Patient-derived urachal cancer organoids for personalized drug screening

Patient-derived urachal cancer organoids for personalized drug screening | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Patient-derived urachal cancer organoids for personalized drug screening Kuangen Zhang, Xinyi LI, Zhenting Zhang, Ning zhang, Xin Yao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8245003/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Urachal cancer (UrC) is a rare, aggressive malignancy typically diagnosed at advanced stages, where systemic treatment becomes necessary. However, cytotoxic chemotherapy offers limited efficacy, and prospective clinical trials are exceedingly difficult due to the rarity of the disease. Thus, robust in vitro models are urgently needed to support precision medicine approaches for UrC. Methods Fresh UrC tumor samples were collected from patients undergoing en bloc resection and cultured to generate PDOs. These organoids were subjected to drug screening using standard chemotherapeutic agents. Whole-exome sequencing (WES) and RNA sequencing (RNA-seq) were conducted to compare the molecular profiles of the PDOs with their corresponding parental tumors. Associations between drug responses and genomic/transcriptomic features were analyzed. Student’s t -test was used for statistical assessment. Results The established UrC PDOs faithfully reproduced the genomic and transcriptomic landscapes of the original tumors, including intratumoral heterogeneity, and demonstrated consistent drug response profiles. Molecular characterization further revealed actionable targets within the RAS/MAPK and PI3K/AKT/mTOR pathways, as well as immune-related targets such as PD-L1. These findings highlight the utility of PDOs in modeling rare cancers and guiding personalized therapeutic strategies. Conclusions UrC PDOs recapitulate the phenotypic and molecular features of their parental tumors, capturing critical heterogeneity. As such, they represent a valuable platform for reflecting treatment responses, investigating resistance mechanisms, and developing individualized therapeutic regimens. Biological sciences/Cancer Biological sciences/Computational biology and bioinformatics Health sciences/Oncology Urachal cancer organoid drug screening personalized medicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Urachal cancer (UrC) is a rare and aggressive tumor originating from the urachus, a vestigial structure connecting the fetal allantois to the bladder dome 1 . It accounts for less than 1% of all bladder cancers 2 , 3 , with over 90% of cases presenting as adenocarcinomas that share histopathological features with both primary bladder adenocarcinoma and colorectal adenocarcinoma 4 . In localized disease, the standard of care involves partial cystectomy with en bloc resection of the urachal ligament and umbilicus 5 . However, for recurrent or metastatic disease, systemic therapies—such as 5-fluorouracil-based regimens, cisplatin combinations, or hyperthermic intraperitoneal chemotherapy—are employed with limited efficacy 6 – 8 . Radiotherapy remains largely ineffective, and due to the rarity of UrC, there are no standardized chemotherapy protocols or prospective clinical trials for advanced disease, resulting in poor survival outcomes 9 . The lack of randomized studies and consensus treatment guidelines underscores the urgent need for robust preclinical models to facilitate research and inform therapeutic decision-making. Patient-derived organoids (PDOs) have emerged as promising preclinical tools capable of modeling tumor heterogeneity and enabling individualized drug screening across multiple cancer types 10 – 13 . Notably, PDOs preserve the genomic landscape and histological architecture of the primary tumor while providing predictive insight into treatment responses 14 , highlighting their potential in personalized oncology. This is particularly crucial for rare malignancies, where low incidence rates and ethical constraints limit the feasibility of clinical trials. Organoid technology thus offers a powerful solution by enabling high-fidelity modeling of tumor biology and patient-specific drug responses in vitro . In this study, we established four PDO lines from a single UrC patient and demonstrated their capacity to replicate the phenotypic and genomic heterogeneity of the corresponding tumors. These PDOs exhibited strong concordance with clinical responses to 5-fluorouracil (5-FU) and cisplatin, two standard chemotherapeutic agents. Furthermore, based on insights from whole-exome and transcriptomic profiling, we evaluated several FDA recently approved or investigational compounds and identified potential therapeutic candidates targeting the RAS/MAPK pathway, the PI3K/AKT/mTOR signaling pathway, and immune checkpoints in UrC organoids, and found this UrC patient is potentially sensitive to these inhibitors’ treatment. Collectively, our findings provide compelling evidence that UrC PDOs can serve as a translational platform for precision oncology, supporting their use in personalized treatment development and research for rare cancers. Materials and Methods Human tissue collection. Fresh tumor and matched normal tissues from patients diagnosed with urachal carcinoma (UrC) or bladder cancer (BC) were obtained from the Department of Urology at Tianjin Medical University Cancer Institute and Hospital. Detailed bladder cancer organoids clinicopathological data are summarized in Table S1. All procedures were approved by the Peking University First Hospital and Tianjin Medical University Cancer Institute review board (approval number: 2023051-001), and written informed consent was obtained from each participant.All research was performed in accordance with relevant guidelines/regulations. Research involving human research participants have been performed in accordance with the Declaration of Helsinki. Establishment of UrC and BC organoids. Tumor specimens were transported on ice to the laboratory and immediately processed. Resected tissues were minced and enzymatically digested using Collagenase I (1 mg/mL, Worthington), Collagenase II (1 mg/mL, Worthington), and Hyaluronidase (100 U/mL, Sigma-Aldrich) at 37 °C for 30–60 minutes. The tissue suspension was further digested in TrypLE Express (Invitrogen) at 37 °C for 10 minutes. Following filtration and washing, cells were resuspended in growth factor–reduced Matrigel (Corning), seeded into 6-well plates (Thermo Fisher Scientific), and overlaid with culture medium. The organoid medium, adapted from previously reported protocols for BC organoids 15 , consisted of Advanced DMEM/F12 supplemented with 1× HEPES, 1× GlutaMax (Invitrogen), 1× Primocin (InvivoGen), 1× B27 Supplement (Gibco), 0.5 μM A83-01 (Selleck), 10 μM Y27632 (Selleck), 0.1 μg/mL FGF-10 (Novoprotein), 1.25 mM N-acetylcysteine (Sigma-Aldrich), and 12.5 mM nicotinamide (Sigma-Aldrich). Medium was refreshed every 2–3 days. For passaging, organoids were first released from Matrigel using 1 mg/mL dispase (STEMCELL Technologies) for 60 minutes at 37 °C, then dissociated into single cells with TrypLE Express. For cryopreservation, organoids were dissociated into single cells or small clusters and frozen in cryopreservation medium (Corning). Histology and immunohistochemistry. Organoids and tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed using the Roche Ventana Discovery Ultra automated system after heat-induced antigen retrieval. Primary antibodies used were: anti-MLH1 (1:100, ZM-0152), anti-PMS2 (1:100, ZA-0542), anti-MSH2 (1:100, ZA-0622), anti-MSH6 (1:100, ZA-0541), and anti-CK20 (1:100, ZA-0574), all obtained from Zhongshan Golden Bridge Biotechnology (Beijing, China). Two board-certified pathologists independently confirmed all histopathological evaluations in accordance with standard diagnostic criteria. Organoid drug screening. Organoids were enzymatically dissociated and passed through a 70 μm strainer (Corning) to remove large aggregates. The resulting suspension (20,000–50,000 organoids/mL in 5% Matrigel-containing medium) was plated in ultralow-attachment 384-well plates (Corning) in triplicate. After 24 hours, a dilution series of each drug was added. Concentration ranges varied depending on compound properties: 100 μM to 12.5 μM, 100 μM to 0.78 μM, or 20 μM to 2.5 μM (maximum DMSO concentration: 1%). After 6 days of incubation, cell viability was assessed using CellTiter-Glo 3D (Promega), following the manufacturer’s instructions. Data were normalized to vehicle controls, and IC 50 and AUC values were calculated using nonlinear regression (log[inhibitor] vs . normalized response, variable slope) in GraphPad Prism 10.0b. Immune stimulation assays in organoids. Organoids and peripheral blood mononuclear cells (PBMCs) were co-cultured at a 1:5 ratio in medium supplemented with 100 U/mL IL-2, 10 ng/mL IL-7, and 10 ng/mL IL-15 (all from Peprotech). A total of 500 organoids and 2,500 PBMCs were seeded per well in 384-well plates and treated with either 20 μg/mL atezolizumab (Selleck) or IgG1 isotype control. After 72 hours, cell viability was measured using CellTiter-Glo 3D. Quantitative real-time PCR. Total RNA was extracted using the TRIzol reagent and reverse-transcribed to cDNA with a commercial RT-PCR kit (TianGen, Beijing, China). Quantitative PCR was performed on an AriaMx Real-Time PCR System (Agilent Technologies) using SYBR Green (TianGen) according to the manufacturer’s protocol. Primer sequences are listed in Supplementary Table S2. siRNA Transfection. Silencer Select predesigned siRNAs targeting PCSK1 (GM-SI-147903) and CLUL1 (GM-SI-147979), as well as non-targeting negative control siRNA, were purchased from Genomeditech (Shanghai, China). Organoids were dissociated into single cells and transfected using Lipofectamine RNAiMAX (Invitrogen), following minor modifications to the manufacturer's protocol. Following siRNA transfection, cells were subjected to drug treatment for 6 days, and viability was assessed using CellTiter-Glo 3D. Whole-exome sequencing and analysis. Genomic DNA was extracted using the DNeasy & RNeasy kit (Qiagen). Exonic regions were captured with Agilent SureSelectXT Human All Exon V6 probes, and libraries were sequenced on the Illumina NovaSeq 6000 platform (150 bp paired-end reads). Median sequencing depth was ×300 for tumor tissues and PDOs (passage1), and ×100 for normal tissues. Quality control of raw reads was performed using FastQC (v0.11.9), Cutadapt (v2.5), and Trimmomatic (v0.39). Clean reads were aligned to the UCSC human reference genome (hg19) using bwa-mem2 (v2.0). BAM file processing—including sorting, merging, and indexing—was performed with Samtools (v1.10), and PCR duplicates were removed using GATK (v4.1.2.0). Coverage statistics were calculated using Samtools based on Sure Select All ExonV6r2 BED file coordinates. Somatic point mutations and indels were called using GATK Mutect2 (v4.1.2.0) in paired tumor–normal mode. Phylogenetic tree construction. Phylogenetic relationships among multi-regional organoids were inferred as described previously 16 . Genomic sequences (± 20 bp surrounding mutation sites) were extracted and used to construct phylogenetic trees using MEGA5 software with the maximum parsimony algorithm. Driver mutations were annotated along the tree’s root, branches, and leaves to depict clonal evolution. Differential gene expression and gene set enrichment analysis. Differentially expressed genes (DEGs) between experimental groups were identified using DESeq2, applying an absolute fold-change cutoff > 2 and an adjusted P- value < 0.05. The ranked gene lists were used for gene set enrichment analysis (GSEA) with a previously published stem cell gene set and the GO_BP gene sets from MSigDB (v7.5.1). A q-value < 0.25 was considered statistically significant. KEGG and GO pathway enrichment analyses 17,18 were conducted using the clusterProfiler R package. (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html) We get permission to use the KEGG software from the Kanehisa laboratory. RNA sequencing and quantification . Total RNA was isolated from NEN organoids (passage1) by using the DNeasy & RNeasy kit (Qiagen), and poly-T oligo-attached magnetic beads were used for mRNA purification. Sequencing libraries were prepared and sequenced on the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. Quality control of raw reads was performed with FastQC (v0.11.9), Cutadapt (v2.5, using Illumina universal adapters), and Trimmomatic (v0.39, parameters: PE, MINLEN = 36). Reads were aligned to the human reference genome (hg19) using STAR (v2.7.3a) with default settings. Gene-level counts were generated using HTSeq-count with GENCODE annotations, and transcript per million (TPM) values were calculated via RSEM . Unless otherwise stated, mRNA expression is presented as log₂(TPM + 1). Correlation heatmaps comparing organoids and matched tumor tissues were generated as previously described 11 , and statistical significance for expression variance across samples was assessed using one-way ANOVA. Statistical analysis. Statistical comparisons were performed using Student’s t -test in GraphPad Prism 10.0b. Data are presented as mean ± standard deviation (SD). A P -value < 0.05 was considered statistically significant. Results Case presentation A 31-year-old female presented in November 2023 for urological evaluation following six months of intermittent, painless gross hematuria accompanied by mucous discharge. Initial ultrasonography of the urinary tract identified a 3.2 × 3.1 × 3.5 cm hypoechoic nodule with indistinct margins along the anterior bladder wall. Cystoscopy confirmed the presence of a sessile, non-papillary lesion at the same anatomical site. Contrast-enhanced computed tomography (CT) further characterized a solid, calcified mass measuring 4.2 × 2.9 cm in the anterior bladder wall (Figs. 1A–B). To exclude metastatic dissemination from gastrointestinal origins, a colonoscopy and biopsy were performed (Fig. S1A), which revealed a low-grade tubular adenoma (Fig. S1B), supporting the diagnosis of a primary urachal carcinoma (UrC) and excluding a metastasis from an intestinal tumor. Intraoperative exploration revealed a broad-based, well-demarcated 3.2 × 3.1 cm mass on the anterior bladder wall, distinct from surrounding mucosa. Guided by cystoscopy, an en bloc resection was performed, which included the tumor, adjacent normal mucosa, partial umbilical tissue, urachal remnant, and adherent peritoneal structures (Fig. 1C). Histopathological analysis confirmed the diagnosis of urachal adenocarcinoma. Establishment of multi-regional urachal carcinoma patient-derived organoids To model intratumoral heterogeneity, fresh surgical specimens were subjected to immediate multi-regional sampling for PDO establishment and multi-omics analysis (Fig. 1D). Four spatially distinct tumor fragments were cultured to generate organoids, T1 and T2 derived from histologically invasive regions, and T3 and T4 from morphologically superficial, non-invasive areas, were confirmed by histopathological mapping.Using this approach, we have generated independent four urachal organoid lines. These lines have been propagated by serial passaging (up to passage 4) (Fig. S2). Urachal organoids retain histological, genomic, and transcriptomic features of parental tumors To assess fidelity between organoids and their parental tumors, we performed hematoxylin and eosin (H&E) staining and immunohistochemistry for canonical UrC markers, including cytokeratin 20 (CK20) and the mismatch repair (MMR) proteins, MSH2, MSH6, PMS2, and MLH1. As shown in Fig. 2A, the organoids exhibited a histological architecture comparable to that of the parental tumor. Both the original tumor and derived PDOs demonstrated a mismatch repair-deficient (d-MMR) phenotype, evidenced by the selective loss of PMS2 expression while retainment of MLH1, MSH2, and MSH6. Additionally, periodic acid–Schiff (PAS) staining confirmed mucin secretion capacity of the organoids (Fig. S3), consistent with their adenocarcinomatous identity. Thus, the established UrC PDOs faithfully mirrored both the histopathological and molecular features of the parental tumors. Whole-exome sequencing (WES) was conducted on four UrC tumor-organoid pairs (median depth: 300×), two bladder cancer (BC) tumor-organoid pairs (median depth: 300×), and one matched normal skin sample as a somatic control (median depth: 100×). Detailed bladder cancer organoids clinicopathological data are summarized in Table S1 .In line with previous genomic analyses of UrC 13,19 , recurrent mutations in TP53 and KRAS were identified in all UrC samples (Fig. 2B). Immunohistochemistry and WES consistently confirmed MMR deficiency due to a somatic missense mutation in PMS2 . Notably, UrC tumors and organoids shared a median of 78.07% of somatic cancer-related variants (Fig. 2C). In contrast, BC samples displayed distinct mutational signatures with a higher burden of alterations in epigenetic regulators, such as EP300 , KMT2D , and KDM6A (Fig. 2B). Transcriptomic profiling via RNA-seq revealed high transcriptional concordance between tumor-organoid pairs (average Pearson’s r = 0.77). Hierarchical clustering demonstrated distinct clustering between UrC and BC samples, further supporting tumor-type specificity (Fig. 2D). Collectively, these multi-omics data confirm that UrC PDOs faithfully recapitulate the genomic and transcriptional landscapes of their tumors of origin and provide a robust platform for modeling disease heterogeneity. Urachal carcinoma organoids recapitulate tumor heterogeneity To investigate the organoids’ capacity to preserve intra-tumoral heterogeneity, we analyzed four PDOs generated from anatomically distinct regions of the tumor mass. Histological analysis of the primary tumor revealed pronounced heterogeneity, including superficial intestinal-type adenocarcinoma and deeper mucinous adenocarcinoma invading the muscularis propria (Fig. 3A). Phylogenetic tree reconstruction based on genomic data demonstrated both branching and parallel evolutionary patterns among the sampled tumor regions (Fig. 3B). Morphologically, PDOs also reflected their site of origin. Organoids T1 and T2, derived from invasive regions, displayed irregular contours with stellate projections—features previously associated with aggressive phenotypes and epithelial-mesenchymal transition (EMT) 20,21 . In contrast, T3 and T4 organoids from superficial areas retained smooth borders. To evaluate EMT status, RNA-seq analysis compared gene expression profiles between T1/T2 (invasive) and T3/T4 (superficial) PDOs. Mesenchymal markers ( TWIST1 , VIM ) were enriched in T1/T2, while epithelial markers ( CLDN3 , CDH1 , CLDN4 ) were upregulated in T3/T4 (Fig. 3C). Differential gene expression and pathway enrichment analyses further identified activation of EMT-related pathways in T1/T2 organoids, including ECM-receptor interaction 22-24 , Wnt/β-catenin, and PI3K-AKT-mTOR signaling (Fig. S4A). Gene Set Enrichment Analysis (GSEA) validated significant Wnt pathway activation in the invasive PDOs (Fig. S4B). Together, these findings demonstrate that UrC PDOs not only recapitulate the molecular and morphological diversity of the parental tumor but also maintain regional differences in EMT-associated signaling and invasive potential, reinforcing their value for studying intratumoral heterogeneity. Screening of clinically relevant agents in urachal carcinoma organoids reflects patient response The patient in this study underwent partial cystectomy with en bloc resection of the urachal ligament and umbilicus in November 2023. Postoperatively, a 5-fluorouracil (5-FU)-based chemotherapy regimen combined with cisplatin was administered. However, follow-up computed tomography (CT) revealed disease progression, accompanied by a marked elevation in serum CA72-4 levels (Fig. 4A). To evaluate the predictive capacity of patient-derived organoids (PDOs) in therapeutic response, we screened the four UrC PDOs established from surgical tumor specimens and two bladder cancer (BC) PDOs against six commonly used chemotherapeutic agents, including 5-FU and cisplatin. As shown in Fig. 4B, while BC PDOs were sensitive to cisplatin, all four UrC PDOs demonstrated complete resistance. Notably, two UrC PDOs (T2 and T4) displayed relative sensitivity to 5-FU, exhibiting IC 50 values below the predefined efficacy threshold of 50 μM 25,26 . However, the remaining two PDOs were resistant. Considering that therapeutic resistance is often driven by the most refractory subclones within a tumor, our findings suggest an overall chemoresistant phenotype in this case, aligning with the patient's clinical outcome (Fig. 4A). These results underscore the utility of UrC PDOs in accurately reflecting patient-specific drug responses. To further explore the molecular underpinnings of 5-FU response, we conducted transcriptomic profiling comparing the 5-FU-sensitive and -resistant UrC PDOs. As shown in Fig. 4C, PCSK1 and CLUL1 were significantly upregulated in the resistant group. Importantly, targeted knockdown of PCSK1 or CLUL1 in T1 and T3 PDOs, respectively, resulted in increased sensitivity to 5-FU treatment (Figs. 4D–E). These findings suggest a functional role for both genes in modulating chemoresistance of UrC to 5-FU treatment. Of particular note, PCSK1 overexpression has previously been associated with poor prognosis and therapeutic resistance in rectal adenocarcinoma 27 , indicating a potentially conserved mechanism across epithelial malignancies. Altogether, these results identify candidate biomarkers predictive of 5-FU response and offer a framework for the mechanistic dissection of chemoresistance in UrC. Drug response profiling of Urachal carcinoma organoids Given the patient’s resistance to standard chemotherapy (Fig. 4), we next assessed the efficacy of targeted therapies based on insights from whole-exome and transcriptomic profiling. Comparative transcriptomic analysis between two normal bladder urothelial PDOs and four UrC PDOs revealed significant dysregulation in several oncogenic pathways in UrC, including PD-L1/PD-1 checkpoint signaling, Ras signaling, and MAPK cascades (Fig. 5A). Functional drug screening demonstrated that UrC PDOs were significantly more sensitive than a BC PDO (lacking MAPK-activating mutations such as PIK3CA or FGFR3 ) to the PI3K inhibitor pictilisib, the mTOR inhibitor rapamycin, and the MEK inhibitor trametinib (Fig. 5B). These responses strongly correlated with the transcriptional profiles of the PDOs, highlighting the mechanistic relevance of pathway activation to therapeutic vulnerability. Moreover, whole-exome sequencing revealed KRAS and PMS2 mutations in the UrC PDOs (Fig. 2B), and transcriptomic data confirmed elevated expression of KRAS and PD-L1 (Fig. 5C), supporting their potential as therapeutic targets. We therefore evaluated the efficacy of the KRAS inhibitor BI-2865 and the PD-L1 inhibitor atezolizumab. Dose-response assays showed that the IC 50 values of UrC PDOs to BI-2865 ranged from 12.62 μM to 23.92 μM, markedly lower than those of BC PDOs (54.89 μM to 112.4 μM), indicating enhanced KRAS inhibitor sensitivity in UrC organoids (Fig. 5C). Furthermore, treatment with atezolizumab led to significant cytotoxic responses in UrC PDOs that cell viability decreased by 53.42% ± 4.11% and 45.28% ± 2.19% in T1 and T2 PDOs, respectively (Fig. 5D). Collectively, these results demonstrate that molecular profiling of UrC PDOs enables the identification of alternative, actionable therapeutic targets. This approach supports the integration of PDO-based drug screening into personalized treatment strategies for rare malignancies such as urachal carcinoma. Discussion Systemic therapy is frequently required in urachal carcinoma (UrC) due to its late-stage presentation or progression following locoregional interventions. However, treatment options remain limited. Chemotherapy efficacy data are largely confined to retrospective studies, as the rarity of UrC precludes prospective clinical trials. Current treatment strategies rely on empirical use of conventional chemotherapies, which often yield suboptimal outcomes. Moreover, evidence supporting targeted therapies is scarce, and no validated alternatives exist once resistance develops. Therefore, there is a pressing need for reliable experimental models to address the clinical challenges unique to UrC. In this study, we successfully established four UrC patient-derived organoid (PDO) lines from a 31-year-old female patient carrying somatic PMS2 mutations. Histological and immunohistochemical analyses confirmed that the PDOs preserved key features of the parental tumors, including mucin production and expression of canonical markers such as CK20 and MLH1 (Fig. 2 ). Genomic and transcriptomic profiling further demonstrated high fidelity between organoids and corresponding tumor tissues (Fig. 2 ), supporting the robustness of these models for translational applications. PDO platforms represent a transformative tool in precision oncology, enabling the integration of mutational and transcriptomic data to identify therapeutic vulnerabilities. In our study, differentially expressed gene (DEG) analysis and KEGG pathway enrichment revealed activation of the RAS/MAPK, PI3K/AKT/mTOR, and PD-L1 signaling pathways as potential therapeutic targets (Fig. 5 A). Whole-exome sequencing identified a KRAS G12V driver mutation, consistent with constitutive activation of MAPK signaling 28 , providing a mechanistic basis for sensitivity to pathway-specific inhibitors. Functional validation using targeted therapies confirmed that drug responses were in strong concordance with molecular profiles (Fig. 5 B), highlighting the predictive value of PDO-based pharmacotyping. Intratumoral heterogeneity (ITH) presents a major barrier to achieving durable therapeutic responses 29 . Our multi-regional sampling approach captured spatial heterogeneity within the tumor, reflected in differential drug sensitivity across UrC PDOs (Fig. 4 ). While dependency on the MAPK pathway was predominant, subpopulations exhibiting resistance via pathway-independent mechanisms were also observed (Fig. 5 ). These findings emphasize the importance of integrating spatially resolved tumor sampling with PDO pharmacotyping to inform more comprehensive and effective therapeutic strategies. Immune checkpoint blockade targeting PD-1/PD-L1 is emerging as a promising therapeutic approach, particularly in tumors with microsatellite instability(MSI) status 30 . The PDOs in our study exhibited dMMR/MSI features, including loss of PMS2 and elevated PD-L1 expression. Mechanistically, MSI tumors accumulate neoantigens that enhance immunogenicity and sensitize tumors to immune checkpoint inhibitors 31 – 33 . Consistent with this, in vitro drug assays demonstrated that PD-L1 blockade via atezolizumab augmented T cell-mediated cytotoxicity. These results align with recent clinical reports, including a case of stable disease in a UrC patient treated with atezolizumab 19 , and broader evidence supporting the efficacy of PD-1/PD-L1 inhibitors in MSI tumors. Overall, our data underscore the potential of combining histological, genomic, and transcriptomic insights with functional drug screening to advance precision medicine in UrC. This study has several limitations. First, the small sample size reflects the rarity of UrC, which limits the statistical power and generalizability of the findings. Second, while our PDO-based platform identifies promising therapeutic candidates, clinical validation through prospective trials—particularly for novel chemotherapeutics, targeted agents, and immune checkpoint inhibitors—is essential. Third, current PDO models do not incorporate immune components, limiting their utility for evaluating immunotherapy. The development of tumor–immune co-culture systems will be crucial to overcome this limitation. Finally, although drug sensitivity findings are compelling, they have not yet been translated into clinical treatment decisions. Interventional studies are needed to confirm their clinical utility and drive bench-to-bedside translation. To address these challenges, we are launching a longitudinal biospecimen collection and functional validation pipeline to extend this research and enable future clinical implementation. Conclusions In summary,UrC PDOs reproduce the phenotypic and molecular features of their parental tumors, capturing intratumoral heterogeneity. Thus, they serve as a valuable platform for reflecting treatment responses, investigating resistance mechanisms, and developing personalized therapeutic regimens. Declarations Acknowledgements We thank the patients and their families for donating tumor samples and the surgical teams for their collaboration. Funding This work was financially supported by the National Natural Science Foundation of China (No. 82588201 and 82341005). Author contributions L., N.Z., and X.Y. supervised this study. K.Z., performed experiments. K.Z. and Z.Z. collected the samples and clinical information. X.L. performed genomic analyses and interpreted the analysis results. K.Z., drafted the manuscript. R.L. and N.Z. revised the manuscript. All authors commented on the manuscript and approved this final version. Data availability The datasets generated and/or analysed during the current study are available in the Genome Sequence Archive for Human PRJCA053683/HRA015623 repository, (https://ngdc.cncb.ac.cn/gsa-human/). Footnote Conflicts of Interest: These authors have no conflicts of interests to declare. Competing Interests: The authors have declared no competing interest. References Schubert, G. E., Pavkovic, M. B. & Bethke-Bedürftig, B. A. Tubular urachal remnants in adult bladders. J Urol 127 , 40-42 (1982). https://doi.org/10.1016/s0022-5347(17)53595-8 Paner, G. P., Lopez-Beltran, A., Sirohi, D. & Amin, M. B. Updates in the Pathologic Diagnosis and Classification of Epithelial Neoplasms of Urachal Origin. Adv Anat Pathol 23 , 71-83 (2016). https://doi.org/10.1097/pap.0000000000000110 Loizzo, D. et al. Current Management of Urachal Carcinoma: An Evidence-based Guide for Clinical Practice. Eur Urol Open Sci 39 , 1-6 (2022). https://doi.org/10.1016/j.euros.2022.02.009 Szarvas, T. et al. Clinical, prognostic, and therapeutic aspects of urachal carcinoma-A comprehensive review with meta-analysis of 1,010 cases. Urol Oncol 34 , 388-398 (2016). https://doi.org/10.1016/j.urolonc.2016.04.012 Hamilou, Z. et al. Management of urachal cancer: A consensus statement by the Canadian Urological Association and Genitourinary Medical Oncologists of Canada. Can Urol Assoc J 14 , E57-e64 (2020). https://doi.org/10.5489/cuaj.5946 Yazawa, S. et al. Surgical and chemotherapeutic options for urachal carcinoma: report of ten cases and literature review. Urol Int 88 , 209-214 (2012). https://doi.org/10.1159/000334414 Siefker-Radtke, A. O. et al. Multimodality management of urachal carcinoma: the M. D. Anderson Cancer Center experience. J Urol 169 , 1295-1298 (2003). https://doi.org/10.1097/01.ju.0000054646.49381.01 Galsky, M. D. et al. Prospective trial of ifosfamide, paclitaxel, and cisplatin in patients with advanced non-transitional cell carcinoma of the urothelial tract. Urology 69 , 255-259 (2007). https://doi.org/10.1016/j.urology.2006.10.029 Collins, D. C. et al. National Incidence, Management and Survival of Urachal Carcinoma. Rare Tumors 8 , 6257 (2016). https://doi.org/10.4081/rt.2016.6257 Lee, S. H. et al. Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer. Cell 173 , 515-528.e517 (2018). https://doi.org/10.1016/j.cell.2018.03.017 Kopper, O. et al. An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity. Nat Med 25 , 838-849 (2019). https://doi.org/10.1038/s41591-019-0422-6 Vlachogiannis, G. et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359 , 920-926 (2018). https://doi.org/10.1126/science.aao2774 Jacob, F. et al. A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity. Cell 180 , 188-204.e122 (2020). https://doi.org/10.1016/j.cell.2019.11.036 Yan, H. H. N. et al. A Comprehensive Human Gastric Cancer Organoid Biobank Captures Tumor Subtype Heterogeneity and Enables Therapeutic Screening. Cell Stem Cell 23 , 882-897.e811 (2018). https://doi.org/10.1016/j.stem.2018.09.016 Mullenders, J. et al. Mouse and human urothelial cancer organoids: A tool for bladder cancer research. Proc. Natl. Acad. Sci. U. S. A. 116 , 4567-4574 (2019). https://doi.org/10.1073/pnas.1803595116 Xue, R. et al. Genomic and Transcriptomic Profiling of Combined Hepatocellular and Intrahepatic Cholangiocarcinoma Reveals Distinct Molecular Subtypes. Cancer Cell 35 , 932-947.e938 (2019). https://doi.org/10.1016/j.ccell.2019.04.007 Kanehisa, M., Sato, Y., Kawashima, M., Furumichi, M. & Tanabe, M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res 44 , D457-462 (2016). https://doi.org/10.1093/nar/gkv1070 Ogata, H. et al. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 27 , 29-34 (1999). https://doi.org/10.1093/nar/27.1.29 Kardos, J. et al. Comprehensive Molecular Characterization of Urachal Adenocarcinoma Reveals Commonalities With Colorectal Cancer, Including a Hypermutable Phenotype. JCO Precis Oncol 1 (2017). https://doi.org/10.1200/po.17.00027 Cheung, K. J., Gabrielson, E., Werb, Z. & Ewald, A. J. Collective invasion in breast cancer requires a conserved basal epithelial program. Cell 155 , 1639-1651 (2013). https://doi.org/10.1016/j.cell.2013.11.029 Padmanaban, V. et al. E-cadherin is required for metastasis in multiple models of breast cancer. Nature 573 , 439-444 (2019). https://doi.org/10.1038/s41586-019-1526-3 Lambert, A. W. & Weinberg, R. A. Linking EMT programmes to normal and neoplastic epithelial stem cells. Nat Rev Cancer 21 , 325-338 (2021). https://doi.org/10.1038/s41568-021-00332-6 Pastushenko, I. & Blanpain, C. EMT Transition States during Tumor Progression and Metastasis. Trends Cell Biol 29 , 212-226 (2019). https://doi.org/10.1016/j.tcb.2018.12.001 Nieto, M. A., Huang, R. Y., Jackson, R. A. & Thiery, J. P. EMT: 2016. Cell 166 , 21-45 (2016). https://doi.org/10.1016/j.cell.2016.06.028 van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161 , 933-945 (2015). https://doi.org/10.1016/j.cell.2015.03.053 Zhao, Y. et al. Personalized drug screening using patient-derived organoid and its clinical relevance in gastric cancer. Cell Rep Med 5 , 101627 (2024). https://doi.org/10.1016/j.xcrm.2024.101627 Chou, C. L. et al. PCSK1 Overexpression in Rectal Cancer Correlates with Poor Response to Preoperative Chemoradiotherapy and Prognosis. Onco Targets Ther 13 , 3141-3150 (2020). https://doi.org/10.2147/ott.S243750 Singhal, A., Li, B. T. & O'Reilly, E. M. Targeting KRAS in cancer. Nat Med 30 , 969-983 (2024). https://doi.org/10.1038/s41591-024-02903-0 Dagogo-Jack, I. & Shaw, A. T. Tumour heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol 15 , 81-94 (2018). https://doi.org/10.1038/nrclinonc.2017.166 Le, D. T. et al. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med 372 , 2509-2520 (2015). https://doi.org/10.1056/NEJMoa1500596 Giannakis, M. et al. Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma. Cell Rep 15 , 857-865 (2016). https://doi.org/10.1016/j.celrep.2016.03.075 Llosa, N. J. et al. The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. Cancer Discov 5 , 43-51 (2015). https://doi.org/10.1158/2159-8290.Cd-14-0863 Saeterdal, I. et al. Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer. Proc. Natl. Acad. Sci. U. S. A. 98 , 13255-13260 (2001). https://doi.org/10.1073/pnas.231326898 Additional Declarations No competing interests reported. Supplementary Files s1.tif s2.tif s3.tif s4.tif Supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8245003","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":575451235,"identity":"10a691b9-9421-4295-8999-2a685cf44d81","order_by":0,"name":"Kuangen Zhang","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kuangen","middleName":"","lastName":"Zhang","suffix":""},{"id":575451236,"identity":"ccbcae46-129b-45fc-9797-8a2f892e4dc3","order_by":1,"name":"Xinyi LI","email":"","orcid":"","institution":"Peking University","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"LI","suffix":""},{"id":575451237,"identity":"190da96f-4294-4e05-9acc-d1a3d46123b7","order_by":2,"name":"Zhenting Zhang","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhenting","middleName":"","lastName":"Zhang","suffix":""},{"id":575451239,"identity":"9e4163ac-b4b9-453c-bced-de1635d2ba08","order_by":3,"name":"Ning zhang","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"zhang","suffix":""},{"id":575451241,"identity":"8236abb6-2ea8-4caf-b135-0aaf30be0b9c","order_by":4,"name":"Xin Yao","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Yao","suffix":""},{"id":575451244,"identity":"fa7b16e1-d31d-4607-8c49-5b33e1a27c51","order_by":5,"name":"Rong Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYBACPmYILcfAw8DwmSgtbFAtxkAtjLOJ0wKlExuI18LOY/bh447a9O08ZwybCxjs5HQbCDqMx3jmzDPHc3f29hg2z2BINjY7QIQWZt62Y7kbzvOYP+ZhOJC4jVgt6QbneQybSdFSk2BwtodoLWzFjDPbDhju7DlW2MxjQIRf+PkPb2b42FYnb86TvLGZp8JOjqAWKDjMYACmDYhTDgJ1pCgeBaNgFIyCkQYA3So4MfRqZBAAAAAASUVORK5CYII=","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":true,"prefix":"","firstName":"Rong","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-12-01 01:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8245003/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8245003/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100582938,"identity":"84781b02-6194-4bee-8547-2013724ec573","added_by":"auto","created_at":"2026-01-19 11:28:49","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5406986,"visible":true,"origin":"","legend":"","description":"","filename":"F1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/805486c9c4e89f42a9e9077d.tif"},{"id":100583030,"identity":"33295804-2a36-4723-969f-3349d8d7d5b5","added_by":"auto","created_at":"2026-01-19 11:29:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11802964,"visible":true,"origin":"","legend":"","description":"","filename":"revisedManuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/68e0a07735cbb73c5cbdccb2.docx"},{"id":100582951,"identity":"88364516-1df8-4d12-8980-574623097362","added_by":"auto","created_at":"2026-01-19 11:28:53","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26110304,"visible":true,"origin":"","legend":"","description":"","filename":"F2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/71accf3a653e32646392641e.tif"},{"id":100596138,"identity":"298af1e4-f637-4811-8ddd-721306626c22","added_by":"auto","created_at":"2026-01-19 13:53:14","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"F3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/986c4bfdacdd055ac4812a38.tif"},{"id":100596113,"identity":"52e4d6c6-98ec-4793-80ef-a8014d884962","added_by":"auto","created_at":"2026-01-19 13:52:09","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5752598,"visible":true,"origin":"","legend":"","description":"","filename":"F4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/9922fe951eda694cbe265fd3.tif"},{"id":100582931,"identity":"e0dc5ed6-8ac1-406d-a85d-9045fcdb4587","added_by":"auto","created_at":"2026-01-19 11:28:47","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7094680,"visible":true,"origin":"","legend":"","description":"","filename":"F5.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/f412382031ee9ca54fd8a7c2.tif"},{"id":100583025,"identity":"39a5b9f9-af8b-4e36-af9b-3806406097cc","added_by":"auto","created_at":"2026-01-19 11:29:27","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2574386,"visible":true,"origin":"","legend":"","description":"","filename":"s1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/3bb3583b6741301b481deec8.tif"},{"id":100582956,"identity":"04d8c289-f9e4-4f2c-a51b-aac0460104b9","added_by":"auto","created_at":"2026-01-19 11:28:57","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":30380100,"visible":true,"origin":"","legend":"","description":"","filename":"s2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/3019ccaac672ccf2dd3215d2.tif"},{"id":100582954,"identity":"265b8a93-adc9-4daa-9baf-72227ce71dff","added_by":"auto","created_at":"2026-01-19 11:28:56","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1958010,"visible":true,"origin":"","legend":"","description":"","filename":"s3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/ae9a30b48f26ff8a81e82faf.tif"},{"id":100596148,"identity":"7aa152ac-acef-41c4-afcf-bbf80f01ea47","added_by":"auto","created_at":"2026-01-19 13:53:31","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"s4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/91ed85e5ab1911315aa91be3.tif"},{"id":100596157,"identity":"bed168f0-155c-44be-9844-fed428de9706","added_by":"auto","created_at":"2026-01-19 13:53:57","extension":"json","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7590,"visible":true,"origin":"","legend":"","description":"","filename":"565705e4cbc74a63994a93159d6900af.json","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/5bdb8797fb8d38686aa8077f.json"},{"id":100582924,"identity":"c9f1ca95-89f6-485e-a335-fbfbdc35226d","added_by":"auto","created_at":"2026-01-19 11:28:45","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104321,"visible":true,"origin":"","legend":"","description":"","filename":"565705e4cbc74a63994a93159d6900af1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/2ee89f7b642d78bf30b4e19d.xml"},{"id":100582905,"identity":"2a24af9f-a4fa-489a-ba1e-338ac3878797","added_by":"auto","created_at":"2026-01-19 11:28:42","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5406986,"visible":true,"origin":"","legend":"","description":"","filename":"F1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/9d86438bae90d7c372323bbd.tif"},{"id":100582928,"identity":"0bbdbb3b-99d4-49c5-bb5b-cb90fec2b09e","added_by":"auto","created_at":"2026-01-19 11:28:46","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26110304,"visible":true,"origin":"","legend":"","description":"","filename":"F2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/3394091e4c6fc497d07d5bdd.tif"},{"id":100582900,"identity":"fca6010a-661f-46d6-92e8-4a8ee3d6f565","added_by":"auto","created_at":"2026-01-19 11:28:36","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"F3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/53dd53092db6437b9c3f07c8.tif"},{"id":100583031,"identity":"fff273d5-9d53-4061-8f56-60c5e6be4161","added_by":"auto","created_at":"2026-01-19 11:29:34","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5752598,"visible":true,"origin":"","legend":"","description":"","filename":"F4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/03bab446172024d08c7d44de.tif"},{"id":100583012,"identity":"4506110f-9048-4c6e-84aa-6de0785f79df","added_by":"auto","created_at":"2026-01-19 11:29:22","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7094680,"visible":true,"origin":"","legend":"","description":"","filename":"F5.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/d73a07e9898d6db2ce6f3067.tif"},{"id":100596090,"identity":"bd2a54e6-f8a8-41f7-a2d3-0b3b17652389","added_by":"auto","created_at":"2026-01-19 13:51:11","extension":"jpeg","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/2aa138e002088fded66a2cb4.jpeg"},{"id":100582925,"identity":"cab0a2d1-290a-45c7-8a7c-7abef0d94bd3","added_by":"auto","created_at":"2026-01-19 11:28:46","extension":"jpeg","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/4579a9e300a036d58f4d71b0.jpeg"},{"id":100596046,"identity":"f93046ab-a0ae-44b9-8a03-0cfb89d96212","added_by":"auto","created_at":"2026-01-19 13:50:26","extension":"jpeg","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"F3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/e08e45a021e7d8df2f066cde.jpeg"},{"id":100596134,"identity":"6dad5183-7b0d-4423-b0e7-ae926bcb8584","added_by":"auto","created_at":"2026-01-19 13:52:54","extension":"jpeg","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5752598,"visible":true,"origin":"","legend":"","description":"","filename":"F4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/4c89cc6d3896f1d829f776f2.jpeg"},{"id":100583039,"identity":"960888a7-0a15-4944-b5f5-1a3fc998a049","added_by":"auto","created_at":"2026-01-19 11:29:41","extension":"jpeg","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7094680,"visible":true,"origin":"","legend":"","description":"","filename":"F5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/cea4843632356eff0b8e238e.jpeg"},{"id":100582979,"identity":"dcba5cf8-6d55-41fd-b428-f25783ab8fb3","added_by":"auto","created_at":"2026-01-19 11:29:03","extension":"jpeg","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":557668,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/248b998a6f48d3dd7d62bf1e.jpeg"},{"id":100596093,"identity":"362e412d-25e0-4433-95c1-87703635624a","added_by":"auto","created_at":"2026-01-19 13:51:13","extension":"jpeg","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":483305,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/96f54dcb4cc09b8da412cdb8.jpeg"},{"id":100596128,"identity":"4333908b-f624-4b5a-9229-f834c2f7424c","added_by":"auto","created_at":"2026-01-19 13:52:45","extension":"jpeg","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/c147051f53c74a87653997ec.jpeg"},{"id":100583051,"identity":"792c86cb-0b29-491a-845c-9f17559d393d","added_by":"auto","created_at":"2026-01-19 11:29:58","extension":"jpeg","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":637559,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/27f6092ba2803b54d0462ddb.jpeg"},{"id":100583032,"identity":"6f7fe14b-563e-4e1e-9e62-a466b4b1c61b","added_by":"auto","created_at":"2026-01-19 11:29:34","extension":"tif","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2574386,"visible":true,"origin":"","legend":"","description":"","filename":"s1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/db84dd0f58826f77e6efc76e.tif"},{"id":100583009,"identity":"4c0ac8fd-8bbc-42f8-84ee-d00b69ef8371","added_by":"auto","created_at":"2026-01-19 11:29:22","extension":"tif","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":30380100,"visible":true,"origin":"","legend":"","description":"","filename":"s2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/04d65cdf73c1cf8997f76582.tif"},{"id":100583033,"identity":"9fad59ce-18d6-4b34-916e-2397a6b6b1b9","added_by":"auto","created_at":"2026-01-19 11:29:35","extension":"tif","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1958010,"visible":true,"origin":"","legend":"","description":"","filename":"s3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/168f46d9bc75d998507f5fee.tif"},{"id":100582930,"identity":"966c982f-602b-4bcb-a097-f2fa5b06800b","added_by":"auto","created_at":"2026-01-19 11:28:47","extension":"tif","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"s4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/58e4000acaf3d57bb147b9f5.tif"},{"id":100582960,"identity":"226d41d5-2842-43d8-9410-6f09a21c6ded","added_by":"auto","created_at":"2026-01-19 11:28:59","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":279816,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineF1.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/8cc9d1097364bfa891f1d2e5.png"},{"id":100582950,"identity":"14aa5c8c-1b18-4fa7-9afd-116af642b1d7","added_by":"auto","created_at":"2026-01-19 11:28:53","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":425847,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineF2.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/8adbc7d16b67709ee4fe5ae6.png"},{"id":100582986,"identity":"d732fbfa-a9a2-453c-8ebc-2e3ce10eebf0","added_by":"auto","created_at":"2026-01-19 11:29:07","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130578,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineF3.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/834d4564be9752a42288c750.png"},{"id":100582890,"identity":"c5febbe1-76ec-4064-8632-0df06625bbbf","added_by":"auto","created_at":"2026-01-19 11:28:24","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102696,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineF4.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/b651f10285c46d4bb62889ea.png"},{"id":100596152,"identity":"8de5e269-b41c-4e3f-a342-b762e15c8a06","added_by":"auto","created_at":"2026-01-19 13:53:39","extension":"png","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128729,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineF5.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/8cb5c1a00510a43d9655d111.png"},{"id":100596102,"identity":"5dada688-0dca-470c-9ad3-cb3140044614","added_by":"auto","created_at":"2026-01-19 13:51:35","extension":"png","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":273269,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/1873d2b0da7b30b97e80fb3d.png"},{"id":100583007,"identity":"6f40f6f8-61b2-4d0b-b939-f19cb9fc518f","added_by":"auto","created_at":"2026-01-19 11:29:21","extension":"png","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140913,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/e123195ff9a96467aa9498cd.png"},{"id":100582981,"identity":"03ffd42f-a251-45a2-a736-bf6e86c32747","added_by":"auto","created_at":"2026-01-19 11:29:04","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130578,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/8e6995f98f4f5fc45556ec8c.png"},{"id":100582957,"identity":"4a80f4cf-2d0b-4403-8ff9-10ced969de62","added_by":"auto","created_at":"2026-01-19 11:28:58","extension":"png","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102696,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/4da78aa6c2c64b340b7101e6.png"},{"id":100596114,"identity":"3417f1fe-6606-49e1-9894-a818210dc3f8","added_by":"auto","created_at":"2026-01-19 13:52:09","extension":"png","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128729,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/054ba27c8d32a77d5f7a5ab5.png"},{"id":100596147,"identity":"5e83a007-870a-4523-9d75-3d06a15ca9d5","added_by":"auto","created_at":"2026-01-19 13:53:30","extension":"png","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":229332,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/1e8b2983bca6fa3c2a868663.png"},{"id":100582985,"identity":"b1f6ad81-b0ff-4637-af2c-da8d1e5544ab","added_by":"auto","created_at":"2026-01-19 11:29:05","extension":"png","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":238537,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/212e0504f5ad9a73c9b499d8.png"},{"id":100582893,"identity":"429ea42f-d652-4677-a693-c7efd1c985a3","added_by":"auto","created_at":"2026-01-19 11:28:26","extension":"png","order_by":42,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79426,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/13d39dce6230d584e7aff450.png"},{"id":100582895,"identity":"2c594093-9c54-4387-b46f-31f94d96ead9","added_by":"auto","created_at":"2026-01-19 11:28:28","extension":"png","order_by":43,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112118,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/70c88e3d9c78cf8de3006bf2.png"},{"id":100582901,"identity":"55116b73-1c10-452e-80cd-f66789f62afe","added_by":"auto","created_at":"2026-01-19 11:28:36","extension":"png","order_by":44,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":156062,"visible":true,"origin":"","legend":"","description":"","filename":"Onlines1.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/970d288c20ef91cf32dc712f.png"},{"id":100583041,"identity":"66135022-cfb6-49c4-a3ac-021f3cc3fef7","added_by":"auto","created_at":"2026-01-19 11:29:42","extension":"png","order_by":45,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3688423,"visible":true,"origin":"","legend":"","description":"","filename":"Onlines2.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/c1fea18660e03d38f2e8de7b.png"},{"id":100582929,"identity":"6974eb1e-582d-4ab5-8a03-efbd4dee0f33","added_by":"auto","created_at":"2026-01-19 11:28:47","extension":"png","order_by":46,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75164,"visible":true,"origin":"","legend":"","description":"","filename":"Onlines3.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/9b396592e8e599ec72dce687.png"},{"id":100583013,"identity":"1c837dca-687c-49ef-80fc-0c5a8e107353","added_by":"auto","created_at":"2026-01-19 11:29:22","extension":"png","order_by":47,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74916,"visible":true,"origin":"","legend":"","description":"","filename":"Onlines4.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/7522f84bff7a01104df6b200.png"},{"id":100582894,"identity":"5817218c-b29a-4766-a112-e52e4694f4fb","added_by":"auto","created_at":"2026-01-19 11:28:26","extension":"xml","order_by":48,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102519,"visible":true,"origin":"","legend":"","description":"","filename":"565705e4cbc74a63994a93159d6900af1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/ca6fb7c8be3dea40dd99bb20.xml"},{"id":100583045,"identity":"7af10d61-c420-4a90-82d2-3e784d957fc3","added_by":"auto","created_at":"2026-01-19 11:29:51","extension":"html","order_by":49,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116593,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/891388cccceb0325ef59038c.html"},{"id":100596137,"identity":"e3934147-27bc-4790-a0ac-349ede3154fe","added_by":"auto","created_at":"2026-01-19 13:53:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4769588,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment of multi-regional urachal carcinoma (UrC) patient-derived organoids.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Ultrasonography revealed a protruding lesion measuring 3.2 × 3.1 × 3.5 cm on the anterior bladder wall.\u003c/p\u003e\n\u003cp\u003e(B) Contrast-enhanced CT confirmed a solid anterior bladder wall mass of 4.2 × 2.9 cm.\u003c/p\u003e\n\u003cp\u003e(C) Gross morphology of the surgically resected UrC specimen.\u003c/p\u003e\n\u003cp\u003e(D) Bright-field images of patient-derived organoids (PDOs) passage 0, from spatially distinct tumor regions. Organoid:T1and T3 at day 5 ,Organoid:T2 and T4 at day 10.Scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/b454f08a7d3ce483a18026e0.png"},{"id":100582995,"identity":"eb3a4cbd-030d-4530-968b-194df27ded23","added_by":"auto","created_at":"2026-01-19 11:29:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13321881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUrC organoids preserve the histological, genomic, and transcriptomic features of parental tumors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Histological and immunohistochemical comparison of UrC organoids and matched tumor tissues, showing staining for mismatch repair proteins (PMS2, MLH1, MSH2, MSH6) and luminal marker CK20. Organoids: passage 1,Scale bars: 50 μm, 100 μm.\u003c/p\u003e\n\u003cp\u003e(B) Whole-exome sequencing (WES) reveals mutational profiles of four UrC organoid-tumor pairs, with two bladder cancer (BC) organoids as controls.Organoids: passage 1.\u003c/p\u003e\n\u003cp\u003e(C) Concordance analysis of cancer-related somatic variants between UrC tumors and their corresponding organoids. Median concordance is indicated.\u003c/p\u003e\n\u003cp\u003e(D) Heatmap of Spearman correlation coefficients for transcriptomic profiles among UrC (\u003cem\u003en\u003c/em\u003e= 4), BC (\u003cem\u003en\u003c/em\u003e = 2) organoids, and parental tumors.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/bd08c7222a7a90d001188bd8.png"},{"id":100582984,"identity":"83d5d960-5648-47ae-a4e9-92e1c2c064d3","added_by":"auto","created_at":"2026-01-19 11:29:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2262802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUrachal carcinoma organoids recapitulate intratumoral heterogeneity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Histological analysis shows distinct morphologies in superficial (T3, T4) versus invasive (T1, T2) tumor regions: intestinal-type adenocarcinoma vs. mucinous adenocarcinoma, respectively.\u003c/p\u003e\n\u003cp\u003e(B) Phylogenetic reconstruction of multi-regional UrC tissues based on WES.\u003c/p\u003e\n\u003cp\u003e(C) RNA-seq-based heatmap of epithelial–mesenchymal transition (EMT) gene expression profiles across UrC organoids.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/48a8ce19c25cc345cf5f4943.png"},{"id":100596149,"identity":"236d6504-5790-48ce-8769-baaefeb9fe3f","added_by":"auto","created_at":"2026-01-19 13:53:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2162556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUrC organoids predict patient response to clinically relevant chemotherapeutics.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Postoperative imaging and CA-724 serum marker levels in the UrC patient.\u003cbr\u003e\n(B) Dose–response curves for 5-FU, cisplatin, and other drugs in four UrC and two BC organoids. Viability was measured after 6 days using CellTiter-Glo.\u003cbr\u003e\n(C) Volcano plot of DEGs distinguishing 5-FU-sensitive (T2, T4) \u003cem\u003evs\u003c/em\u003e. 5-FU-resistant (T1, T3) UrC organoids.\u003cbr\u003e\n(D, E) siRNA-mediated knockdown of \u003cem\u003ePCSK1\u003c/em\u003eand \u003cem\u003eCLUL1\u003c/em\u003e sensitized T1 and T3 organoids to 5-FU. Data are presented as mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001,****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001, two-tailed Student's \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/943dd6510532ddf23b69ae1d.png"},{"id":100582991,"identity":"69a1693b-49db-4505-baa5-f9f0dbe3acbd","added_by":"auto","created_at":"2026-01-19 11:29:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2909739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular profiling reveals potential therapeutic targets in UrC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) KEGG (www.kegg.jp/kegg/kegg1.html) enrichment analysis of DEGs between four UrC organoids and two normal urothelial organoids.\u003c/p\u003e\n\u003cp\u003e(B) Dose–response curves of selected inhibitors (targeting PI3K, mTOR, MEK) tested in UrC and BC organoids. Viability normalized to DMSO controls. Data represent means ± SD from triplicates.\u003c/p\u003e\n\u003cp\u003e(C) Left: RNA-seq-derived heatmap showing expression of candidate therapeutic targets (e.g., \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003ePD-L1\u003c/em\u003e) in UrC organoids. Right: Dose–response curves of KRAS inhibitor BI-2865 in UrC and BC organoids.\u003c/p\u003e\n\u003cp\u003e(D) PD-L1 blockade enhances cytotoxicity in UrC organoids. T cell co-cultures treated with 20 μg/mL atezolizumab or isotype control were assessed for viability at 72 hours. Error bars: mean ± SD, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001; \u003cem\u003et\u003c/em\u003e-test, respectively.\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/dd761b70dfd5aa665489e9c2.png"},{"id":104401572,"identity":"79d0081a-d31e-44e4-b82f-0f8cda17c32e","added_by":"auto","created_at":"2026-03-11 12:13:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27608228,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/04844b81-b8ce-4add-a9b1-15469a7c1275.pdf"},{"id":100582891,"identity":"7e779ee2-f5c9-4ac0-a7e1-92772c4622b4","added_by":"auto","created_at":"2026-01-19 11:28:24","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":757637,"visible":true,"origin":"","legend":"","description":"","filename":"s1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/cc9f4d24b743333ca3d46e70.tif"},{"id":100596095,"identity":"3ccec450-fd0f-4e68-9ca4-f43bb11b5020","added_by":"auto","created_at":"2026-01-19 13:51:18","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7753580,"visible":true,"origin":"","legend":"","description":"","filename":"s2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/978c425ef4dc8325de861198.tif"},{"id":100583049,"identity":"53ba201d-e2f0-4831-b5dc-65f3a28214fd","added_by":"auto","created_at":"2026-01-19 11:29:56","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":460893,"visible":true,"origin":"","legend":"","description":"","filename":"s3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/9f1ebb0a3289eed482781cc8.tif"},{"id":100582940,"identity":"c53c54cf-49f0-45cd-bf80-a6a051f0db72","added_by":"auto","created_at":"2026-01-19 11:28:52","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":271450,"visible":true,"origin":"","legend":"","description":"","filename":"s4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/63e8941319fe6aab954f6898.tif"},{"id":100582933,"identity":"bf47f31e-9358-488e-9561-ef20e47e7b17","added_by":"auto","created_at":"2026-01-19 11:28:48","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":17132,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8245003/v1/450e65a4523e6289f5aaa151.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Patient-derived urachal cancer organoids for personalized drug screening","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUrachal cancer (UrC) is a rare and aggressive tumor originating from the urachus, a vestigial structure connecting the fetal allantois to the bladder dome \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It accounts for less than 1% of all bladder cancers \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, with over 90% of cases presenting as adenocarcinomas that share histopathological features with both primary bladder adenocarcinoma and colorectal adenocarcinoma \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In localized disease, the standard of care involves partial cystectomy with en bloc resection of the urachal ligament and umbilicus \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, for recurrent or metastatic disease, systemic therapies\u0026mdash;such as 5-fluorouracil-based regimens, cisplatin combinations, or hyperthermic intraperitoneal chemotherapy\u0026mdash;are employed with limited efficacy \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Radiotherapy remains largely ineffective, and due to the rarity of UrC, there are no standardized chemotherapy protocols or prospective clinical trials for advanced disease, resulting in poor survival outcomes \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The lack of randomized studies and consensus treatment guidelines underscores the urgent need for robust preclinical models to facilitate research and inform therapeutic decision-making.\u003c/p\u003e \u003cp\u003ePatient-derived organoids (PDOs) have emerged as promising preclinical tools capable of modeling tumor heterogeneity and enabling individualized drug screening across multiple cancer types \u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Notably, PDOs preserve the genomic landscape and histological architecture of the primary tumor while providing predictive insight into treatment responses \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, highlighting their potential in personalized oncology. This is particularly crucial for rare malignancies, where low incidence rates and ethical constraints limit the feasibility of clinical trials. Organoid technology thus offers a powerful solution by enabling high-fidelity modeling of tumor biology and patient-specific drug \u003cem\u003eresponses in vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we established four PDO lines from a single UrC patient and demonstrated their capacity to replicate the phenotypic and genomic heterogeneity of the corresponding tumors. These PDOs exhibited strong concordance with clinical responses to 5-fluorouracil (5-FU) and cisplatin, two standard chemotherapeutic agents. Furthermore, based on insights from whole-exome and transcriptomic profiling, we evaluated several FDA recently approved or investigational compounds and identified potential therapeutic candidates targeting the RAS/MAPK pathway, the PI3K/AKT/mTOR signaling pathway, and immune checkpoints in UrC organoids, and found this UrC patient is potentially sensitive to these inhibitors\u0026rsquo; treatment. Collectively, our findings provide compelling evidence that UrC PDOs can serve as a translational platform for precision oncology, supporting their use in personalized treatment development and research for rare cancers.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eHuman tissue collection.\u003c/strong\u003e Fresh tumor and matched normal tissues from patients diagnosed with urachal carcinoma (UrC) or bladder cancer (BC) were obtained from the Department of Urology at Tianjin Medical University Cancer Institute and Hospital. Detailed bladder cancer organoids clinicopathological data are summarized in Table S1. All procedures were approved by the Peking University First Hospital and Tianjin Medical University Cancer Institute \u0026nbsp; review board (approval number: 2023051-001), and written informed consent was obtained from each participant.All research was performed in accordance with relevant guidelines/regulations. Research involving human research participants have been performed in accordance with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of UrC and BC organoids.\u0026nbsp;\u003c/strong\u003eTumor specimens were transported on ice to the laboratory and immediately processed. Resected tissues were minced and enzymatically digested using Collagenase I (1 mg/mL, Worthington), Collagenase II (1 mg/mL, Worthington), and Hyaluronidase (100 U/mL, Sigma-Aldrich) at 37 \u0026deg;C for 30\u0026ndash;60 minutes. The tissue suspension was further digested in TrypLE Express (Invitrogen) at 37 \u0026deg;C for 10 minutes. Following filtration and washing, cells were resuspended in growth factor\u0026ndash;reduced Matrigel (Corning), seeded into 6-well plates (Thermo Fisher Scientific), and overlaid with culture medium. The organoid medium, adapted from previously reported protocols for BC organoids \u003csup\u003e15\u003c/sup\u003e, consisted of Advanced DMEM/F12 supplemented with 1\u0026times; HEPES, 1\u0026times; GlutaMax (Invitrogen), 1\u0026times; Primocin (InvivoGen), 1\u0026times; B27 Supplement (Gibco), 0.5 \u0026mu;M A83-01 (Selleck), 10 \u0026mu;M Y27632 (Selleck), 0.1 \u0026mu;g/mL FGF-10 (Novoprotein), 1.25 mM N-acetylcysteine (Sigma-Aldrich), and 12.5 mM nicotinamide (Sigma-Aldrich). Medium was refreshed every 2\u0026ndash;3 days. For passaging, organoids were first released from Matrigel using 1 mg/mL dispase (STEMCELL Technologies) for 60 minutes at 37 \u0026deg;C, then dissociated into single cells with TrypLE Express. For cryopreservation, organoids were dissociated into single cells or small clusters and frozen in cryopreservation medium (Corning).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology and immunohistochemistry.\u0026nbsp;\u003c/strong\u003eOrganoids and tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H\u0026amp;E). Immunohistochemistry (IHC) was performed using the Roche Ventana Discovery Ultra automated system after heat-induced antigen retrieval. Primary antibodies used were: anti-MLH1 (1:100, ZM-0152), anti-PMS2 (1:100, ZA-0542), anti-MSH2 (1:100, ZA-0622), anti-MSH6 (1:100, ZA-0541), and anti-CK20 (1:100, ZA-0574), all obtained from Zhongshan Golden Bridge Biotechnology (Beijing, China). Two board-certified pathologists independently confirmed all histopathological evaluations in accordance with standard diagnostic criteria.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrganoid drug screening.\u0026nbsp;\u003c/strong\u003eOrganoids were enzymatically dissociated and passed through a 70 \u0026mu;m strainer (Corning) to remove large aggregates. The resulting suspension (20,000\u0026ndash;50,000 organoids/mL in 5% Matrigel-containing medium) was plated in ultralow-attachment 384-well plates (Corning) in triplicate. After 24 hours, a dilution series of each drug was added. Concentration ranges varied depending on compound properties: 100 \u0026mu;M to 12.5 \u0026mu;M, 100 \u0026mu;M to 0.78 \u0026mu;M, or 20 \u0026mu;M to 2.5 \u0026mu;M (maximum DMSO concentration: 1%). After 6 days of incubation, cell viability was assessed using CellTiter-Glo 3D (Promega), following the manufacturer\u0026rsquo;s instructions. Data were normalized to vehicle controls, and IC\u003csub\u003e50\u003c/sub\u003e and AUC values were calculated using nonlinear regression (log[inhibitor] \u003cem\u003evs\u003c/em\u003e. normalized response, variable slope) in GraphPad Prism 10.0b.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmune stimulation assays in organoids.\u0026nbsp;\u003c/strong\u003eOrganoids and peripheral blood mononuclear cells (PBMCs) were co-cultured at a 1:5 ratio in medium supplemented with 100 U/mL IL-2, 10 ng/mL IL-7, and 10 ng/mL IL-15 (all from Peprotech). A total of 500 organoids and 2,500 PBMCs were seeded per well in 384-well plates and treated with either 20 \u0026mu;g/mL atezolizumab (Selleck) or IgG1 isotype control. After 72 hours, cell viability was measured using CellTiter-Glo 3D. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR.\u0026nbsp;\u003c/strong\u003eTotal RNA was extracted using the TRIzol reagent and reverse-transcribed to cDNA with a commercial RT-PCR kit (TianGen, Beijing, China). Quantitative PCR was performed on an AriaMx Real-Time PCR System (Agilent Technologies) using SYBR Green (TianGen) according to the manufacturer\u0026rsquo;s protocol. Primer sequences are listed in Supplementary Table S2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNA Transfection.\u0026nbsp;\u003c/strong\u003eSilencer Select predesigned siRNAs targeting PCSK1 (GM-SI-147903) and CLUL1 (GM-SI-147979), as well as non-targeting negative control siRNA, were purchased from Genomeditech (Shanghai, China). Organoids were dissociated into single cells and transfected using Lipofectamine RNAiMAX (Invitrogen), following minor modifications to the manufacturer\u0026apos;s protocol. Following siRNA transfection, cells were subjected to drug treatment for 6 days, and viability was assessed using CellTiter-Glo 3D. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole-exome sequencing and analysis.\u0026nbsp;\u003c/strong\u003eGenomic DNA was extracted using the DNeasy \u0026amp; RNeasy kit (Qiagen). Exonic regions were captured with Agilent SureSelectXT Human All Exon V6 probes, and libraries were sequenced on the Illumina NovaSeq 6000 platform (150 bp paired-end reads). Median sequencing depth was \u0026times;300 for tumor tissues and PDOs (passage1), and \u0026times;100 for normal tissues. Quality control of raw reads was performed using FastQC (v0.11.9), Cutadapt (v2.5), and Trimmomatic (v0.39). Clean reads were aligned to the UCSC human reference genome (hg19) using bwa-mem2 (v2.0). BAM file processing\u0026mdash;including sorting, merging, and indexing\u0026mdash;was performed with Samtools (v1.10), and PCR duplicates were removed using GATK (v4.1.2.0). Coverage statistics were calculated using Samtools based on Sure Select All ExonV6r2 BED file coordinates. Somatic point mutations and indels were called using GATK Mutect2 (v4.1.2.0) in paired tumor\u0026ndash;normal mode.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic tree construction.\u0026nbsp;\u003c/strong\u003ePhylogenetic relationships among multi-regional organoids were inferred as described previously \u003csup\u003e16\u003c/sup\u003e. Genomic sequences (\u0026plusmn; 20 bp surrounding mutation sites) were extracted and used to construct phylogenetic trees using MEGA5 software with the maximum parsimony algorithm. Driver mutations were annotated along the tree\u0026rsquo;s root, branches, and leaves to depict clonal evolution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential gene expression and gene set enrichment analysis.\u0026nbsp;\u003c/strong\u003eDifferentially expressed genes (DEGs) between experimental groups were identified using DESeq2, applying an absolute fold-change cutoff \u0026gt; 2 and an adjusted \u003cem\u003eP-\u003c/em\u003evalue \u0026lt; 0.05. The ranked gene lists were used for gene set enrichment analysis (GSEA) with a previously published stem cell gene set and the GO_BP gene sets from MSigDB (v7.5.1). A q-value \u0026lt; 0.25 was considered statistically significant. KEGG and GO pathway enrichment analyses \u003csup\u003e17,18\u003c/sup\u003ewere conducted using the clusterProfiler R package.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We get permission to use the KEGG software from the Kanehisa laboratory.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing and quantification\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eTotal RNA was isolated from NEN organoids (passage1) by using the DNeasy \u0026amp; RNeasy kit (Qiagen), and poly-T oligo-attached magnetic beads were used for mRNA purification. Sequencing libraries were prepared and sequenced on the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. Quality control of raw reads was performed with FastQC (v0.11.9), Cutadapt (v2.5, using Illumina universal adapters), and Trimmomatic (v0.39, parameters: PE, MINLEN = 36). Reads were aligned to the human reference genome (hg19) using STAR (v2.7.3a) with default settings. Gene-level counts were generated using HTSeq-count with GENCODE annotations, and transcript per million (TPM) values were calculated via RSEM . Unless otherwise stated, mRNA expression is presented as log₂(TPM + 1). Correlation heatmaps comparing organoids and matched tumor tissues were generated as previously described \u003csup\u003e11\u003c/sup\u003e, and statistical significance for expression variance across samples was assessed using one-way ANOVA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u0026nbsp;\u003c/strong\u003eStatistical comparisons were performed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test in GraphPad Prism 10.0b. Data are presented as mean \u0026plusmn; standard deviation (SD). A \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05 was considered statistically significant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCase presentation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 31-year-old female presented in November 2023 for urological evaluation following six months of intermittent, painless gross hematuria accompanied by mucous discharge. Initial ultrasonography of the urinary tract identified a 3.2 \u0026times; 3.1 \u0026times; 3.5 cm hypoechoic nodule with indistinct margins along the anterior bladder wall. Cystoscopy confirmed the presence of a sessile, non-papillary lesion at the same anatomical site. Contrast-enhanced computed tomography (CT) further characterized a solid, calcified mass measuring 4.2 \u0026times; 2.9 cm in the anterior bladder wall (Figs. 1A\u0026ndash;B). To exclude metastatic dissemination from gastrointestinal origins, a colonoscopy and biopsy were performed (Fig. S1A), which revealed a low-grade tubular adenoma (Fig. S1B), supporting the diagnosis of a primary urachal carcinoma (UrC) and excluding a metastasis from an intestinal tumor.\u003c/p\u003e\n\u003cp\u003eIntraoperative exploration revealed a broad-based, well-demarcated 3.2 \u0026times; 3.1 cm mass on the anterior bladder wall, distinct from surrounding mucosa. Guided by cystoscopy, an en bloc resection was performed, which included the tumor, adjacent normal mucosa, partial umbilical tissue, urachal remnant, and adherent peritoneal structures (Fig. 1C). Histopathological analysis confirmed the diagnosis of urachal adenocarcinoma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of multi-regional urachal carcinoma patient-derived organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo model intratumoral heterogeneity, fresh surgical specimens were subjected to immediate multi-regional sampling for PDO establishment and multi-omics analysis (Fig. 1D). Four spatially distinct tumor fragments were cultured to generate organoids, T1 and T2 derived from histologically invasive regions, and T3 and T4 from morphologically superficial, non-invasive areas, were confirmed by histopathological mapping.Using this approach, we have generated independent four urachal organoid lines. These lines have been propagated by serial passaging (up to passage 4) (Fig. S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUrachal\u0026nbsp;organoids retain\u0026nbsp;histological,\u0026nbsp;genomic, and transcriptomic features of parental tumors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess fidelity between organoids and their parental tumors, we performed hematoxylin and eosin (H\u0026amp;E) staining and immunohistochemistry for canonical UrC markers, including cytokeratin 20 (CK20) and the mismatch repair (MMR) proteins, MSH2, MSH6, PMS2, and MLH1. As shown in Fig. 2A, the organoids exhibited a histological architecture comparable to that of the parental tumor. Both the original tumor and derived PDOs demonstrated a mismatch repair-deficient (d-MMR) phenotype, evidenced by the selective loss of PMS2 expression while retainment of MLH1, MSH2, and MSH6. Additionally, periodic acid\u0026ndash;Schiff (PAS) staining confirmed mucin secretion capacity of the organoids (Fig. S3), consistent with their adenocarcinomatous identity. Thus, the established UrC PDOs faithfully mirrored both the histopathological and molecular features of the parental tumors.\u003c/p\u003e\n\u003cp\u003eWhole-exome sequencing (WES) was conducted on four UrC tumor-organoid pairs (median depth: 300\u0026times;), two bladder cancer (BC) tumor-organoid pairs (median depth: 300\u0026times;), and one matched normal skin sample as a somatic control (median depth: 100\u0026times;). Detailed bladder cancer organoids clinicopathological data are summarized in \u003ca href=\"https://pmc.ncbi.nlm.nih.gov/articles/PMC11186361/#SM0\"\u003eTable S1\u003c/a\u003e.In line with previous genomic analyses of UrC \u003csup\u003e13,19\u003c/sup\u003e, recurrent mutations in \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eKRAS\u003c/em\u003e were identified in all UrC samples (Fig. 2B). Immunohistochemistry and WES consistently confirmed MMR deficiency due to a somatic missense mutation in \u003cem\u003ePMS2\u003c/em\u003e. Notably, UrC tumors and organoids shared a median of 78.07% of somatic cancer-related variants (Fig. 2C). In contrast, BC samples displayed distinct mutational signatures with a higher burden of alterations in epigenetic regulators, such as \u003cem\u003eEP300\u003c/em\u003e, \u003cem\u003eKMT2D\u003c/em\u003e, and \u003cem\u003eKDM6A\u003c/em\u003e (Fig. 2B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTranscriptomic profiling via RNA-seq revealed high transcriptional concordance between tumor-organoid pairs (average Pearson\u0026rsquo;s \u003cem\u003er\u003c/em\u003e = 0.77). Hierarchical clustering demonstrated distinct clustering between UrC and BC samples, further supporting tumor-type specificity (Fig. 2D). Collectively, these multi-omics data confirm that UrC PDOs faithfully recapitulate the genomic and transcriptional landscapes of their tumors of origin and provide a robust platform for modeling disease heterogeneity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUrachal carcinoma organoids recapitulate tumor heterogeneity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the organoids\u0026rsquo; capacity to preserve intra-tumoral heterogeneity, we analyzed four PDOs generated from anatomically distinct regions of the tumor mass. Histological analysis of the primary tumor revealed pronounced heterogeneity, including superficial intestinal-type adenocarcinoma and deeper mucinous adenocarcinoma invading the muscularis propria (Fig. 3A). Phylogenetic tree reconstruction based on genomic data demonstrated both branching and parallel evolutionary patterns among the sampled tumor regions (Fig. 3B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMorphologically, PDOs also reflected their site of origin. Organoids T1 and T2, derived from invasive regions, displayed irregular contours with stellate projections\u0026mdash;features previously associated with aggressive phenotypes and epithelial-mesenchymal transition (EMT) \u003csup\u003e20,21\u003c/sup\u003e. In contrast, T3 and T4 organoids from superficial areas retained smooth borders. To evaluate EMT status, RNA-seq analysis compared gene expression profiles between T1/T2 (invasive) and T3/T4 (superficial) PDOs. Mesenchymal markers (\u003cem\u003eTWIST1\u003c/em\u003e, \u003cem\u003eVIM\u003c/em\u003e) were enriched in T1/T2, while epithelial markers (\u003cem\u003eCLDN3\u003c/em\u003e, \u003cem\u003eCDH1\u003c/em\u003e, \u003cem\u003eCLDN4\u003c/em\u003e) were upregulated in T3/T4 (Fig. 3C). Differential gene expression and pathway enrichment analyses further identified activation of EMT-related pathways in T1/T2 organoids, including ECM-receptor interaction \u003csup\u003e22-24\u003c/sup\u003e, Wnt/\u0026beta;-catenin, and PI3K-AKT-mTOR signaling (Fig. S4A). Gene Set Enrichment Analysis (GSEA) validated significant Wnt pathway activation in the invasive PDOs (Fig. S4B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, these findings demonstrate that UrC PDOs not only recapitulate the molecular and morphological diversity of the parental tumor but also maintain regional differences in EMT-associated signaling and invasive potential, reinforcing their value for studying intratumoral heterogeneity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening of clinically relevant agents in urachal carcinoma organoids reflects patient response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patient in this study underwent partial cystectomy with en bloc resection of the urachal ligament and umbilicus in November 2023. Postoperatively, a 5-fluorouracil (5-FU)-based chemotherapy regimen combined with cisplatin was administered. However, follow-up computed tomography (CT) revealed disease progression, accompanied by a marked elevation in serum CA72-4 levels (Fig. 4A).\u003c/p\u003e\n\u003cp\u003eTo evaluate the predictive capacity of patient-derived organoids (PDOs) in therapeutic response, we screened the four UrC PDOs established from surgical tumor specimens and two bladder cancer (BC) PDOs against six commonly used chemotherapeutic agents, including 5-FU and cisplatin. As shown in Fig. 4B, while BC PDOs were sensitive to cisplatin, all four UrC PDOs demonstrated complete resistance. Notably,\u0026nbsp;two UrC PDOs (T2 and T4)\u0026nbsp;displayed relative sensitivity to 5-FU, exhibiting IC\u003csub\u003e50\u003c/sub\u003e values below the predefined efficacy threshold of 50 \u0026mu;M \u003csup\u003e25,26\u003c/sup\u003e. However, the remaining two PDOs were resistant. Considering that therapeutic resistance is often driven by the most refractory subclones within a tumor, our findings suggest an overall chemoresistant phenotype in this case, aligning with the patient\u0026apos;s clinical outcome (Fig. 4A). These results underscore the utility of UrC PDOs in accurately reflecting patient-specific drug responses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further explore the molecular underpinnings of 5-FU response, we conducted transcriptomic profiling comparing the 5-FU-sensitive and -resistant UrC PDOs. As shown in Fig. 4C, \u003cem\u003ePCSK1\u003c/em\u003e and \u003cem\u003eCLUL1\u003c/em\u003e were significantly upregulated in the resistant group. Importantly, targeted knockdown of \u003cem\u003ePCSK1\u003c/em\u003e or \u003cem\u003eCLUL1\u003c/em\u003e in T1 and T3 PDOs, respectively, resulted in increased sensitivity to 5-FU treatment (Figs. 4D\u0026ndash;E). These findings suggest a functional role for both genes in modulating chemoresistance of UrC to 5-FU treatment. Of particular note, \u003cem\u003ePCSK1\u003c/em\u003e overexpression has previously been associated with poor prognosis and therapeutic resistance in rectal adenocarcinoma \u003csup\u003e27\u003c/sup\u003e, indicating a potentially conserved mechanism across epithelial malignancies. Altogether, these results identify candidate biomarkers predictive of 5-FU response and offer a framework for the mechanistic dissection of chemoresistance in UrC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug response profiling of Urachal carcinoma organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the patient\u0026rsquo;s resistance to standard chemotherapy (Fig. 4), we next assessed the efficacy of targeted therapies based on insights from whole-exome and transcriptomic profiling. Comparative transcriptomic analysis between two normal bladder urothelial PDOs and four UrC PDOs revealed significant dysregulation in several oncogenic pathways in UrC, including PD-L1/PD-1 checkpoint signaling, Ras signaling, and MAPK cascades (Fig. 5A). Functional drug screening demonstrated that UrC PDOs were significantly more sensitive than a BC PDO (lacking MAPK-activating mutations such as \u003cem\u003ePIK3CA\u003c/em\u003e or \u003cem\u003eFGFR3\u003c/em\u003e) to the PI3K inhibitor pictilisib, the mTOR inhibitor rapamycin, and the MEK inhibitor trametinib (Fig. 5B). These responses strongly correlated with the transcriptional profiles of the PDOs, highlighting the mechanistic relevance of pathway activation to therapeutic vulnerability.\u003c/p\u003e\n\u003cp\u003eMoreover, whole-exome sequencing revealed \u003cem\u003eKRAS\u003c/em\u003e and \u003cem\u003ePMS2\u003c/em\u003e mutations in the UrC PDOs (Fig. 2B), and transcriptomic data confirmed elevated expression of \u003cem\u003eKRAS\u003c/em\u003e and \u003cem\u003ePD-L1\u003c/em\u003e (Fig. 5C), supporting their potential as therapeutic targets. We therefore evaluated the efficacy of the KRAS inhibitor BI-2865 and the PD-L1 inhibitor atezolizumab. Dose-response assays showed that the IC\u003csub\u003e50\u003c/sub\u003e values of UrC PDOs to BI-2865 ranged from 12.62 \u0026mu;M to 23.92 \u0026mu;M, markedly lower than those of BC PDOs (54.89 \u0026mu;M to 112.4 \u0026mu;M), indicating enhanced KRAS inhibitor sensitivity in UrC organoids (Fig. 5C). Furthermore, treatment with atezolizumab led to significant cytotoxic responses in UrC PDOs that cell viability decreased by 53.42% \u0026plusmn; 4.11% and 45.28% \u0026plusmn; 2.19% in T1 and T2 PDOs, respectively (Fig. 5D).\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that molecular profiling of UrC PDOs enables the identification of alternative, actionable therapeutic targets. This approach supports the integration of PDO-based drug screening into personalized treatment strategies for rare malignancies such as urachal carcinoma.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSystemic therapy is frequently required in urachal carcinoma (UrC) due to its late-stage presentation or progression following locoregional interventions. However, treatment options remain limited. Chemotherapy efficacy data are largely confined to retrospective studies, as the rarity of UrC precludes prospective clinical trials. Current treatment strategies rely on empirical use of conventional chemotherapies, which often yield suboptimal outcomes. Moreover, evidence supporting targeted therapies is scarce, and no validated alternatives exist once resistance develops. Therefore, there is a pressing need for reliable experimental models to address the clinical challenges unique to UrC.\u003c/p\u003e \u003cp\u003eIn this study, we successfully established four UrC patient-derived organoid (PDO) lines from a 31-year-old female patient carrying somatic \u003cem\u003ePMS2\u003c/em\u003e mutations. Histological and immunohistochemical analyses confirmed that the PDOs preserved key features of the parental tumors, including mucin production and expression of canonical markers such as CK20 and MLH1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Genomic and transcriptomic profiling further demonstrated high fidelity between organoids and corresponding tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e), supporting the robustness of these models for translational applications.\u003c/p\u003e \u003cp\u003ePDO platforms represent a transformative tool in precision oncology, enabling the integration of mutational and transcriptomic data to identify therapeutic vulnerabilities. In our study, differentially expressed gene (DEG) analysis and KEGG pathway enrichment revealed activation of the RAS/MAPK, PI3K/AKT/mTOR, and PD-L1 signaling pathways as potential therapeutic targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Whole-exome sequencing identified a \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12V\u003c/em\u003e\u003c/sup\u003e driver mutation, consistent with constitutive activation of MAPK signaling \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, providing a mechanistic basis for sensitivity to pathway-specific inhibitors. Functional validation using targeted therapies confirmed that drug responses were in strong concordance with molecular profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), highlighting the predictive value of PDO-based pharmacotyping.\u003c/p\u003e \u003cp\u003eIntratumoral heterogeneity (ITH) presents a major barrier to achieving durable therapeutic responses \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Our multi-regional sampling approach captured spatial heterogeneity within the tumor, reflected in differential drug sensitivity across UrC PDOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e). While dependency on the MAPK pathway was predominant, subpopulations exhibiting resistance via pathway-independent mechanisms were also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings emphasize the importance of integrating spatially resolved tumor sampling with PDO pharmacotyping to inform more comprehensive and effective therapeutic strategies.\u003c/p\u003e \u003cp\u003eImmune checkpoint blockade targeting PD-1/PD-L1 is emerging as a promising therapeutic approach, particularly in tumors with microsatellite instability(MSI) status \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The PDOs in our study exhibited dMMR/MSI features, including loss of PMS2 and elevated PD-L1 expression. Mechanistically, MSI tumors accumulate neoantigens that enhance immunogenicity and sensitize tumors to immune checkpoint inhibitors \u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Consistent with this, \u003cem\u003ein vitro\u003c/em\u003e drug assays demonstrated that PD-L1 blockade via atezolizumab augmented T cell-mediated cytotoxicity. These results align with recent clinical reports, including a case of stable disease in a UrC patient treated with atezolizumab \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and broader evidence supporting the efficacy of PD-1/PD-L1 inhibitors in MSI tumors. Overall, our data underscore the potential of combining histological, genomic, and transcriptomic insights with functional drug screening to advance precision medicine in UrC.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, the small sample size reflects the rarity of UrC, which limits the statistical power and generalizability of the findings. Second, while our PDO-based platform identifies promising therapeutic candidates, clinical validation through prospective trials\u0026mdash;particularly for novel chemotherapeutics, targeted agents, and immune checkpoint inhibitors\u0026mdash;is essential. Third, current PDO models do not incorporate immune components, limiting their utility for evaluating immunotherapy. The development of tumor\u0026ndash;immune co-culture systems will be crucial to overcome this limitation. Finally, although drug sensitivity findings are compelling, they have not yet been translated into clinical treatment decisions. Interventional studies are needed to confirm their clinical utility and drive bench-to-bedside translation. To address these challenges, we are launching a longitudinal biospecimen collection and functional validation pipeline to extend this research and enable future clinical implementation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary,UrC PDOs reproduce the phenotypic and molecular features of their parental tumors, capturing intratumoral heterogeneity. Thus, they serve as a valuable platform for reflecting treatment responses, investigating resistance mechanisms, and developing personalized therapeutic regimens.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe thank the patients and their families for donating tumor samples and the surgical teams for their collaboration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (No. 82588201 and 82341005).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL., N.Z., and X.Y. supervised this study. K.Z., performed experiments. K.Z. and Z.Z. collected the samples and clinical information. X.L. performed genomic analyses and interpreted the analysis results. K.Z., drafted the manuscript. R.L. and N.Z. revised the manuscript. All authors commented on the manuscript and approved this final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the Genome Sequence Archive for Human \u0026nbsp;PRJCA053683/HRA015623 repository, (https://ngdc.cncb.ac.cn/gsa-human/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFootnote\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConflicts of Interest:\u003c/em\u003e These authors have no conflicts of interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared no competing interest.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchubert, G. E., Pavkovic, M. B. \u0026amp; Bethke-Bed\u0026uuml;rftig, B. A. Tubular urachal remnants in adult bladders. \u003cem\u003eJ Urol\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 40-42 (1982). https://doi.org/10.1016/s0022-5347(17)53595-8\u003c/li\u003e\n\u003cli\u003ePaner, G. P., Lopez-Beltran, A., Sirohi, D. \u0026amp; Amin, M. B. Updates in the Pathologic Diagnosis and Classification of Epithelial Neoplasms of Urachal Origin. \u003cem\u003eAdv Anat Pathol\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 71-83 (2016). https://doi.org/10.1097/pap.0000000000000110\u003c/li\u003e\n\u003cli\u003eLoizzo, D.\u003cem\u003e et al.\u003c/em\u003e Current Management of Urachal Carcinoma: An Evidence-based Guide for Clinical Practice. \u003cem\u003eEur Urol Open Sci\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 1-6 (2022). https://doi.org/10.1016/j.euros.2022.02.009\u003c/li\u003e\n\u003cli\u003eSzarvas, T.\u003cem\u003e et al.\u003c/em\u003e Clinical, prognostic, and therapeutic aspects of urachal carcinoma-A comprehensive review with meta-analysis of 1,010 cases. \u003cem\u003eUrol Oncol\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 388-398 (2016). https://doi.org/10.1016/j.urolonc.2016.04.012\u003c/li\u003e\n\u003cli\u003eHamilou, Z.\u003cem\u003e et al.\u003c/em\u003e Management of urachal cancer: A consensus statement by the Canadian Urological Association and Genitourinary Medical Oncologists of Canada. \u003cem\u003eCan Urol Assoc J\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, E57-e64 (2020). https://doi.org/10.5489/cuaj.5946\u003c/li\u003e\n\u003cli\u003eYazawa, S.\u003cem\u003e et al.\u003c/em\u003e Surgical and chemotherapeutic options for urachal carcinoma: report of ten cases and literature review. \u003cem\u003eUrol Int\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 209-214 (2012). https://doi.org/10.1159/000334414\u003c/li\u003e\n\u003cli\u003eSiefker-Radtke, A. O.\u003cem\u003e et al.\u003c/em\u003e Multimodality management of urachal carcinoma: the M. D. Anderson Cancer Center experience. \u003cem\u003eJ Urol\u003c/em\u003e \u003cstrong\u003e169\u003c/strong\u003e, 1295-1298 (2003). https://doi.org/10.1097/01.ju.0000054646.49381.01\u003c/li\u003e\n\u003cli\u003eGalsky, M. D.\u003cem\u003e et al.\u003c/em\u003e Prospective trial of ifosfamide, paclitaxel, and cisplatin in patients with advanced non-transitional cell carcinoma of the urothelial tract. \u003cem\u003eUrology\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 255-259 (2007). https://doi.org/10.1016/j.urology.2006.10.029\u003c/li\u003e\n\u003cli\u003eCollins, D. C.\u003cem\u003e et al.\u003c/em\u003e National Incidence, Management and Survival of Urachal Carcinoma. \u003cem\u003eRare Tumors\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 6257 (2016). https://doi.org/10.4081/rt.2016.6257\u003c/li\u003e\n\u003cli\u003eLee, S. H.\u003cem\u003e et al.\u003c/em\u003e Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e173\u003c/strong\u003e, 515-528.e517 (2018). https://doi.org/10.1016/j.cell.2018.03.017\u003c/li\u003e\n\u003cli\u003eKopper, O.\u003cem\u003e et al.\u003c/em\u003e An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 838-849 (2019). https://doi.org/10.1038/s41591-019-0422-6\u003c/li\u003e\n\u003cli\u003eVlachogiannis, G.\u003cem\u003e et al.\u003c/em\u003e Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e359\u003c/strong\u003e, 920-926 (2018). https://doi.org/10.1126/science.aao2774\u003c/li\u003e\n\u003cli\u003eJacob, F.\u003cem\u003e et al.\u003c/em\u003e A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e180\u003c/strong\u003e, 188-204.e122 (2020). https://doi.org/10.1016/j.cell.2019.11.036\u003c/li\u003e\n\u003cli\u003eYan, H. H. N.\u003cem\u003e et al.\u003c/em\u003e A Comprehensive Human Gastric Cancer Organoid Biobank Captures Tumor Subtype Heterogeneity and Enables Therapeutic Screening. \u003cem\u003eCell Stem Cell\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 882-897.e811 (2018). https://doi.org/10.1016/j.stem.2018.09.016\u003c/li\u003e\n\u003cli\u003eMullenders, J.\u003cem\u003e et al.\u003c/em\u003e Mouse and human urothelial cancer organoids: A tool for bladder cancer research. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e116\u003c/strong\u003e, 4567-4574 (2019). https://doi.org/10.1073/pnas.1803595116\u003c/li\u003e\n\u003cli\u003eXue, R.\u003cem\u003e et al.\u003c/em\u003e Genomic and Transcriptomic Profiling of Combined Hepatocellular and Intrahepatic Cholangiocarcinoma Reveals Distinct Molecular Subtypes. \u003cem\u003eCancer Cell\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 932-947.e938 (2019). https://doi.org/10.1016/j.ccell.2019.04.007\u003c/li\u003e\n\u003cli\u003eKanehisa, M., Sato, Y., Kawashima, M., Furumichi, M. \u0026amp; Tanabe, M. KEGG as a reference resource for gene and protein annotation. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, D457-462 (2016). https://doi.org/10.1093/nar/gkv1070\u003c/li\u003e\n\u003cli\u003eOgata, H.\u003cem\u003e et al.\u003c/em\u003e KEGG: Kyoto Encyclopedia of Genes and Genomes. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 29-34 (1999). https://doi.org/10.1093/nar/27.1.29\u003c/li\u003e\n\u003cli\u003eKardos, J.\u003cem\u003e et al.\u003c/em\u003e Comprehensive Molecular Characterization of Urachal Adenocarcinoma Reveals Commonalities With Colorectal Cancer, Including a Hypermutable Phenotype. \u003cem\u003eJCO Precis Oncol\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e (2017). https://doi.org/10.1200/po.17.00027\u003c/li\u003e\n\u003cli\u003eCheung, K. J., Gabrielson, E., Werb, Z. \u0026amp; Ewald, A. J. Collective invasion in breast cancer requires a conserved basal epithelial program. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e155\u003c/strong\u003e, 1639-1651 (2013). https://doi.org/10.1016/j.cell.2013.11.029\u003c/li\u003e\n\u003cli\u003ePadmanaban, V.\u003cem\u003e et al.\u003c/em\u003e E-cadherin is required for metastasis in multiple models of breast cancer. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e573\u003c/strong\u003e, 439-444 (2019). https://doi.org/10.1038/s41586-019-1526-3\u003c/li\u003e\n\u003cli\u003eLambert, A. W. \u0026amp; Weinberg, R. A. Linking EMT programmes to normal and neoplastic epithelial stem cells. \u003cem\u003eNat Rev Cancer\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 325-338 (2021). https://doi.org/10.1038/s41568-021-00332-6\u003c/li\u003e\n\u003cli\u003ePastushenko, I. \u0026amp; Blanpain, C. EMT Transition States during Tumor Progression and Metastasis. \u003cem\u003eTrends Cell Biol\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 212-226 (2019). https://doi.org/10.1016/j.tcb.2018.12.001\u003c/li\u003e\n\u003cli\u003eNieto, M. A., Huang, R. Y., Jackson, R. A. \u0026amp; Thiery, J. P. EMT: 2016. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e166\u003c/strong\u003e, 21-45 (2016). https://doi.org/10.1016/j.cell.2016.06.028\u003c/li\u003e\n\u003cli\u003evan de Wetering, M.\u003cem\u003e et al.\u003c/em\u003e Prospective derivation of a living organoid biobank of colorectal cancer patients. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e161\u003c/strong\u003e, 933-945 (2015). https://doi.org/10.1016/j.cell.2015.03.053\u003c/li\u003e\n\u003cli\u003eZhao, Y.\u003cem\u003e et al.\u003c/em\u003e Personalized drug screening using patient-derived organoid and its clinical relevance in gastric cancer. \u003cem\u003eCell Rep Med\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 101627 (2024). https://doi.org/10.1016/j.xcrm.2024.101627\u003c/li\u003e\n\u003cli\u003eChou, C. L.\u003cem\u003e et al.\u003c/em\u003e PCSK1 Overexpression in Rectal Cancer Correlates with Poor Response to Preoperative Chemoradiotherapy and Prognosis. \u003cem\u003eOnco Targets Ther\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 3141-3150 (2020). https://doi.org/10.2147/ott.S243750\u003c/li\u003e\n\u003cli\u003eSinghal, A., Li, B. T. \u0026amp; O\u0026apos;Reilly, E. M. Targeting KRAS in cancer. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 969-983 (2024). https://doi.org/10.1038/s41591-024-02903-0\u003c/li\u003e\n\u003cli\u003eDagogo-Jack, I. \u0026amp; Shaw, A. T. Tumour heterogeneity and resistance to cancer therapies. \u003cem\u003eNat Rev Clin Oncol\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 81-94 (2018). https://doi.org/10.1038/nrclinonc.2017.166\u003c/li\u003e\n\u003cli\u003eLe, D. T.\u003cem\u003e et al.\u003c/em\u003e PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e372\u003c/strong\u003e, 2509-2520 (2015). https://doi.org/10.1056/NEJMoa1500596\u003c/li\u003e\n\u003cli\u003eGiannakis, M.\u003cem\u003e et al.\u003c/em\u003e Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 857-865 (2016). https://doi.org/10.1016/j.celrep.2016.03.075\u003c/li\u003e\n\u003cli\u003eLlosa, N. J.\u003cem\u003e et al.\u003c/em\u003e The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. \u003cem\u003eCancer Discov\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 43-51 (2015). https://doi.org/10.1158/2159-8290.Cd-14-0863\u003c/li\u003e\n\u003cli\u003eSaeterdal, I.\u003cem\u003e et al.\u003c/em\u003e Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, 13255-13260 (2001). https://doi.org/10.1073/pnas.231326898 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Urachal cancer, organoid, drug screening, personalized medicine","lastPublishedDoi":"10.21203/rs.3.rs-8245003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8245003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUrachal cancer (UrC) is a rare, aggressive malignancy typically diagnosed at advanced stages, where systemic treatment becomes necessary. However, cytotoxic chemotherapy offers limited efficacy, and prospective clinical trials are exceedingly difficult due to the rarity of the disease. Thus, robust \u003cem\u003ein vitro\u003c/em\u003e models are urgently needed to support precision medicine approaches for UrC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFresh UrC tumor samples were collected from patients undergoing en bloc resection and cultured to generate PDOs. These organoids were subjected to drug screening using standard chemotherapeutic agents. Whole-exome sequencing (WES) and RNA sequencing (RNA-seq) were conducted to compare the molecular profiles of the PDOs with their corresponding parental tumors. Associations between drug responses and genomic/transcriptomic features were analyzed. Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used for statistical assessment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe established UrC PDOs faithfully reproduced the genomic and transcriptomic landscapes of the original tumors, including intratumoral heterogeneity, and demonstrated consistent drug response profiles. Molecular characterization further revealed actionable targets within the RAS/MAPK and PI3K/AKT/mTOR pathways, as well as immune-related targets such as PD-L1. These findings highlight the utility of PDOs in modeling rare cancers and guiding personalized therapeutic strategies.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eUrC PDOs recapitulate the phenotypic and molecular features of their parental tumors, capturing critical heterogeneity. As such, they represent a valuable platform for reflecting treatment responses, investigating resistance mechanisms, and developing individualized therapeutic regimens.\u003c/p\u003e","manuscriptTitle":"Patient-derived urachal cancer organoids for personalized drug screening","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 11:22:49","doi":"10.21203/rs.3.rs-8245003/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9089d561-849b-4b40-976d-2bf50360872e","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61226864,"name":"Biological sciences/Cancer"},{"id":61226865,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":61226866,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-03-04T12:55:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 11:22:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8245003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8245003","identity":"rs-8245003","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}