly regulated proteins and phospho-proteins, but also regulate a large number uniquely. AKT1/AKT2 double-KD cells show a strongly dysregulated replication machinery, as well as a decrease in cell cycle activity and stem-cell-associated processes, underlining the non-redundant role of AKT isoforms.

Extracellular vesicles (EVs) are membranous structures that cells release into the extracellular space. EVs carry various molecules such as proteins, lipids, and nucleic acids, and serve as specialized transporters to influence other cells. In the central nervous system, EVs have been linked to many important processes, including intercellular communication, but molecular details of their physiological functions are not fully understood. Our study aimed to investigate how EVs are released by neuronal cells, and how they affect the neuronal activity of other recipient neurons. We show that mature primary cortical neurons release EVs from both their soma and dendrites. EVs released from neurons closely resemble non-neuronal EVs regarding size and marker proteins, and proteomic analyses showed that neuronally released EVs contain proteins typically acting in pre- and post-synaptic compartments. Interestingly, our analysis revealed that EVs alter spontaneous activity in target neurons by increasing the amplitude of postsynaptic potentials. In summary, our findings elaborate on the role of EVs in synaptic activity modulation in neurons mediated by glutamate receptors.

A critical step in the metastatic cascade is the survival of circulating tumor cells (CTCs) within the bloodstream. Although interactions between CTCs and various hematopoietic cells have been described, the role of red blood cells (RBCs) remains underexplored. This study investigated the interactions between tumor cells and RBCs from breast and lung cancer patients, revealing significant phenotypic and functional changes in tumor cells, unlike interactions with RBCs from healthy donors. Tumor cell and patient-derived RBC co-cultures increased tumor cell attachment and induced morphological changes. RBC-primed tumor cells showed increased adhesion, disruption of the endothelial barrier, and invasiveness, both in vitro and in vivo. Global proteome changes, including actin remodeling and VASP accumulation at cell edges, promote directional migration. RBCs from patients with metastatic breast cancer also upregulate PAK4, enhancing migration and epithelial-mesenchymal transition, whereas PAK4 inhibition reduces these effects. Clinically, a higher red blood cell distribution width (RDW) in patients with metastasis is associated with increased CTC counts and poor outcomes. This study highlights the previously unrecognized role of RBCs in promoting metastatic behavior in cancer cells and suggests potential therapeutic targets, such as PAK4, to counteract these effects.

Despite advancements in cancer therapies, bacterial complications remain a major challenge, delaying treatment and worsening outcomes. While immunosuppressive therapies and prolonged hospitalizations contribute, they do not fully explain the elevated infection risk in cancer patients. Here we show that tumors producing high levels of granulocyte colony-stimulating factor (G-CSF) promote the persistence of Gram-negative pathogens in head and neck squamous cell carcinoma due to neutrophil reprogramming. Mechanistically, we identify tumor-driven activation of the G-CSF / nicotinamide phosphoribosyltransferase (NAMPT) signaling axis in neutrophil progenitors, resulting in impaired antibacterial functions, such as phagocytosis and neutrophil extracellular traps formation, and development of tissue-damaging neutrophil subsets. This disrupts lung tissue integrity and facilitates bacterial persistence. Importantly, targeting the G-CSF/NAMPT pathway prevents the generation of dysfunctional neutrophils and improves bacterial clearance in vivo. Our findings reveal tumor-induced, NAMPT-dependent neutrophil reprogramming as a central driver of compromised antimicrobial defenses in cancer. Therapeutic strategies aimed at modulating G-CSF/NAMPT signaling could enhance infection control and survival for cancer patients.