Abstract Extracellular vesicles (EVs) are nanosized, membrane-bound particles released by all cell types. They carry proteins, nucleic acids, and lipids reflective of their cellular origin and have emerged as promising non-invasive biomarkers for cancer diagnosis and monitoring of therapy response. However, the clinical translation of EV-based assays remains limited by heterogeneous isolation methods, a lack of standardization of the clinical workflows, and insufficient validation in large patient cohorts. To address these challenges, our group at the National Center for Tumor Diseases in Heidelberg, Germany, has developed an EV profiling framework compliant with MISEV2023 recommendations. We systematically benchmark isolation and pre-processing procedures to ensure reproducibility and compatibility with high-throughput liquid biopsy workflows within the prospective EValuate study (S-773/2021). Blood samples are collected via the NCT Cell and Liquid Biobank, processed within 30 minutes, and stored as serum and plasma aliquots at -80 °C for longitudinal analyses. To enable large-cohort EV analyses, we also characterized a miniaturized size-exclusion chromatography protocol using low-volume serum or plasma, which requires no specialized equipment and complements conventional differential centrifugation workflows. To demonstrate the feasibility of the pipeline, we collected and analyzed a total of 125 serum samples - 109 from 24 patients with hepatocellular carcinoma (HCC) undergoing immune checkpoint inhibitor therapy and 16 quality-control samples from four healthy donors. EVs were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, Western blotting, quantitative protein assays, and subsequently profiled proteomically to identify EV-derived protein signatures associated with disease course, radiological treatment response (RECIST), and survival. This standardized, high-throughput EV workflow bridges biobanking, analytical validation, and clinical correlation, providing a robust and scalable framework for integrating EV-based liquid biopsy assays into precision oncology. Citation Format: Antonia Schubert, Nadine Winkler, Robert Ihnatko, Joscha Kraske, Sunanjay Bajaj, Michelle Neßling, Karsten Richter, Dirk Jäger, Guy Ungerechts, Oliver Sedlaczek, Jeroen Krijgsveld, Thomas Walle, Michael Boutros. Toward large-scale clinical implementation of extracellular vesicle profiling for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3340.

Current drug discovery and development approaches rely heavily on cell line models, which often limit the translation of discoveries into novel therapeutics. Recently, patient-derived organoids (PDOs) have been developed that more closely resemble disease tissues. To facilitate drug discovery, in vitro expanded patient-derived organoids can be exposed to hundreds of compounds to assess drug effects. These organoids can be analyzed in detail using high-content imaging. Imaging screening generates multi-parametric data at the single cell or organoid level and can provide insights into patient-specific drug effects and drug mode of action. Additionally, image-based approaches benefit co-culture assays by distinguishing perturbation effects on different cell types. Challenges, such as quality control, automation, and data analysis, will increase as organoid models become more complex to reconstitute the in vivo tissue microenvironment, for example, when combining tumor and stromal cells to model drug action. In this review, we discuss the state of the art in using image-based profiling of organoids to drive innovation in drug discovery.

Current drug discovery and development approaches rely heavily on cell line models, which often limit the translation of discoveries into novel therapeutics. Recently, patient-derived organoids (PDOs) have been developed that more closely resemble disease tissues. To facilitate drug discovery, in vitro expanded patient-derived organoids can be exposed to hundreds of compounds to assess drug effects. These organoids can be analyzed in detail using high-content imaging. Imaging screening generates multi-parametric data at the single cell or organoid level and can provide insights into patient-specific drug effects and drug mode of action. Additionally, image-based approaches benefit co-culture assays by distinguishing perturbation effects on different cell types. Challenges, such as quality control, automation, and data analysis, will increase as organoid models become more complex to reconstitute the in vivo tissue microenvironment, for example, when combining tumor and stromal cells to model drug action. In this review, we discuss the state of the art in using image-based profiling of organoids to drive innovation in drug discovery.

The intestinal interphase is where epithelial renewal and tissue maintenance are balanced alongside immunological regulation. How these functions integrate with cellular signalling is under investigation. Here, we studied the role of the evolutionarily conserved innate immune Toll/NF-κB pathway in Drosophila intestinal regeneration. We found that the core components of the canonical Toll pathway were necessary for intestinal stem cell (ISC) mitosis in homeostasis and upon infection. Toll activation was sufficient to push ISCs into mitosis and the enteroblast (EB) fate, but blocked EB differentiation resulting in ISC and EB accumulation. This was mediated by JNK and Akt/TOR signalling. When JNKK, JNK, Akt or TOR activity was reduced in gut progenitors, ISC mitosis was suppressed. Toll activation also triggered suppression of antimicrobial lysozyme and amidase genes, which led to increased gut bacterial density. Our results identify Toll as necessary and sufficient for ISC mitosis. Our model is that the Toll pathway acts as a regulator of the intestinal landscape integrating JNK and Akt signals to achieve gut tissue renewal and control of commensal bacteria density.

Tumor-associated macrophages (TAMs) and monocytes that accumulate in colorectal cancer (CRC) play a crucial role in shaping the tumor microenvironment (TME) and anti-tumor immune responses. Although TAMs have been linked to both pro- and anti-tumor functions, our understanding of the cues instructing their heterogeneous phenotypes and function in cancer patients remains limited. Here, we established co-cultures comprising primary human monocytes and patient-derived organoids (PDOs) from patients with microsatellite-stable CRC to emulate myeloid/tumor cell interactions in vitro. Upon encountering PDOs, monocytes acquire phenotypic changes that are distinct from those induced by typical polarization protocols. Single-cell RNA sequencing revealed that PDO-exposed monocytes transcriptionally resembled IL1B-programmed monocytes previously identified in the tumor tissues of CRC patients. This phenotype emerged independently of tumor mutational profiles or consensus molecular subtypes. Mechanistically, soluble PDO-derived mediators induced the production of CXCL2, CXCL5 and CXCL7 chemokines, whereas the phagocytic uptake of tumor debris impaired the MHC class II-mediated antigen presentation capabilities of monocytes in co-culture. In addition, our in vitro system allowed functional assessment of PDO-exposed monocytes demonstrating a compromised capacity to mount an inflammatory response upon TLR stimulation. Together, PDO-monocyte co-cultures offer a platform to dissect the interplay between cancer cells and monocytes, and advance our understanding of myeloid plasticity and function in cancer patients.

Abstract The vascular endothelium forms a systemically disseminated organ that translates micromilieu factors into instructive cues, designated as angiocrine signalling. While vascular control of organ function mechanisms have been discovered in essentially all major organs, the mechanisms of translating milieu factors into angiocrine signalling mechanisms remain largely elusive. Here, focussing on the well-established angiocrine Wnt signalling-regulated liver metabolic zonation paradigm, we identified blood flow-induced haemodynamic stress as a biophysical sensor that governs the angiocrine expression profile of liver sinusoidal endothelial cells (LSEC). Combining single-cell RNA sequencing with spatial proteomics, we generated a high-resolution crosstalk map of LSEC and hepatocytes from LSEC Wnt-deficient mutant mice and littermate controls. Intriguingly, vascular Wnt receptors, in parallel with angiocrine Wnt ligands, were specifically enriched in pericentral LSEC, enforcing the spatially coordinated vascular Wnt functions. Consequently, vascular Wnt further modulated angiocrine gene signatures, LSEC morphology, and the expression of gap junctional molecules in an autocrine manner. Taken together, the data define LSEC as a dynamic cellular decoder that translates biophysical forces into instructive angiocrine signals via activating promiscuous vascular Wnt factors.

Cell extrusion is an essential mechanism for controlling cell density in epithelial tissues. Another essential element of epithelia is curvature, which is required to achieve complex shapes, like in the lung or intestine. Here we introduce a three-dimensional bubbly vertex model to study the interplay between extrusion and curvature. We find a generic cellular bulging instability at topological defects which is much stronger than for standard vertex models. Analyzing cell shapes in three-dimensional imaging data of spherical mouse colon organoids, we infer that pentagonal cells have an increased basal interfacial tension, suggesting that cells at topological defects react to the different force conditions. Using the bubbly vertex model, we show that such basal tensions stabilize against the predicted instability and result in better cell shape control than tissue-scale mechanisms such as lumen pressure and spontaneous curvature. Our theory suggests that epithelial curvature naturally leads to bulged and extrusion-like cell shapes because the interfacial curvature of individual cells at the defects strongly amplifies buckling effected by tissue-scale topological defects in elastic sheets. Our results highlight the complex interplay of forces across scales in three-dimensional tissue organization.

CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and undesired repair outcomes. Here, we present a Cas12a-based system in Drosophila that targets each gene with four sgRNAs to overcome these limitations. Multiplexed sgRNAs act through redundancy and synergism, frequently creating deletions between target sites and increasing the fraction of loss-of-function mutations. We show that multiplexed gene targeting is well tolerated and does not cause widespread proximity effects. To visualize CRISPR-nuclease activity in living animals, we developed a screening assay and used it to assess Cas12a activity across 33% of the Drosophila genome in combination with over 2000 sgRNAs. This revealed remarkably high on-target (>99%) and very low (<1%) off-target activity of multiplexed Cas12a sgRNA arrays. Quantitative side-by-side comparisons with current Cas9-based systems targeting over 100 genes in parallel demonstrate that multiplexed Cas12a gene targeting achieves superior performance and reveals phenotypes missed by established methods. The system described here provides a framework for reliable gene knock-out in multicellular systems.

BACKGROUND & AIMS:BRAF-mutant colorectal cancer (CRC) is a clinically aggressive subtype arising from the serrated pathway and is associated with poor prognosis and therapy resistance. The mechanisms driving malignant transformation in microsatellite-stable (MSS) BRAF-mutant CRC remain incompletely understood. We aimed to define the role of WNT pathway activation in serrated CRC progression and tumor-immune interactions. METHODS:We generated multiple genetically engineered mouse models of BRAF-mutant MSS CRC and complementary organoid-based transplantation models. Genetic alterations in WNT pathway components were functionally interrogated. Tumor development and immune microenvironment remodeling were analyzed using bulk RNA sequencing, single-cell RNA sequencing, Cellular Indexing of Transcriptomes and Epitopes by Sequencing, and functional in vivo assays. RESULTS:WNT pathway activation via APC or CTNNB1 mutations, but not RNF43 loss, was required for tumor initiation in BRAF-mutant CRC models. WNT activation induced a molecular subtype shift and suppressed immune response pathways. Mechanistically, WNT signaling suppressed C-C motif chemokine ligand 20 expression and remodeled the tumor microenvironment by promoting immunosuppressive myeloid populations and altering T-cell states. Functional assays demonstrated that WNT activation enhances tumor progression in immunocompetent settings, indicating immune evasion as a key driver of malignant progression. CONCLUSIONS:WNT pathway activation is a critical determinant of malignant transformation in BRAF-mutant MSS CRC by enabling immune escape. These findings identify WNT signaling as a central regulator of tumor-immune interactions and a potential therapeutic target in this aggressive CRC subtype.

PRECISE is a European initiative to move cancer research beyond descriptive atlases toward predictive, mechanistically informed models of tumor vulnerability. By integrating patient cohorts, disease-relevant model systems, perturbation biology, multimodal profiling and artificial intelligence-driven inference, the PRECISE consortium aims to learn generalizable and predictive rules that govern genetic dependencies and synthetic lethality.

Intestinal stem cells (ISCs) continuously renew the gut epithelium by producing specialised cell types, yet the mechanisms that couple ISC renewal with lineage commitment remain poorly characterised. Here, we identify a self-limiting transcriptional program, mediated by the zinc-finger transcription factor Chronophage (Cph), that promotes both ISC maintenance and differentiation into enteroendocrine (EE) cells in the Drosophila midgut. Cph expression is transiently induced by the proneural factor scute at the onset of ISC-to-EE specification. Genetic and single-cell transcriptomic approaches revealed that Cph is required to reprogramme ISCs and sustain normal lifespan. Cph binds to genes involved in proliferation and differentiation, and directly represses its own expression. This autoinhibitory feedback safeguards ISCs from accumulating autophagosomes and undergoing cell death, thus preserving ISC function. Our findings uncover a key regulatory mechanism that balances stem cell maintenance and differentiation, highlighting principles relevant to regenerating tissues.

Abstract Background AXIN1 is a central regulatory hub of many oncogenic pathways in colorectal cancer (CRC). As the main scaffold protein and least abundant component of the beta-catenin destruction complex, changes in AXIN1 levels tightly control Wnt signaling activity. How other cancer pathways beyond Wnt signaling regulate cellular AXIN1 levels is incompletely understood. Methods Colorectal cancer cell lines, murine and patient-derived intestinal and cancer organoids were used as model systems. Changes in AXIN1 levels upon drug perturbation were profiled by immunoblot, qPCR and RNA-seq. Ubiquitin-affinity immunoprecipitation assays and mass spectrometry were used to determine mechanisms of AXIN1 loss. To characterize effects on protein synthesis, we performed polysome and ribosome profiling (Ribo-seq). Results We show that targeting the Ras-MAPK pathway using clinically approved MEK1/2 inhibitors induces AXIN1 loss across a panel of CRC cell lines and patient-derived organoids. In contrast to GSK3 inhibitors, MEK1/2 inhibition neither affects protein stability nor post-translational modifications of AXIN1 and only caused a minor reduction of AXIN1 transcript levels. Co-treatment with tankyrase inhibitors could partially prevent loss of AXIN1 upon MEK1/2 inhibition. Using isogenic CRC cell lines and murine intestinal organoids, we show that APC truncations strongly reduce basal cellular AXIN1 levels, but do not alter dynamics of AXIN1 loss after MEK1/2 inhibition. Polysome profiling and Ribo-seq revealed that MEK1/2 inhibitors reduce global protein synthesis via an mTOR associated pathway. This translational repression is sufficient to cause significant AXIN1 loss, as treatment with mTOR or S6K inhibitors phenocopies the effect of MEK1/2 inhibitors. Conclusion Our study demonstrates that AXIN1 protein homeostasis is critically controlled by Ras-MAPK signaling at the level of protein synthesis, and that MEK1/2 inhibitors cause AXIN1 loss by global translational repression.

CRISPR screens coupled with single-cell RNA sequencing are transforming high-throughput functional genomics. However, applications in vivo remain limited and are confounded by difficulties in identifying and genetically characterizing edited cells. Here we present scPT-seq, a single-cell RNA assay that resolves CRISPR-induced mutations at base-pair resolution and captures transcriptional responses in the same single cells in vivo. scPT-seq comes with a computational analysis suite enabling haplotype-resolved mutation detection and characterization of complex editing outcomes, including splice-junction variation. Applied to the Drosophila intestine, a highly regenerative tissue, scPT-seq distinguishes cell-autonomous from environmental effects by identifying mutant and wild-type cells within tissues, and reveals spatially organized compensatory mechanisms in response to mutations. By using editing outcomes as heritable clonal markers, we identified distinct intestinal stem cell populations with specialized differentiation trajectories. In summary, scPT-seq provides a versatile technology for dissecting gene function and lineage dynamics in complex tissues. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 810296

The intestinal epithelium in vertebrates has a characteristic architecture of protruding villi and receding crypts that enables nutrient absorption and cellular turnover. Intestinal organoids recapitulate its development and can be used as disease models, but the underlying mechanical processes are not fully understood yet. Here we combine advanced image processing and the bubbly vertex model for epithelial cell shape to achieve a fully three-dimensional reconstruction of cell shapes and forces during the development of mouse intestinal organoids. We show that the transition to budded morphologies is caused by a global increase in apical tension, which however is not maintained after budding, suggesting ratchet-like non-reversibility. We further demonstrate that luminal pressure decreases and basal line tensions increase during development, thus facilitating budding on the apical side, but at the same time mechanically stabilizing the system at the basal side, for example against cell extrusion. Our approach demonstrates how one can achieve a complete mechanical analysis of a complex tissue-like system. ### Competing Interest Statement The authors have declared no competing interest.

Single-cell RNA-sequencing has provided intriguing new insights into research areas such as developmental processes and tumor heterogeneity. Most approaches, however, rely on the availability of fresh surgical specimens, thereby dramatically reducing the ability to profile particularly rare tissue types. Here, we optimized a method to isolate intact nuclei from long-term frozen pediatric glioma tissues. We performed a technical comparison between different single-nucleus RNA-sequencing (snRNA-seq) systems and applied the established nucleus isolation method to analyze frozen primary glioma tissues. The results show that our fast, simple and low-cost nuclear isolation protocol provides intact nuclei, which can be used in both droplet- and plate-based single-cell sequencing platforms - allowing the identification of distinct tumor cell populations and infiltrating microglia. Additional optimization to include shorter RNA fragments in the 3' sequencing library improved gene detection and cell type annotation. Taken together, the method dramatically increases the potential of studying rare tumor entities and is specifically tailored for using frozen brain tumor tissue.

WNT signaling governs development, homeostasis, and aging of cells and tissues, and is frequently dysregulated in pathophysiological processes such as cancer. WNT proteins are hydrophobic and traverse the intercellular space between the secreting and receiving cells on various carriers, including extracellular vesicles (EVs). Here, we address the relevance of different EV fractions and other vehicles for WNT5a protein, a non‐canonical WNT ligand that signals independently of beta‐catenin. Its highly context‐dependent roles in cancer (either tumor‐suppressive or tumor‐promoting) have been attributed to two distinct isoforms, WNT5a Short (WNT5aS) and WNT5a Long (WNT5aL), resulting from different signal peptide cleavage sites. To explore possible differences in secretion and extracellular transport, we developed fusion constructs with the fluorescent proteins (FPs) mScarlet and mOxNeonGreen. Functional reporter assays revealed that both WNT5a isoforms inhibit canonical WNT signaling, and EVs produced by WNT5a‐bearing tumor cells, carrying either of the WNT5a isoforms, induced invasiveness of the luminal A breast cancer cell line MCF7. We used fluorescence intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS) to characterize at single‐molecule sensitivity WNT5aL‐bearing entities secreted by HEK293T cells. Importantly, we found that most WNT5aL proteins remained monomeric in the supernatant after ultracentrifugation; only a minor fraction was EV‐bound. We further determined the average sizes of the EV fractions and the average number of WNT5aL proteins per EV. Our detailed biophysical analysis of the physical nature of the EV populations is an important step toward understanding context‐dependent WNT cargo loading and signaling in future studies.

Wnt signaling controls cell proliferation, differentiation, and migration, with critical roles in diseases such as cancer. It was proposed that Wnt signaling relies on the formation of dynamic biomolecular condensates, such as the “signalosome” and the “destruction complex”, with a potential to modify the spatial and temporal regulation of signaling outcomes. To investigate the role of phase separation in Wnt signaling, we generated a cell line with endogenously tagged Dvl2, a central Wnt scaffolding protein, using the mEos3.2 fluorophore. Applying live-cell and single-molecule localization microscopy, we could demonstrate that Dvl2 forms condensates at the centrosome in a Wnt- and cell-cycle-dependent manner in HEK293T cells (Schubert et al. PNAS, 2022). Here, we used image-enabled cell sorting to separate cell pools based on these endogenous Dvl2 condensates, followed by temporal and phosphoproteomic analysis to further understand their functional significance. Condensate-positive cells were enriched with proteins associated with planar cell polarity (PCP) Wnt signaling and proteins of the G2/M cell cycle phase were differentially phosphorylated, suggesting distinct signaling and cell-cycle states. We then conducted a high-throughput, image-based screen of more than 600 kinase inhibitors to identify modulators of Dvl2 condensate dynamics. Several kinases influenced Dvl2 phase separation, revealing a correlation of condensate formation and canonical Wnt signaling activation. Notably, a candidate inhibitor disrupted both Dvl2 condensates and Wnt signaling, affecting cell division, most likely through microtubule regulation. Further phosphoproteomic and chemical-genetic interaction analysis applying CRISPR/Cas9 targeting the Wnt scaffolds APC, Axin1, and MCC, alongside kinase inhibition, revealed distinct roles for these scaffolds in condensate regulation and, interestingly, pathway outcomes. This work underscores the regulatory complexity of Wnt condensates, highlighting post-translational modifications, cellular context, and scaffold roles in Wnt signaling. Our study introduces a high-content screening approach to intracellularly study phase-separated systems. Targeting condensates may represent a novel therapeutic strategy in cancers with aberrant Wnt signaling. Antonia Schubert, Florian Heigwer, Christian Scheeder, Oksana Voloshanenko, Dominique Kranz, Thilo Miersch, Barbara Schmitt, Melanie Kuhse, Franziska Ragaller, Nadine Winkler, Anastasija Paneva, Daniel Gimenes, Diana Ordoñez-Rueda, Jennifer Schwarz, Frank Stein, Dirk Jäger, Ulrike Engel, Michael Boutros. High-content screening reveals kinase regulators of Dvl2 phase separation in Wnt signaling: Implications for targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 183.

Wnt proteins are evolutionarily conserved signaling molecules that control cell-cell communication in health and disease. While different Wnt proteins share a requirement for palmitoleoylation and the cargo receptor Evi/Wls for their secretion, they interact with diverse signaling receptors and co-receptors to elicit distinct outcomes in signal-receiving cells. The molecular mechanisms underlying this contrast between conserved post-translational modification and secretion and diverse receptor interactions remain poorly understood. Here, we demonstrate that Wnt11 maintains partial secretion capability in the absence of Evi/Wls. Unexpectedly, the secreted protein is also non-palmitoleoylated, yet retains its ability to activate downstream signaling in receiving cells through the Frizzled 6 receptor. We identify the N-terminus as the mediator of this unusual behavior, as Wnt3, which strictly requires Evi/Wls, becomes Evi/Wls-independent when its N-terminus is replaced with that of Wnt11. In different cell lines and in Xenopus laevis embryos, the chimeric Wnt11Nterm-Wnt3 protein recapitulates known Wnt11 phenotypes, demonstrating that the N-terminal portion of Wnt11 is sufficient to determine signaling specificity. Our findings reveal novel determinants of Wnt protein specificity and provide mechanistic insights into the molecular basis of Wnt secretion and downstream signaling. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, SFB 1324, TRR 186

Wnt signaling pathways are involved in various developmental and tissue maintenance functions, whereas deregulated Wnt signaling is closely linked to human cancer. Recent work revealed that loss of Wnt signaling impairs mitosis and causes abnormal microtubule growth at the mitotic spindle resulting in chromosome missegregation and aneuploidy, both of which are hallmarks of cancer cells exhibiting chromosomal instability (CIN). Here, we show that upon DNA replication stress, a condition typically associated with CIN, Wnt10b acts to prevent increased microtubule dynamics from the S phase until mitosis, thereby ensuring faithful chromosome segregation. Interestingly, replication stress-induced chromosomal breaks are also efficiently suppressed by Wnt10b. Thus, our results show that Wnt10b signaling regulates replication stress-induced chromosome missegregation and breakage, and hence is a determinant for broad genome instability in cancer cells.

Protein kinases control most cellular processes and aberrant kinase activity is involved in numerous diseases. Here we introduce molecular recorders of kinase activities for later analysis to investigate the link between specific kinase activities and cellular phenotypes in heterogeneous cell populations and in vivo. Based on split-HaloTag and a phosphorylation-dependent molecular switch, our recorders become rapidly labeled in the presence of a specific kinase activity and a fluorescent HaloTag substrate. The kinase activity in a given cell controls the degree of fluorescent labeling, whereas the recording window is set by the presence of the fluorescent substrate. We designed specific recorders for four protein kinases, including protein kinase A. We apply our protein kinase A recorder to sort heterogeneous cell populations for subsequent transcriptome analysis, in genome-wide CRISPR screens to discover regulators of PKA activity and to track neuromodulation in freely moving mice.