Antibody-drug conjugates (ADCs), particularly enfortumab vedotin (EV) in combination with pembrolizumab, have emerged as transformative treatments for advanced bladder cancer (BC). However, platinum-based chemotherapy, especially gemcitabine-cisplatin (GC), remains a widely used, first-line standard globally. This study investigated the impact of GC-therapy on subsequent ADC responsiveness. Using novel, patient-derived BC models, in vitro selection for acquired GC-resistance consistently resulted in downregulation of the EV target, Nectin-4, and corresponding EV cross-resistance in GC-resistant (GC/R) cells. This decrease in membranous Nectin-4 expression was validated in two independent human BC cohorts: (1) matched transurethral resections and radical cystectomy/lymph node specimens during neoadjuvant therapy and (2) primary tumors and distant metastases during adjuvant GC chemotherapy. Transcriptomic profiling indicated that GC-resistance and Nectin-4 downregulation were linked to a transcriptional shift toward an epithelial-to-mesenchymal transition (EMT)-like phenotype. Large-scale drug screening and transcriptomic data highlighted potential vulnerabilities in GC/R cells, notably associated with the transforming growth factor beta (TGF-beta) signaling pathway. These findings suggest that platinum-based chemotherapy may promote EMT-like transcriptional reprogramming in BC, associated with Nectin-4 loss and EV resistance. As such, assessing membranous Nectin-4 expression prior to EV therapy might be particularly relevant in postplatinum settings. Additionally, identifying alternative, chemotherapy-induced druggable vulnerabilities can inform more effective treatment strategies.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney malignancy. Yet, no rapid, non-invasive biomarkers are available for diagnosis or screening. Urine represents an ideal analyte matrix due to its accessibility, low invasiveness, longitudinal sampling, and the kidney’s central role in filtration. Here, we integrated proteomic, lipidomic, and metabolomic analyses of urine from ccRCC patients and controls to identify diagnostic biomarkers. Multi-omics profiling revealed urogenital metabolic dysregulation in ccRCC, including increased lipid metabolism, altered mitochondrial respiration signatures, and elevated urinary lipid content. We identified three urinary protein biomarkers: serum amyloid A1 (SAA1), haptoglobin (HP), and lipocalin 15 (LCN15). Using a parallel reaction monitoring mass spectrometry workflow, we developed a rapid and sensitive assay and combined these markers into a diagnostic UrineScore. The UrineScore achieved 0.96 in an area under the receiver operating characteristic curve analysis in the discovery cohort, and 0.95 in an independent validation cohort. Together, these results support the feasibility of multi-omics-guided urinary biomarker discovery and represent a step toward accessible diagnostic platforms for ccRCC. Urine multi-omics profiling of ccRCC patients identifies a three-protein diagnostic signature (HP, SAA1, and LCN15) that forms a composite UrineScore with an AUROC of 96

Supratentorial ependymomas are aggressive childhood brain cancers that retain features of neurodevelopmental cell types1 and segregate into molecularly and clinically distinct subgroups2,3, suggesting different developmental roots. The developmental signatures, as well as microenvironmental factors, underlying aberrant cellular transformation and behaviour across each supratentorial ependymoma subgroup are unclear. Here we integrated single-cell and spatial transcriptomics, as well as in vitro and in vivo live-cell imaging, to define supratentorial ependymoma cell states, spatial organization and dynamic behaviour within the neural microenvironment. We find that individual tumour subgroups have two distinct progenitor-like cell states-neuroepithelial-like and embryonic-like-that are reminiscent of early human brain development and diverge in the extent of their neuronal or ependymal differentiation. We further identify several modes of spatial organization of these tumours, including a high-order architecture that is influenced by mesenchymal and hypoxia signatures, and local neighbourhood structures. Finally, we identify a role for brain-resident cells in shifting supratentorial ependymoma cellular heterogeneity towards neuronal-like cells that co-opt immature neuronal morphology and migratory mechanisms, and a subset of neuroepithelial-like cells that are both proliferative and highly migratory. Collectively, these findings provide a multidimensional framework to integrate transcriptional and phenotypic characterization of tumour heterogeneity in supratentorial ependymoma and its potential clinical implications.

Pineoblastoma is a clinically aggressive childhood brain tumor composed of distinct molecular subgroups with divergent driver genes, demographics, and clinical outcomes. To identify developmental origins and mechanisms governing disease pathogenesis, we derive single-cell transcriptomes from pineal parenchymal tumors, aligning malignant cells with developmental counterparts to retrace cellular origins. Integrative computational analyses map pineoblastoma origins to transient, cycling pinealocyte progenitors during development. Lineage-specific perturbation of suspected drivers in the early pineal gland yields preclinical models representative of consensus molecular subgroups. Multi-omic characterization of patient tumors and these models uncover a tumor-associated photoreceptor signature (TAPS) common to pineoblastoma, retinoblastoma, and Group 3 medulloblastoma. Transcriptional activity of this signature within respective cellular origins establishes a developmental basis for molecular similarities between entities. Photoreceptor signature constituents are selective dependencies across these anatomically distinct central nervous system malignancies, motivating future studies evaluating developmentally encoded programs of malignancy as potential therapeutic liabilities.

Pediatric high-grade gliomas (pHGGs) are among the most lethal childhood tumors. While therapeutic approaches were largely adapted from adult treatment regime, significant biological differences between pediatric and adult gliomas exist, which influence the immune microenvironment and may contribute to the limited response to current pHGG treatment strategies. We provide a comprehensive transcriptomic analysis of the pHGG immune landscape using single-cell RNA sequencing and spatial transcriptomics. We analyze matched malignant, myeloid, and T cells from patients with pediatric diffuse high-grade glioma (HGG) or high-grade ependymoma, examining immune microenvironment distinctions after chemo-/radiotherapy, immune checkpoint inhibition treatment, and by age. Our analysis reveals differences in the proportions of pediatric myeloid subpopulations compared to adult counterparts. Additionally, we observe significant shifts toward immune-suppressive environments following cancer therapy. Our findings offer valuable insights into potential immunotherapy targets and serve as a robust resource for understanding immune microenvironmental variations across HGG age groups and treatment regimens.

Embryonal tumor with multilayered rosettes (ETMR) is a pediatric brain tumor with dismal prognosis. Characteristic alterations of the chromosome 19 microRNA cluster (C19MC) are observed in most ETMR; however, the ramifications of C19MC activation and the complex cellular architecture of ETMR remain understudied. Here we analyze 11 ETMR samples from patients using single-cell transcriptomics and multiplexed spatial imaging. We reveal a spatially distinct cellular hierarchy that spans highly proliferative neural stem-like cells and more differentiated neuron-like cells. C19MC is predominantly expressed in stem-like cells and controls a transcriptional network governing stemness and lineage commitment, as resolved by genome-wide analysis of microRNA-mRNA binding. Systematic analysis of receptor-ligand interactions between malignant cell types reveals fibroblast growth factor receptor and Notch signaling as oncogenic pathways that can be successfully targeted in preclinical models and in one patient with ETMR. Our study provides fundamental insights into ETMR pathobiology and a powerful rationale for more effective targeted therapies.

Pineoblastoma (PB) is a clinically aggressive embryonal central nervous system (CNS) tumor composed of distinct molecular subgroups. The cellular and biological basis of PB remains poorly defined, limiting the development of more effective therapeutic strategies. To identify the cellular composition, origins, and drivers of PB, we derived single-cell transcriptomes from patient-derived pineal tumors (n=38) and integrated malignant cells with a novel transcriptional atlas of mouse pineal gland development (E13-P21). Application of a series of computational strategies pinpointed transient, cycling pinealocyte progenitors as the likely cellular origin of PB, irrespective of molecular subgroup. To functionally validate these findings, we generated lineage-specific genetically engineered mouse (GEM) models representing distinct PB subgroups with high temporal, anatomic, and phenotypic fidelity. Transcriptomic analysis further substantiated the accuracy of novel GEM models which converged on cell-cycle dysregulation, highlighting a core pathogenic mechanism across PB subgroups. Relative to the developing pineal gland, photoreceptor transcription factors were aberrantly activated in PB GEM models, suggesting photoreceptor involvement in tumor initiation and/or maintenance. Multi-omic analysis across a large range of diverse CNS cancers revealed an oncogenic photoreceptor program specific to PB, retinoblastoma (RETB), and Group 3 medulloblastoma (G3-MB). This shared transcriptional program was active within respective cellular origins, establishing a unified mechanistic basis across these anatomically distinct CNS tumors. CRISPR-based functional studies confirmed selective dependencies of photoreceptor transcription factors (NRL, CRX, OTX2) in PB, RETB and G3-MB, demonstrating the essentiality of this photoreceptor program. These discoveries not only resolve longstanding uncertainties regarding PB pathogenesis but also highlight conserved molecular signatures and therapeutic vulnerabilities spanning different pediatric CNS malignancies. Our results underscore the potential of targeting developmentally regulated programs and master transcription factors as novel therapeutic strategies for PB, RETB, and G3-MB, offering critical insights and preclinical models for future translational research.

PURPOSE OF REVIEW:This review aims to summarize the biology and clinical relevance of Nectin-4 in patients with advanced bladder cancer, with a focus on its role as a therapeutic target and predictive biomarker. RECENT FINDINGS:Nectin-4 is consistently overexpressed in advanced bladder cancer, facilitating tumor progression and survival signaling. Its cell surface localization makes it an ideal target for antibody-drug conjugates (ADCs), such as enfortumab vedotin, which has shown excellent efficacy in both first-line treatment, post platinum-based chemotherapy, and following immune checkpoint inhibitors. Recent insights highlight Nectin-4 as potential predictive biomarker and a target for advanced medical imaging strategies. Resistance mechanisms and new combination approaches are currently being investigated. SUMMARY:Nectin-4 is a key therapy target in patients with advanced bladder cancer, as demonstrated by the success of enfortumab vedotin. Emerging ADC technologies and Nectin-4-targeted imaging tools could improve efficacy and patient selection, redefining both diagnostic and therapeutic approaches.

Posterior fossa type A (PF-EPN-A, PFA) ependymoma are aggressive tumors that mainly affect children and have a poor prognosis. Histopathology shows significant intratumoral heterogeneity, ranging from loose tissue to often sharply demarcated, extremely cell-dense tumor areas. To determine molecular differences in morphologically different areas and to understand their clinical significance, we analyzed 113 PF-EPN-A samples, including 40 corresponding relapse samples. Cell-dense areas ranged from 0 to 100% of the tumor area and displayed a higher proportion of proliferating tumor cells ( p < 0.01). Clinically, cell density was associated with poor progression-free and overall survival ( p PFS = 0.0026, p OS < 0.01). Molecularly, tumor areas with low and high cell density showed diverging DNA methylation profiles regarding their similarity to distinct previously discovered PF-EPN-A subtypes in 9/21 cases. Prognostically relevant chromosomal changes at 1q and 6q showed spatial heterogeneity within single tumors and were significantly enriched in cell-dense tumor areas as shown by single-cell RNA (scRNA)-sequencing as well as copy number profiling and fluorescence in situ hybridization (FISH) analyses of different tumor areas. Finally, spatial transcriptomics revealed cell-dense areas of different tumors to be more similar than various different areas of the same tumor. High-density areas distinctly overexpressed genes encoding histone proteins, WNT5A, TGFB1, or IGF2. Relapsing tumors displayed a higher proportion of cell-dense areas ( p = 0.036), a change in PF-EPN-A methylation subtypes (13/32 patients), and novel chromosome 1q gains and 6q losses (12/32 cases) compared to corresponding primary tumors. Our data suggest that PF-EPN-A ependymomas habor a previously unrecognized intratumoral heterogeneity with clinical implications, which has to be accounted for when selecting diagnostic material, inter alia, by histological evaluation of the proportion of cell-dense areas.

Background and objective:The role of genetic variants in response to systemic therapy in muscle-invasive bladder cancer (MIBC) is still elusive. We assessed variations in genes involved in DNA damage repair (DDR) before and after cisplatin-based neoadjuvant chemotherapy (NAC) and correlation of alteration patterns with DNA damage and response to therapy. Methods:Matched tissue from 46 patients with MIBC was investigated via Ion Torrent-based next-generation sequencing using a self-designed panel of 30 DDR genes. Phosphorylation of γ-histone 2A.X (H2AX) was analyzed via immunohistochemistry to evaluate DNA damage. Genetic variants were analyzed along with clinical data and quantitative phospho-H2AX data using the Kaplan-Meier method, Cox regression analysis, and factor analysis of mixed data. Key findings and limitations:Twenty-five patients (54%) had a response (<pT2 pN0 cM0) to NAC. Responders had more somatic DDR gene variants in preNAC (53 vs 11; p < 0.001) and postNAC (51 vs 9; p = 0.038) tumor tissue in comparison to nonresponders, as well as significantly greater phosphorylation of H2AX after NAC. ERCC2 was significantly co-mutated with REV3L among responders. Owing to the small cohort, no specific mutation was significantly positively associated with therapy response. However, accumulation of CDK12, NBN, MSH3, MLH1, ATR, BRCA1, BRCA2, REVL3L, and SLX4 variants was observed for responders. Conclusions and clinical implications:Patients with MIBC who responded to cisplatin-based NAC had more somatic DDR gene variants than nonresponders. Moreover, responders exhibited significantly greater DNA damage after NAC. Patient summary:Patients with muscle-invasive bladder cancer who have mutations in genes that are involved in repair of DNA damage are more likely to respond to cisplatin-based chemotherapy. Testing to identify these gene mutations could help in selecting the patients who are most likely to benefit from this treatment.

Nintedanib (NIN), a multi-tyrosine kinase inhibitor clinically approved for idiopathic pulmonary fibrosis and lung cancer, is characterized by protonation-dependent lysosomotropic behavior and appearance of lysosome-specific fluorescence emission properties. Here we investigate whether spontaneous formation of a so far unknown NIN matter within the acidic cell compartment is underlying these unexpected emissive properties and investigate the consequences on lysosome functionality. Lysosomes of cells treated with NIN, but not non-protonatable NIN derivatives, exhibited lysosome-associated birefringence signals co-localizing with the NIN-derived fluorescence emission. Sensitivity of both parameters towards vATPase inhibitors confirmed pH-dependent, spontaneous adoption of novel crystalline NIN structures in lysosomes. Accordingly, NIN crystallization from buffer solutions resulted in formation of multiple crystal polymorphs with pH-dependent fluorescence properties. Cell-free crystals grown at lysosomal-like pH conditions resembled NIN-treated cell lysosomes concerning fluorescence pattern, photobleaching dynamics, and Raman spectra. However, differences in birefringence intensity and FAIM-determined anisotropy, as well as predominant association with (intra)lysosomal membrane structures, suggested formation of a semi-solid NIN crystalline matter in acidic lysosomes. Despite comparable target kinase inhibition, NIN, but not its non-protonatable derivatives, impaired lysosomal functionality, mediated massive cell vacuolization, enhanced autophagy, deregulated lipid metabolism, and induced atypical phospholipidosis. Moreover, NIN exerted distinct phototoxicity, strictly dependent on lysosomal microcrystallization events. The spontaneous formation of NIN crystalline structures was also observable in the gut mucosa of orally NIN-treated mice. Summarizing, the here-described kinase inhibition-independent impact of NIN on lysosomal functionality mediates several of its cell biological activities and might contribute to NIN adverse effects.

In recent years, we have witnessed the emergence of transformative new modalities for the treatment of bladder cancer, including antibody–drug conjugates and immune checkpoint inhibitors (ICIs). The approval of Enfortumab vedotin, especially in combination with ICIs like pembrolizumab, will likely reshape therapeutic sequences, introducing hope for more durable patient outcomes. However, clear limitations remain. Success of the existing therapeutic armamentarium, though expanding, is mitigated by the complex interplay of genetic, transcriptional, and epigenetic deregulation, as well as tumor microenvironmental (TME) cues that altogether foster cancer cell resistance and drive eventual tumor relapse or treatment refractoriness. This section will cover recent advances regarding the establishment and characterization of cell lines, patient-derived low-passage models, three-dimensional models, xenografts (PDXs), etc., to study basic biology and novel therapy approaches for bladder cancer; advances in identification of druggable tumor markers in primary tumors and metastases, as well as (pre)clinical development of therapeutic antibodies and targeted small molecule approaches, etc. for bladder cancer therapy; and preclinical contributions of omics-based efforts to assess the evolution of intratumoral heterogeneity along therapy and in the course of metastatic spread with the aim to formulate new rationale therapeutic approaches. As the scientific community aims at resolving the biological complexity of bladder cancer, it becomes increasingly clear that defeating it requires a fine-tuned understanding of its highly heterogeneous nature. Innovations such as patient-derived, adherent cell cultures or organoids and PDXs have emerged as faithful model systems of the original tumor's molecular character, providing more precise platforms for drug development and predictive medicine. The advent of noninvasive 'urinoids' represents a promising approach with the potential to enable longitudinal monitoring and evaluation of therapy. Together, these refined model systems not only offer a more faithful mimicry of the native TME but also provide platforms for high-throughput perturbation screens, revealing potential drug candidates and advancing the frontier of precision medicine. Chemoresistance continues to be a major hurdle for long-lasting therapeutic success. Insights into the mechanisms underpinning resistance, such as the role of unique gene expression patterns in tumor heterogeneity and cancer cell plasticity have yielded potential novel drug targets. These include, for example, Clofarabine as a candidate for drug repurposing and innovative inhibitors designed to induce cancer cell differentiation and outmaneuver resistance networks. Further, sophisticated high-throughput screens, for example, in conjunction with the CRISPR/Cas9 technology, have led to interesting discoveries that suggest alternative intervention strategies. Organoid models have also been utilized for the development of novel immunotherapeutics, elucidating immune pathways and responses that are key to designing more effective treatment strategies. Yet, these models face challenges in replicating the full biological fidelity of tumors and their interaction with the immune system. The integration of these rapidly developing insights with ongoing clinical efforts holds promise to significantly advance translatability of preclinical bladder cancer research, where optimization of therapeutic sequences aligns more closely with each patient's unique molecular profile. The continuous push for innovation within bladder cancer research, including the successful translation of these advances into clinical applications will be of utmost importance. Such endeavors demand united efforts – an interdisciplinary collaboration of scientists and clinicians focused on harnessing the full therapeutic potential of precision medicine. Undoubtedly, a striking development in the bladder cancer research field has been the leveraging of single-cell RNA sequencing (scRNA-seq) and spatial omics methodologies. These technologies allow for an unprecedented dissection of the TME at a single-cell resolution, revealing the spatial relationships and interactions that govern the complex biology of bladder cancer. scRNA-seq and spatial profiling offer a revolutionary perspective on the interplay within the TME, highlighting the potential for informing rationally tailored treatments that take the dynamic and evolving disease landscape into account. With the rise of these multifaceted research tools, including multimodal omics, in combination with disease-relevant, personalized models, the scientific community has the potential to shape new therapeutic paradigms for bladder cancer. These methodologies not only underscore the heterogeneity and resilience of bladder cancer but also equip preclinical as well as clinical scientists with the capability to construct more refined, personalized treatments. The fusion of these advanced methodologies promises to improve patient stratification, predict treatment responses with increasing accuracy, and advance drug discovery aimed at comprehensively tackling the molecular and cellular diversity of bladder cancer. Yet, the integration of these sophisticated approaches into clinical practice requires a concerted effort, as current molecular and pathological classification frameworks still struggle to seamlessly translate into refined clinical management. The challenges on the horizon are substantial and include creating sustainable strategies for the validation and integration of scRNA-seq and spatial omics data into clinical decision-making processes. The personalization of bladder cancer management must account for the heterogeneity in both the tumor cells themselves and their interplay within the TME. Immune cells, cancer-associated fibroblasts, and endothelial cells engage in an elaborate cellular interplay with significant implications for therapeutic outcomes. Understanding and harnessing these interactions is crucial for the next generation of immunotherapies and targeted treatments that will ideally offer the opportunity to take into account the unique biological composition of patients' individual tumors. In envisioning the future, the bladder cancer research community harbors a comprehensive set of disease-relevant models together with powerful technologies to tackle the complexities of individual patient biology and the challenging propensity of bladder cancer for therapy resistance and recurrence. Each novel discovery adds to our collective understanding and represents a puzzle piece to expand the repertoire of strategies to combat bladder cancer. The full realization of this scientific potential depends on the active translation of innovative research into cohesive clinical approaches, with an unwavering commitment to overcoming the multifaceted challenges bladder cancer presents. Acknowledgements None. Financial support and sponsorship None. Conflicts of interest There are no conflicts of interest.

Current standard-of-care systemic therapy options for locally advanced and metastatic bladder cancer (BC), which are predominantly based on cisplatin-gemcitabine combinations, are limited by significant treatment failure rates and frailty-based patient ineligibility. We previously addressed the urgent clinical need for better-tolerated BC therapeutic strategies using a drug screening approach, which identified outstanding antineoplastic activity of clofarabine in preclinical models of BC. To further assess clofarabine as a potential BC therapy component, we conducted head-to-head comparisons of responses to clofarabine versus gemcitabine in preclinical in vitro and in vivo models of BC, complemented by in silico analyses. In vitro data suggest a distinct correlation between the two antimetabolites, with higher cytotoxicity of gemcitabine, especially against several nonmalignant cell types, including keratinocytes and endothelial cells. Accordingly, tolerance of clofarabine (oral or intraperitoneal application) was distinctly better than for gemcitabine (intraperitoneal) in patient-derived xenograft models of BC. Clofarabine also exhibited distinctly superior anticancer efficacy, even at dosing regimens optimized for gemcitabine. Neither complete remission nor cure, both of which were observed with clofarabine, were achieved with any tolerable gemcitabine regimen. Taken together, our findings demonstrate that clofarabine has a better therapeutic window than gemcitabine, further emphasizing its potential as a candidate for drug repurposing in BC.Patient summaryWe compared the anticancer activity of clofarabine, a drug used for treatment of leukemia but not bladder cancer, and gemcitabine, a drug currently used for chemotherapy against bladder cancer. Using cell cultures and mouse models, we found that clofarabine was better tolerated and more efficacious than gemcitabine, and even cured implanted tumors in mouse models. Our results suggest that clofarabine, alone or in combination schemes, might be superior to gemcitabine for the treatment of bladder cancer.

Purpose of review Current risk stratification and treatment decision-making for bladder cancer informed by histopathology as well as molecular diagnostics face limitations. This review summarizes recent advancements in single-cell and spatial omics methodologies for understanding bladder cancer biology and their potential impact on development of novel therapeutic strategies. Recent findings Single-cell RNA sequencing and spatial omics techniques offer unprecedented insights into various aspects of tumor microenvironment (TME), bladder cancer heterogeneity, cancer stemness, and cellular plasticity. Studies have identified multiple malignant cell subpopulations within tumors, revealing diverse transcriptional states and clonal evolution. Additionally, intratumor heterogeneity has been linked to tumor progression and therapeutic response. Immune cell composition analysis has revealed immunosuppressive features in the TME, impacting treatment response. Furthermore, studies have elucidated the role of cancer-associated fibroblasts and endothelial cells in shaping the tumor immune landscape and response to therapy. Summary Single-cell and spatial omics technologies have revolutionized our understanding of bladder cancer biology, uncovering previously unseen complexities. These methodologies provide valuable insights into tumor heterogeneity and microenvironmental interactions, with implications for therapeutic development. However, challenges remain in translating research findings into clinical practice and implementing personalized treatment strategies. Continued interdisciplinary collaboration and innovation are essential for overcoming these challenges and leveraging the full potential of single-cell and spatial omics in improving bladder cancer diagnosis and treatment.

Diffuse hemispheric gliomas, H3G34R/V-mutant (DHG-H3G34), are lethal brain tumors lacking targeted therapies. They originate from interneuronal precursors; however, leveraging this origin for therapeutic insights remains unexplored. Here, we delineate a cellular hierarchy along the interneuron lineage development continuum, revealing that DHG-H3G34 mirror spatial patterns of progenitor streams surrounding interneuron nests, as seen during human brain development. Integrating these findings with genome-wide CRISPR-Cas9 screens identifies genes upregulated in interneuron lineage progenitors as major dependencies. Among these, CDK6 emerges as a targetable vulnerability: DHG-H3G34 tumor cells show enhanced sensitivity to CDK4/6 inhibitors and a CDK6-specific degrader, promoting a shift toward more mature interneuron-like states, reducing tumor growth, and prolonging xenograft survival. Notably, a patient with progressive DHG-H3G34 treated with a CDK4/6 inhibitor achieved 17 months of stable disease. This study underscores interneuronal progenitor-like states, organized in characteristic niches, as a distinct vulnerability in DHG-H3G34, highlighting CDK6 as a promising clinically actionable target.