Abstract Background Esophagogastric resection is the curative treatment for upper gastrointestinal (uGI) tumors but carries risks such as anastomotic leakage (AL). While AL is known to negatively influence oncological outcomes in other cancer entities, its impact on long-term survival in uGI tumors remains unclear. The aim of the study was to determine the rates of tumor recurrence (TR), recurrence patterns, and overall survival (OS) in patients who experienced AL after esophagogastric resection. Materials and methods This retrospective cohort study was conducted at the University Hospital Carl Gustav Carus Dresden from January 2013 to December 2019. Patients who underwent esophagectomy or gastrectomy for histologically confirmed carcinoma of the uGI tract were included. After 2:1 propensity score matching, a total of 185 patients with complete 5-year-follow-up were analyzed. Results Overall tumor recurrence rates did not differ significantly between patients with AL (AL+) and without AL (AL-) (46.3% vs. 39.0%, p = 0.386). However, stratified analysis revealed a higher risk of hematogenous recurrence in the AL+ group (HR 1.701, 95% CI 1.051–2.753, p = 0.031) in the cause-specific Cox model, while this association was not significant in the Fine–Gray analysis accounting for competing risks. Median OS was worse in the AL+ group (33 vs. 42 months, p = 0.007). Conclusion AL after esophagogastric resection was associated with worse long-term OS and a higher current risk of hematogenous recurrence. Since this association was observed only in the cause-specific Cox model and not in the Fine–Gray model, results should be interpreted cautiously. Although overall recurrence rates were similar, the pattern of recurrence differed, highlighting the importance of analyzing recurrence types. Strategies to reduce AL and further research into its biological impact are warranted.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, underscoring the urgent need for precise and personalized therapeutic strategies. Globo-H has emerged as a clinically relevant glycan target with promising diagnostic and therapeutic utility across multiple cancer types. In this study, we stratified colorectal cancer patients into Globo-H-high and Globo-H-low groups using a histology-based classification, followed by RNA-sequencing analyses to elucidate the key signaling pathways associated with Globo-H overactivation. Among the 31 genes that were identified to meet the Globo-H histology criterion, DUSP14 (dual specificity phosphatase 14) emerged as a promising pharmacological target associated with Globo-H abundance. DUSP14 is an underexplored but pharmacologically actionable therapeutic target. DUSP14 protein in colon cancer cells is inversely correlated with total Transforming growth factor-β-activated kinase 1 (TAK1) protein. The druggability of DUSP14 was demonstrated through in vitro using cell lines and patient-derived organoids (PDO). These results enhance current diagnostic frameworks and provide a foundation for developing novel targeted therapies. Further, in vivo studies are warranted to evaluate the potential of Globo-H targeting in combination with standard treatment regimens. Overall, our work highlights the value of integrating PDO-based functional assays with molecular profiling to uncover and validate actionable targets for CRC theranostics.

Sarcopenia is an important prognostic factor in gastrointestinal malignancies, but CT-based definitions vary. This study evaluated recently published computed tomography (CT)-based cut-offs for skeletal muscle index (SMI) and psoas muscle thickness normalized to height (PMTH), derived from a young, presumably healthy reference population according to recommendations of the European Working Group on Sarcopenia in Older People (EWGSOP), in patients undergoing surgery for gastric cancer. This retrospective, single-center cohort study included patients who underwent surgery for gastric cancer between 2013 and 2018. Preoperative CT was used to assess SMI and PMTH. The recently published reference cut-offs were compared with the established definitions of Prado et al. (SMI) and Gu et al. (PMTH). Overall survival (OS) and recurrence-free survival (RFS) were the primary and secondary endpoints, respectively. Data from 212 patients with complete 5-year follow-up were analyzed. The reference cut-offs significantly discriminated OS and RFS (SMI, both p < 0.001; PMTH, both p = 0.002), whereas the established cut-offs showed weaker or no discrimination. Patients with both low SMI and low PMTH had the worst OS and RFS (both p < 0.001). Combined low SMI and low PMTH was associated with worse OS in univariable analysis (HR 2.57, 95

Human liver ductal epithelium is morphologically, functionally, and transcriptionally heterogeneous. Understanding the impact of this heterogeneity has been challenging due to the absence of systems that recapitulate this heterogeneity in vitro. Here, we found that human liver cholangiocyte organoids do not retain the complex cellular heterogeneity of the native ductal epithelium. Inspired by the knowledge of the cellular niche, we refined our previous organoid medium to fully capture the in vivo cellular heterogeneity. We employed this refined system to analyze the relationships between human biliary epithelial cell states. In our refined model, cholangiocytes transition toward hepatocyte-like states through a bipotent state. Additionally, inhibiting WNT signaling enhances the differentiation capacity of the cells toward hepatocyte-like states. By capturing the in vivo cholangiocyte heterogeneity, our improved organoid model represents a platform to investigate the impact of the different liver ductal cell states in cell plasticity, regeneration, and disease.

Despite advances in defined culture systems, current organoid models lack programmable control of transcriptomic states beyond fixed genetic constraints or broadly specific microenvironmental conditions. Here, a bottom-up biomaterial-based platform is introduced to program cell state changes in pancreatic cancer organoids by tuning minimal adhesion cues within a synthetic matrix. A Design of Experiments framework is used to systematically model the patient-specific transcriptome-wide impact of matrix-presented adhesion cues. Focusing on epithelial-mesenchymal transition (EMT) as a proof-of-concept cellular program, a multiobjective optimization approach is applied to identify patient-specific matrix compositions that enrich EMT-associated transcriptional programs. Organoids cultured in these optimized matrices exhibit transcriptomic signatures consistent with EMT enrichment and coordinated shift in EMT-associated regulatory signatures. Secretome profiling further reveals changes in cytokines previously linked to EMT-associated inflammatory, hypoxia, and TGF-β signaling. Together, these findings demonstrate that quantitative and targeted modulation of defined adhesion cues enables programmable control of transcriptomic states in pancreatic cancer organoids.

Infectious complications remain a significant cause of morbidity after gastrectomy for gastric cancer. Selective decontamination of the digestive tract (SDD) is effective in colorectal surgery, but its role in upper gastrointestinal procedures is unclear. We conducted a retrospective cohort study of patients undergoing open gastrectomy between 2014 and 2024. Patients treated with perioperative SDD (2018–2024) were compared with a matched historical cohort without SDD (2014–2018) using 1:1 propensity score matching (PSM). The primary endpoint was infectious complications. A total of 108 patients were matched (54 SDD, 54 non-SDD). Rates of major complications (16.7

Macrophages are among the most abundant immune cells in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) and play a key role in regulating the immunosuppressive niche that facilitates tumor growth. Although recent three-dimensional (3D) culture systems using patient-derived materials have advanced our understanding of tumor biology, most models lack key cellular TME components and thus fail to capture tumor-immune cell interactions. To address this gap, we developed an in-vitro 3D co-culture model incorporating PDAC patient-derived organoids (PDOs) and macrophages within a synthetic hydrogel matrix. We optimized culture conditions by tuning medium and matrix conditions to support both cell lineages. Flow cytometry and transcriptomic analyses revealed that initially undifferentiated macrophages adopt an M2-like profile upon exposure to PDAC PDOs in starPEG-heparin hydrogels, mirroring the macrophage phenotypes observed by multiplex immunohistochemistry in the matched primary PDAC tissues. Cytokine secretome profiling revealed PDO-specific differences, indicating distinct underlying macrophage polarization subtypes. Collectively, our starPEG-heparin hydrogel-based 3D co-culture enables hypothesis-driven and physiologically relevant studies of tumor-macrophage interactions and may advance immune-modulatory treatment strategies in patients with PDAC.

Organoide sind dreidimensionale (3-D-)Zellkulturmodelle, die aus Gewebestammzellen von Patienten gewonnen werden und die Struktur und Funktion ihres Ursprungsgewebes nachbilden. Werden Organoide aus Tumorgewebe gewonnen, bilden sie zusätzlich noch das Mutationsspektrum und damit die genetischen Eigenschaften der Ursprungstumore nach. Diese Tumormodelle haben das Potenzial, die Onkologie grundlegend zu verändern, indem sie eine robuste Plattform zur Testung therapeutischer Ansätze bieten und personalisierte Behandlungsstrategien ermöglichen. Patientenabgeleitete Organoide bewahren die Heterogenität des Primärtumors und konnten bereits erfolgreich bei der präklinischen Therapietestung bei verschiedenen Krebsarten, darunter gastrointestinale Tumoren und Brustkrebs, unter Beweis stellen. Neue Fortschritte, wie die Integration von zellulären Komponenten der Tumormikroumgebung und der extrazellulären Matrix sowie die Entwicklung mikrofluidischer Organ-on-Chip-Systeme, erhöhen die physiologische Relevanz dieser Modelle weiter. Herausforderungen wie Standardisierung, Skalierbarkeit und prädiktives Potenzial werden aktiv durch nationale multizentrische Initiativen wie u. a. die Organoid-Plattform des Deutschen Konsortiums für Translationale Krebsforschung (DKTK) adressiert. Neben der möglichen klinischen Anwendung haben Tumororganoide als realitätsnahe Tumormodelle in der Grundlagen- und translationalen Forschung bereits zu erheblichen Fortschritten geführt. Diese Übersicht beleuchtet die aktuelle Rolle von Tumororganoiden in der Onkologie, die Integration mit innovativen Technologien und zukünftige Ansätze zur Weiterentwicklung der Krebstherapie.

In translational medicine, ethics is often treated as a question of approval and something to overcome to be allowed to do research. Given the many moral challenges related to developing biomedicine, we argue for the need to expand our understanding of ethics.

To date, comprehensive omics sequencing has greatly advanced cancer therapies and precision oncology. However, rare cancers remain challenging due to unique genetic profiles and low prevalence, limiting molecular mechanism-oriented treatment strategies. Comprehensive precision oncology programs enable molecularly informed treatments, benefiting many patients with advanced rare cancers. Nevertheless, therapeutic target selection remains complex, particularly in cases with no or multiple targetable alterations. To improve patient stratification for rare cancers, we developed a high-throughput drug testing strategy that rapidly identifies actionable therapeutic vulnerabilities to support molecular sequencing-based target identification in precision oncology. We developed a workflow to generate 3D patient-derived cancer models (PCM) and conduct drug sensitivity testing (DST) from viable patient tissues. Tumor tissue was processed into 3D short-term (ST-PCM) or expandable 3D long-term PCM (LT-PCM), such as organoids and spheroids. Dissociated PCM were seeded on pre-spotted drug test plates containing 87 FDA-approved or clinical trial drugs in five concentrations. Cell viability and drug sensitivity scores (DSS) were assessed after 72 hours for hit identification, followed by discussion in a molecular tumor board. DSS correlation in replicates across entities and PCM types showed consistent hit classification with low variance (∼2.3-8.3%). High DSS values for mutation-targeting drugs validated assay sensitivity, while lack of response in controls confirmed specificity. Screening of >100 PCMs (76 from precision oncology patients) from 17 distinct rare and common tumor entities, including sarcomas, CUP, and others, revealed high feasibility: all LT-PCMs and 97% of ST-PCMs derived from surgical specimens allowed partial or full DST. Despite limited material from small biopsies, i.e., fine needle biopsies, successful screening of drug subsets was achieved in 78% of cases with direct seeding as ST-PCM. Additionally, 24 patient samples were screened after tumor cell expansion in LT-PCM, further expanding the cohort of patients eligible for functional testing. Among all tested PCMs, 91% responded to at least one of the tested drugs ex vivo (range 1-38, median 8). Molecular analysis linked pathway alterations with a higher likelihood of pathway inhibitor response. Importantly, preliminary analysis suggests concordance between ex vivo predictions for drug sensitivity and patient responses. We established a robust DST strategy that identifies potential therapeutic vulnerabilities to guide personalized treatment recommendations in precision oncology. This approach, feasible even with limited material, has the potential to enhance treatment decision-making and improve outcomes for adults with rare solid tumors. Jasmina Paluncic, Zunamys I. Carrero, Juliana Schulz, Attila Jady, Claudia Dagostino, Sára Hrabovská, Lino Möhrmann, Irina Kerle, Maximilian Werner, Dorothea Hanf, Maria Gabriela Pereira dos Santos, Elinor Young, Jessica Pablik, Ivona Mateska, Heike Peterziel, Daniel E. Stange, Alexander A. Wurm, Ina Oehme, Vivek Venkataramani, Christoph Heilig, Simon Kreutzfeldt, Benjamin Schmidt, Peter Horak, Stephan Richter, Jürgen Weitz, Klaus-Dieter Schaser, Daniela Richter, Stefan Froehling, Christoph Heining, Hanno Glimm, Claudia R. Ball. Systematic pan-cancer functional patient stratification to advance therapy personalization in rare solid tumors [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 7203.

Gastroesophageal junction (GEJ) adenocarcinoma is an increasingly common cancer with complex biology and poor prognosis. The treatment strategy for locally advanced tumors involves multimodal treatment with perioperative chemotherapy. However, survival rates remain low, especially for advanced disease. Here, formalin-fixed paraffin-embedded tumor sections from 72 patients with GEJ I and II adenocarcinoma who underwent primary resection or perioperative standard-of-care FLOT treatment were analyzed for their intratumoral T cell composition using multiplex immunohistochemistry. The proportions of T cells and their influence on survival were evaluated using Mann-Whitney U and log rank analyses. A comparison of short- and long-term survivors revealed significant differences in the infiltration of regulatory T cells (Tregs). Tumors after neoadjuvant FLOT treatment presented increased proportions of CD8+ T cells with reduced Granzyme B expression, indicating an altered immune response. Overall survival analysis revealed that high infiltration of Tregs was associated with poor survival. Notably, responders to FLOT therapy had a greater T cell frequency and improved survival, whereas nonresponders presented higher levels of Tregs and CD8+ T cells expressing TIM-3. Overall, GEJ cancer patients had increased CD8+ T cells after neoadjuvant chemotherapy with FLOT, and Tregs were associated with treatment response and reduced survival.

In pancreatic cancer, response to treatment is impaired by the tumor microenvironment (TME), forming a desmoplastic barrier. However, experimental 3D models that adequately model the pancreatic TME are still missing. Here, we employ a matrix metalloproteinase (MMP)-degradable star-shaped poly(ethylene glycol) (star-PEG)-heparin hydrogel matrix to replicate pathophysiological features of the pancreatic environment and investigate the role of the tumor-associated protease kallikrein-related peptidase 6 (KLK6) in 3D. Coupled with CRISPR/Cas9, we found that KLK6 promotes cancer growth. RNA sequencing revealed that KLK6 impairs genes associated with antigen presentation and neutrophil recruitment, all major immune suppressors in pancreatic cancer. In patients, KLK6 expression correlated with neutrophil-recruiting chemokines. Neutrophil recruitment was recapitulated in vitro, which was lower in the absence of KLK6. Treatment of neutrophils with cell-conditioned KLK6 knockout medium reduced the immunosuppressive phenotype, indicated by decreased arginase 1 (Arg1) expression. KLK6 also impacted genes associated with immune checkpoint inhibition and decreased cell responses to anti-PD-1 checkpoint blockade in vitro. Our study identified KLK6 as a drug target as it regulates neutrophil recruitment, immunosuppression, and cell responses to anti-PD-1 therapy in a biomaterial-based disease model.

The development of complex multicellular human in vitro systems holds great promise for modelling disease and advancing drug discovery and tissue engineering 1 . In the liver, despite the identification of key signalling pathways involved in hepatic regeneration 2,3 , in vitro expansion of human hepatocytes directly from fresh patient tissue has not yet been achieved, limiting the possibility of modelling liver composite structures in vitro. Here we first developed human hepatocyte organoids (h-HepOrgs) from 28 different patients. Patient-derived hepatocyte organoids sustained long-term expansion of hepatocytes in vitro and maintained patient-specific gene expression and bile canaliculus features and function of the in vivo tissue. After transplantation, expanded h-HepOrgs rescued the phenotype of a mouse model of liver disease. By combining h-HepOrgs with portal mesenchyme and our previously published cholangiocyte organoids 4–6 , we generated patient-specific periportal liver assembloids that retain the histological arrangement, gene expression and cell interactions of periportal liver tissue, with cholangiocytes and mesenchyme embedded in the hepatocyte parenchyma. We leveraged this platform to model aspects of biliary fibrosis. Our human periportal liver assembloid system represents a novel in vitro platform to investigate human liver pathophysiology, accelerate drug development, enable early diagnosis and advance personalized medicine.

Adenocarcinomas of the gastroesophageal junction exhibit genetic and non-genetic heterogeneity that impact clinical outcomes, though the underlying mechanisms behind drug resistance remain poorly understood. We integrated bulk whole-genome sequencing (WGS) and single cell RNA sequencing (scRNA-seq) data from patient-derived organoid lines generated from drug resistant gastric tumors of three patients before and after chemotherapy with FLOT (5-fluorouracil, leucovorin, oxaliplatin, and docetaxel), investigating both in vivo and ex vivo treatment effects. We found that inter-patient variability of gene expression exceeds intra-patient differences and predominantly shapes the expression profiles. Integration of WGS-inferred cancer phylogenies with scRNA-seq data allowed us to associate genetic clones with the individual cells’ transcriptional program and to track the genetic and transcriptomic history of dominant genetic clones in post-treatment samples relative to the corresponding primary tumor. Notably, in vivo treated samples appeared to be transcriptionally distinct from the untreated counterparts, marked by sustained NF-κB down-regulation, which suggests that they retain an immune-mediated imprint of the prior therapy. Changes in the clonal composition of a tumor alone cannot explain the post-chemotherapy NF-κB-associated transcriptional reprogramming. Instead, non-genetic mechanisms shape the altered transcriptomic landscape, particularly a distinct subpopulation of epithelial cells that specifically express pro-inflammatory cytokines, key components of the NF-κB regulatory network. These observations support a model of transcriptional reprogramming after FLOT treatment, which is most likely independent of genetic evolution and consequently potentially reversible. Downregulated NF-κB signaling may thus represent a candidate pathway change for predictive response assessment and/or NF-κB-stimulating co-therapeutic strategies to overcome FLOT resistance. ### Competing Interest Statement The authors have declared no competing interest.

WNT signaling plays a key role in maintaining the gastric epithelium and promoting tumorigenesis. However, how gastric tumors achieve WNT niche independence remains unclear, as mutations on APC or CTNNB1—common mechanisms of ligand-independent WNT activation in colorectal cancer—are infrequent in gastric cancer. Understanding how WNT self-sufficiency is acquired in the stomach is therefore critical. We analyzed mouse gastric organoids harboring oncogenic KRASG12D with or without RNF43/ZNRF3 (RZ) or CDH1/TP53 (CP) mutations, along with corresponding in vivo mouse models. Niche independence was assessed through growth factor withdrawal, Porcupine and pathway-specific inhibitor treatments, and WNT rescue assays. We performed single-nucleus multiome sequencing (RNA + ATAC) to investigate transcriptional and chromatin dynamics. Findings from mouse models were validated using patient-derived gastric cancer organoids, and pan-cancer cell line datasets were analyzed to evaluate clinical and cross-tissue relevance. Gastric fibroblasts secreted canonical WNT2B to maintain the homeostatic gastric epithelium. Upon KRAS activation, epithelial cells were reprogrammed to secrete WNT ligands independently of additional mutations. Single-nucleus multiome analysis revealed that KRAS-driven MAPK signaling opened SMAD2/3-bound enhancers at the WNT7B locus, leading to the emergence of WNT7B-expressing subpopulations. Inhibition of SMAD2/3 phosphorylation suppressed both organoid growth and WNT7B transcription, whereas exogenous WNT restored organoid proliferation. Patient-derived organoids with HER2 amplification, KRAS amplification, or WNT2 copy-number gain exhibited Porcupine inhibitor-sensitive growth, indicating dependence on WNT secretion from the organoids. Analysis of public transcriptomic datasets further demonstrated that the KRAS–MAPK–WNT7B axis is conserved across other cancer types, including lung cancer. Gastric tumors can bypass niche dependence by acquiring KRAS–MAPK–SMAD2/3-driven epithelial WNT secretion. Targeting this axis—through MAPK inhibition, SMAD2/3 blockade, or suppression of WNT secretion—may represent a therapeutic vulnerability in gastric cancer and other KRAS-high malignancies.

Colorectal cancer (CRC) represents the third-leading cause of cancer-related deaths. Here, we present an in-depth comparative mRNA-seq and microRNA-seq analysis of tissue samples from 32 CRC, pairing tumors with adjacent healthy tissues. The differential expression gene (DEG) analysis revealed an interconnection between nutrients, metabolic programs, and cell cycle pathways. We focused on the impact of overexpressed SLC7A11 (xCT) and SLC3A2 genes which compose the cystine/glutamate transporter (Xc-) system. We applied a knowledge-based approach for analyzing gene perturbations from CRISPR screens across various cell types as well as using a variety of functional assays in five primary patient-derived organoid cell models to functionally verify our hypothesis. We identified a previously undescribed cell surface protein signature predicting chemotherapy resistance and further highlighted the causality and potential of pharmacological blockage of ferroptosis as promising avenue for cancer therapy. Biological processes such as redox homeostasis, ion/amino acid transporters and de novo nucleotide synthesis were associated with these co-dependent genes. This study highlighted overlooked genes as potential clinical targets with focus on SLC7A11 and its associated genes in tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and current therapies show limited efficacy. Ligands and receptors of the TIGIT axis were analyzed using multicolor flow cytometry of tumor and blood samples, IHC from primary tumors, and single-cell RNA-Seq from primary tumors and liver metastasis from patients with various stages of PDAC. The effect of soluble and plate-bound Nectin-4 on T cell function was tested in vitro. Furthermore, patient-derived PDAC organoids were treated with the standard-of-care therapies FOLFIRINOX, gemcitabine plus paclitaxel, or the antibody-drug conjugate enfortumab vedotin. TIGIT expression was increased on tumor-infiltrating conventional T cells and Tregs compared with T cells from matched blood. Nectin-4 but not CD155 expression was associated with poor outcome. Nectin-4 was exclusively expressed by tumor cells and correlated with low immune infiltration. Notably, Nectin-4 inhibited T cell effector cytokine production in vitro. Targeting Nectin-4 with the antibody-drug conjugate enfortumab vedotin inhibited tumor growth in multiple patient-derived PDAC organoids. Collectively, our data underscore Nectin-4 as a potential novel therapeutic target and provide the rationale to test this agent in patients with PDAC.

BackgroundPancreatic ductal adenocarcinoma (PDAC) continues to pose profound challenges within the field of oncology due to its notorious resistance to existing therapies and constant high mortality rates. The recent emergence of three-dimensional patient-derived organoid (PDO) models marks a significant advancement, opening new avenues for exploring cancer biology and assessing therapeutic approaches.AimsThe aim of this study focuses on the innovative use of Fourier-transform infrared (FT-IR) spectroscopy to analyze PDAC organoids, thus illuminating their biochemical intricacies.Materials and MethodsIn this study, PDAC organoids, cultivated from specimens sourced from cancer patients, were subjected to FT-IR spectroscopic imaging. By examining the spectral data within the critical fingerprint region (950-1800 cm-1), and employing principal component analysis (PCA), biochemical disparities were detected and analyzed.ResultsThe results revealed distinct spectral profiles corresponding to different sample preparation techniques, which in turn highlighted variations in protein content and structure. PCA revealed a high homogeneity within classes and minimal passage number influence on spectral profiles, with variations in lipid content and protein profiles. Significantly, the biochemical fingerprint of these PDOs closely mirrored that of the original human tissue samples.ConclusionThis investigation underscores the efficacy of molecular spectroscopy as a non-invasive method for profound characterization of PDAC organoids, enhancing our comprehension of tumor biochemistry. The capacity for swift and precise biochemical profiling of PDOs via molecular spectroscopy heralds a promising future for this technique in the realms of cancer diagnostics and personalized medicine.