Abstract Tumor plasticity and cellular heterogeneity have long been recognized as major contributors to the limited success of cancer therapies, underscoring the need for more physiologically relevant preclinical models. Patient-derived organoids (PDOs) offer a three-dimensional, heterogeneous platform that better reflects tumor complexity compared to traditional cell lines or animal models. Their adoption in drug development pipelines and avatar clinical trials has grown significantly in recent years. In this study, tumor PDOs were characterized using single-cell RNA sequencing, whole-exome sequencing, and sub-lethal therapeutic exposure to assess intra- and inter-patient heterogeneity and adaptive dynamics. PDOs from different donors exhibited patient-specific transcriptional signatures while retaining stem-like features. Mutational landscapes evolved over time, with variants being retained, lost, or acquired relative to the original tissue. Notably, subpopulations persisted through treatment and gave rise to distinct clonal cultures with unique transcriptional profiles upon drug withdrawal. These findings confirm that tumor PDOs preserve cellular heterogeneity and exhibit dynamic adaptability, making them powerful tools for investigating cancer evolution, drug resistance, and mutation dynamics. Inter-patient variability highlights the need for personalized therapeutic strategies, while intra-patient heterogeneity positions PDOs as ideal models for studying complex cellular interactions and mechanisms of pharmacological persistence. Citation Format: Javier Frias Aldeguer, Jasmin Pourfarzad, Alessandro Gregory, Farzin Pourfarzad, Rene Overmeer, Robert G. Vries, Sylvia F. Boj. Dynamic adaptation and heterogeneity in tumor patient-derived organoids: Insights into cancer evolution and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 699.

Abstract Tumor heterogeneity—both within individual tumors and among different patients—poses a significant challenge for effective cancer treatment. To advance precision oncology, preclinical models that capture this complexity are essential. Patient-derived organoids (PDOs) provide a physiologically relevant platform that maintains the architecture and diversity of the original tumor, making them ideal for studying heterogeneity and patient-specific biology. In this study, we established and characterized PDO biobanks from colorectal and bladder cancers to evaluate their ability to represent intra-tumor heterogeneity and patient-specific molecular signatures. We profiled organoids using whole-exome sequencing and bulk RNA sequencing to assess their genomic and transcriptomic fidelity. Our analyses revealed that PDOs retain key mutational profiles and transcriptional programs of the original tumors, including subtype-specific signatures. Importantly, we observed distinct cellular subpopulations within individual PDO cultures that reflect intra-tumor diversity, including stem-like and differentiated phenotypes. These features persisted over time, confirming the stability of heterogeneity in vitro. Across the biobank, PDOs captured the spectrum of molecular subtypes present in colorectal and bladder cancers, enabling subtype-specific drug testing and biomarker discovery. This diversity highlights the potential of PDO platforms to model patient variability and inform personalized therapeutic strategies. Our findings demonstrate that PDOs are not only accurate representations of patient tumors but also dynamic systems that preserve heterogeneity at multiple levels. By integrating genomic and transcriptomic profiling with functional assays, PDO biobanks provide a powerful resource for studying tumor complexity and accelerating precision medicine. Citation Format: Rene Overmeer, Farzin Pourfarzad, Alejandra Hernandez Segura, Merel Derksen, Carla Verissimo, Robert G. Vries, Sylvia F. Boj. Patient-derived organoid biobanks preserve tumor heterogeneity and molecular signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 701.

Abstract Antibody-drug conjugates (ADCs) represent an emerging class of cancer therapies designed to address the limitations of traditional chemotherapy, particularly the toxicity that arises from healthy cells being exposed to cytotoxic agents. ADCs combine a target-specific monoclonal antibody with a cytotoxic payload, enabling selective delivery to cancer cells and significantly reducing adverse effects. HUB Patient-Derived Organoids (PDOs) are advanced 3D in vitro models derived from adult stem cells that faithfully replicate the architecture, genetics, and physiology of original patient tissues. By preserving patient-specific genetic and phenotypic traits, including surface marker expression, HUB has established a comprehensive and well-characterized biobank of tumor-derived PDOs. This biobank provides a powerful platform for translational research, drug discovery, and the development of targeted therapies. In this study, we present an organoid-based, image-based internalization assay that complements standard Cell Titer-Glo viability assays by enabling direct visualization of ADC internalization. This assay facilitates a deeper exploration of the mechanisms of action of ADCs. We pre-selected tumor-derived organoids based on HER2 expression, which was assessed using flow cytometry. After treating the organoids with FDA-approved HER2-targeting ADCs, we utilized the internalization assay to support the viability data and to define key parameters such as the timing of action, the effect of the payload, and whether cell death resulted from ADC internalization. Overall, these results reinforce the value of patient-derived organoids as a physiologically relevant preclinical platform for ADC development. They also highlight the suitability of the described image-based assay for investigating critical stages of ADC mechanisms, including internalization and payload activity. Citation Format: Daniele Mori, Javier Frias Aldeguer, Rene Overmeer, Sylvia F. Boj, . An image-based approach to visualize ADC internalization and cytotoxicity in patient-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5702.

Abstract The Kirsten rat sarcoma (KRAS) gene is a proto-oncogene frequently mutated in colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC), and is often associated with poor clinical outcomes. KRAS has long been considered an elusive and historically “undruggable” target in cancer therapy. However, recent breakthroughs, including the clinical approval of KRAS^G12C inhibitors such as Sotorasib and Adagrasib, have paved the way for novel compounds targeting KRAS^G12C and other KRAS mutations. The development of KRAS inhibitors can be accelerated and de-risked by using patient-relevant preclinical models that enable efficacy screening and safety assessment. Patient-derived organoids (PDOs), or HUB Organoids®, are advanced 3D models derived from adult stem cells of normal and malignant epithelial tissues, including colon, pancreas, and lung. They recapitulate the molecular heterogeneity, morphology, and functionality of the original tissue, accurately reflecting patient-specific responses HUB Organoids support high-throughput screening, providing a powerful, scalable platform for testing drug efficacy, safety, and resistance mechanisms. Their characterized genomic and transcriptomic profiles allow drug responses to be linked to molecular features, enabling patient stratification. In this study We demonstrate the suitability of our PDO-based platform for KRAS inhibitor screening within six weeks. Eight KRAS inhibitors at various clinical development stages were tested on a panel of over 20 PDOs (CRC, PDAC, and NSCLC) harbouring KRAS mutations (G12C, G12D, Q61R, G13C, G12V, and G12S) using an ATP-based viability assay. Responses varied across PDOs. Notably, G12C and G12D mutants showed selective sensitivity to KRAS^G12C and KRAS^G12D inhibitors, respectively. Resistance to these inhibitors was mitigated by combining them with an EGFR inhibitor, consistent with EGF-driven resistance mechanisms. Additionally, KRAS PDO screening enables assessment of drug synergy and safety in normal tissue-derived PDOs. In conclusion, HUB’s KRAS PDO screening platform offers a time-efficient, mutation-specific approach for evaluating compound specificity, potency, and combination strategies for KRAS-targeted therapies. Citation Format: Merel Derksen, Yasmine Abouleila, Mariana Martins Costa Silva, Gerben ten Hag, Rene Overmeer, Farzin Pourfarzad, Fabian Stavenuiter, Robert G. Vries, Sylvia F. Boj, . A patient-derived organoid screening platform for evaluating KRAS inhibitor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3187.

Background & Aims Hepatitis B virus (HBV) drives hepatocellular carcinoma in part through the activity of its X protein (HBx), yet the mechanisms by which HBx alters hepatocyte function remain incompletely understood. Progress has been limited by the lack of relevant human models that support controlled HBx expression in mature hepatocytes. Here, we use an improved hepatocyte-like organoid (HLO) platform that supports enhanced hepatocyte maturation to investigate HBx function in a differentiated hepatocyte context. Methods Adult stem cell-derived HLOs were differentiated using an optimized protocol to generate hepatocyte-like cells with enhanced maturation and transcriptional similarity to primary liver tissue. HBx function was interrogated using both cognate promoter-driven expression and doxycycline-inducible systems across multiple donor-derived organoid lines. Transcriptomic, pathway, and single-cell imaging analyses were performed to assess the impact of HBx expression on hepatocytes. Results HBx expression consistently suppressed apoptosis-associated transcripts and reduced expression of core hepatocyte identity genes, including CYP3A4. Pathway analysis revealed downregulation of liver-specific functions, including metabolism, detoxification, complement, and coagulation. At the single-cell level, higher HBx expression was associated with reduced caspase 3/7 activation following apoptotic challenge and decreased hepatocyte marker expression. Functionally, HBx expression increased resistance to apoptosis and enhanced the ability of differentiated hepatocyte-like cells to revert to a proliferative, less differentiated state. Conclusions HBx expression in differentiated human liver organoids reduces apoptosis and impairs hepatocyte identity, consistently across donors and expression systems. These findings support a model in which HBx promotes a survival-permissive less differentiated state that may contribute to early HBV-driven tumorigenesis. This HLO platform provides a relevant system to dissect HBV-host interactions and reveals a mechanism by which HBV may prime the liver for malignant transformation.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.

Abstract Patient-derived organoids (PDOs), or HUB Organoids®, are advanced 3D models generated from adult stem cells of normal and malignant epithelial tissues and stored in high-quality biobanks to ensure reproducibility. HUB Organoids faithfully recapitulate the physiology, molecular heterogeneity, and morphological and functional characteristics of the original tissue, effectively mimicking patient response and bridging the gap between laboratory research and clinical application—bringing the “patient into the lab.” The rapid development of Petosemtamab (MCLA-158) exemplifies the value of organoid technology in drug development, as this target could not have been identified using conventional 2D models. Further supporting this, recent publications highlight the ability of organoids to predict treatment response in metastatic colorectal cancer. Here, we present our capabilities for medium- to high-throughput screening, enabling the evaluation of over 6,000 compounds across multiple organoid models in parallel. Available readouts include plate reader-based viability assays (CellTiter-Glo 3D) and imaging-based assays (CyQuant). Beyond initial large-scale screening, rapid iterations of follow-up structure-activity relationship (SAR) studies or expanded screening across diverse patient models are facilitated by the precision and reproducibility of our platform. In summary, we offer a robust, clinically relevant, and cost-effective platform to support drug development from early-stage compound screening to advanced validation studies. Citation Format: Rene Overmeer, Mariana Martins Costa Silva, Gerben ten Hag, Mayke Doorn, Yasmine Abouleila, Ricardo Korporaal, Francisco Morales Rodriguez, Merel Derksen, Carla Verissimo, Robert G. Vries, Sylvia F. Boj. A robust organoid-based platform for high-throughput screening and drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6404.

Abstract Therapy resistance in colorectal cancer remains a major clinical challenge. Despite standard-of-care chemotherapies such as FOLFOX and FOLFIRI, tumors frequently develop resistance, leading to treatment failure. A growing body of research implicates drug-tolerant persister (DTP) cells—a reversible, slow-growing population that survives initial therapy—as a critical step in resistance development. These persister cells create a window for cancer to acquire additional adaptations—such as mutations and alternative signaling pathway activation—ultimately leading to irreversible resistance. Understanding this transition from persistence to full resistance is essential for developing therapies that can prevent relapse. To investigate these mechanisms in a clinically relevant context, we developed patient-derived organoid (PDO) models of colorectal cancer capable of surviving FOLFOX or FOLFIRI treatment. These organoids retain the heterogeneity and architecture of the original tumors, providing a physiologically faithful system to study persistence. Because chemotherapy impact varies by mechanism of action, standard assays may not fully capture response diversity. We therefore optimized viability readouts to measure treatment effects across the entire organoid population, capturing both cytotoxicity and partial survival. Using these persistent PDOs, we performed whole-exome sequencing alongside bulk and single-cell RNA sequencing to map the molecular features associated with the DTP state and subsequent resistance. This approach revealed pathways and cellular programs that enable tumor cells to tolerate chemotherapy, highlighting potential targets for overcoming resistance. By modeling clinically relevant persistence, these PDOs offer a platform to test interventions that eliminate persister populations before they evolve into fully resistant tumors. Citation Format: Yasmine Abouleila,Timo Voskuilen,Mayke Doorn,Roel Verkerk,Jasmin Pourfarzad,Joris Maas,Robert G. Vries,Sylvia F. Boj. Modeling chemotherapy persistence in colorectal cancer using patient-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3119.

Abstract Recent advances in cancer immunotherapy have significantly improved patient survival across multiple indications. As new treatment strategies and druggable targets emerge, the number of patients eligible for immunotherapy continues to grow. However, translating promising preclinical findings into clinical success remains challenging, as conventional 2D cancer models offer limited predictive value. We developed an innovative alternative based on the ability of adult stem cells to form three-dimensional organotypic structures within an extracellular matrix. Patient-derived organoids, generated from normal and malignant tissues and stored in high-quality biobanks, yield highly reproducible results. HUB Organoids faithfully recapitulate the complexity of parental tissue, including molecular heterogeneity and morphological and functional traits. Over the last decade, bispecific antibodies (bsAbs) have gained significant attention in cancer treatment, leading to several approved therapies. Bispecific T-cell engagers (BiTEs), a subclass of bsAbs, bring T cells into close proximity with cancer cells and promote immunologic memory, enabling the immune system to more effectively recognize and eliminate tumor cells. To de-risk drug development and accelerate clinical translation, we developed an assay in which labeled tumor organoids are co-cultured with labeled PBMCs and exposed to BiTEs to assess immune cell-mediated cytotoxicity. Fluorescent dyes enable real-time evaluation of cytotoxic activity via high-resolution microscopy, complemented by cytokine secretion analysis to confirm immune cell activation. HUB’s organoid co-culture system is robust and reproducible, providing a dynamic window to evaluate the activity of multiple BiTEs. Organoid-based cytotoxicity assays accurately reflect BiTE-mediated T cell killing, demonstrating that our model captures the expected mechanism of action. This platform offers a powerful tool for developing and validating cancer immunotherapies, helping to de-risk novel strategies through patient-derived models. Citation Format: Cesar Oyarce, Sumeyra Mucuk, Javier Frias Aldeguer, Timo Voskuilen, Merel Derksen, Farzin Pourfarzad, Robert G. Vries, Sylvia F. Boj, . Organoid-T cell co-cultures: A robust platform for bispecific antibody immunotherapy development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5544.

Patient-derived-organoids (PDOs) are valuable tools for predicting individual responses to cancer treatments. However, current screening methods require large numbers of PDOs, resulting in long turnaround times and limiting clinical use. This study aimed to streamline the process by automating PDO seeding with the Yamaha Cell Handler™ (YCH). We optimized the YCH to pick and place up to ten PDOs per well, significantly reducing sample requirements compared to conventional methods. Assay optimization included evaluating seeding densities, devices, readouts, and measurement techniques. We validated the miniaturized assay by comparing it to standard screens and correlating organoid responses with patient outcomes. Our proof-of-concept demonstrated that mCRC PDOs respond to chemotherapy and targeted treatments in a way that closely matches results from traditional assays. This miniaturized automated platform enables efficient, high-quality drug screening with fewer cells, offering promising potential for faster, personalized cancer treatment predictions in clinical settings.

Background: Nonsense variants in CFTR account for ~10% of cystic fibrosis (CF) variants and cannot be treated with approved CFTR modulators. Translational readthrough agents such as ELX-02 offer a potential therapeutic strategy, but clinical trials evaluated mainly in G542X CFTR nonsense variant and underlined limited efficacy. This study aimed to evaluate ELX-02-mediated CFTR rescue across a broad range of nonsense variants using patient-derived intestinal organoids (PDIOs) to define variant-specific determinants of readthrough efficacy and assess its potential across a genetically diverse CF population. Method: The ex vivo response to ELX-02 was assessed in 206 PDIOs carrying heterogeneous nonsense variants. CFTR function was quantified using forskolin-induced swelling (FIS) assay after 48-hour exposure to ELX-02. Responses were analysed by genotype and stop codon identity, with secondary validation performed in a selected subset of PDIOs (n = 60). Results: ELX-02 mediated CFTR rescue varied markedly, ranging from responses approaching those observed with approved CFTR modulators (LUM/IVA) to responses at or below detection limit. Overall, maximal responses were modest and at the lower end of the functional range for CFTR modulators. Rescue was dose-dependent and higher in PDIOs carrying two nonsense variants when compared with PDIOs carrying a single nonsense variant combined with a residual or minimal function variant. Nonsense variants in nucleotide-binding domain 1, including G542X, S466X, G550X and R553X, showed relatively higher responsiveness. Conclusion: ELX-02 induces limited and highly heterogeneous CFTR rescue across nonsense variants. PDIO-based functional screening provides a framework to guide patient selection and stratification for future readthrough therapy trials.

Abstract Patients with relapsed or metastatic colorectal cancer (CRC) face limited treatment options, significant side effects, and prolonged delays in identifying effective therapies. Patient-derived organoids (PDOs; HUB Organoids®) provide a clinically relevant platform that faithfully mirrors individual tumour biology, enabling personalised drug testing. However, conventional drug screening formats typically require several hundred organoids per well, limiting the feasibility of using PDOs to guide real-time treatment decisions at diagnosis or relapse. Translational speed is critical: in metastatic CRC, there is a narrow window to select effective therapy before disease progression or treatment-related toxicity occurs. Traditional preclinical models often take weeks to months, which is too slow to inform immediate patient care. To address this limitation, we developed an automated organoid-handling workflow using the Yamaha CELL HANDLER™ system, enabling precise transfer and image-based quantification while requiring far fewer organoids per well. Miniaturisation reduced input material by 96% (from 250 to 10 PDOs per well) compared to conventional screening. Drug sensitivity of PDOs measured using the miniaturised assay closely mirrored that of conventional screening (R=0.67-0.85, p<0.03). PDO responses in the miniaturised assay also correlated with patient outcomes, including progression-free survival (R = -0.85, p < 0.01). By combining automation, miniaturisation, and quantitative readouts, this platform preserves the predictive power of PDOs, drastically reduces the need for organoids, and shortens turnaround time. Citation Format: Yasmine Abouleila, Roel Verkerk, Mayke Doorn, Timo Voskuilen, Gakuro Harada, Masahiko Watanabe, Lidwien Smabers, Hideaki Kyan, Takahiko Kumagai, Yuichi Hikichi, Rene Overmeer, Jeanine Roodhart, Kiyotaka Matsuno, Carla S. Verissimo, Robert G. Vries, Sylvia F. Boj. Preserving predictive power with minimal PDOs: Accelerated drug testing for personalized therapy in metastatic CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2515.

Schematic overview of the study. Patients underwent a biopsy of a metastasis for PDO culture and WGS, before starting a new line of systemic treatment. Predictors of successful PDO establishment were identified: male sex, increased LDH, biopsy in academic hospitals, optimized culture conditions, and experience. A total of 42 PDOs were screened for standard-of-care treatments, and the patient received standard systemic treatment. PDO response was correlated with patient response, and the association between PDO response and survival was assessed. (Created in BioRender. Roodhart, J. [2025] https://BioRender.com/by1f1uq.)

Loss of the functional Adenomatous Polyposis Coli (APC-LOF) tumor suppressor gene represents the disease-initiating event in most colorectal cancer (CRC) cases. A newly identified dependency between PRMT5 and APC-LOF suggests that inhibiting PRMT5 may help intercept CRC. To circumvent hematological toxicities associated with orally bioavailable first-generation PRMT5 inhibitors, we aimed to limit systemic exposure after oral administration. We describe our efforts, challenges, and compound evaluation workflow resulting in gut-restricted PRMT5 inhibitors. A two-pronged approach was envisioned, consisting of (1) minimizing passive absorption, and (2) maximizing systemic clearance by incorporation of a metabolic "soft spot". This resulted in 9 and 18, displaying low absorption in preclinical species and high first-pass extraction mediated by aldehyde oxidase. 9 and 18 demonstrated in vivo colon pharmacodynamics without signs of systemic on-target toxicity, confirming gut-restriction. Administering 9 to dextran sodium sulfate (DSS)-treated polyp-bearing ApcMin/+ mice significantly reduced polyp number, indicating local treatment efficacy.

Clinical variables related to PDO establishment success: CEA, LDH, mutational status, sex, primary tumor sidedness, prior chemotherapeutic treatment, metastatic site of the biopsy, WGS success, and aggregated biopsy color (brown, pink, red, and white). CEA, carcinoembryonic antigen; mut, mutant.

Kirsten rat sarcoma (KRAS) gene is a proto-oncogene frequently mutated in various cancer types. Upon activation by growth factors such as Epidermal Growth Factor (EGF), KRAS initiates signaling pathways promoting cell proliferation and survival. Activation KRAS mutations are most common in colorectal cancer (CRC), Pancreatic Ductal Adeno Carcinoma (PDAC) and Non-Small Cell Lung Cancer (NSCLC) and are associated with worse disease outcomes. Though KRAS is an interesting target for cancer treatment, the development of specific KRAS inhibitors has, for many years, been challenging due to its small and smooth binding pocket, making KRAS a challenging “druggable” protein. Recently, advances have been made in this area, with the development and approval for clinical use of the KRASG12C inhibitors (Sotorasib and Adagrasib). These developments can pave the way for the design of novel compounds targeting KRAS G12C and other KRAS mutations, which can be accelerated when developed in patient-relevant preclinical models. Patient-derived organoids (PDOs) or HUB Organoids® are generated from the adult stem cells of normal and malignant tissues of epithelial organs, including the colon, pancreas, and lung. HUB Organoids recapitulate tissue physiology, molecular heterogeneity, and morphological and functional characteristics, including mimicking patient response. Organoids are suitable for high-throughput screening and serve as a platform for testing drug efficacy and safety. In this study, we showcase the suitability of our PDO-based platform for screening the efficacy of several KRAS inhibitors in 4 weeks. Eight KRAS inhibitors in different clinical research stages were tested in a panel of over 20 PDOs (including CRC, PDAC and NSCLC) harboring different KRAS mutations (G12C, G12D, Q61R, G13C, G12V and G12S) and using an ATP-based cell viability readout. As expected, various responses were identified among the 20 PDOs. In addition, the results show specific responses of G12C and G12D mutants to KRASG12C and KRASG12D-selective inhibitors, respectively. KRASG12CPDO resistance to KRASG12C inhibitors was alleviated by combinatorial treatment with an EGFR inhibitor, which aligns with the KRASi resistance mechanism driven by EGF signaling. In conclusion, our PDO platform effectively captures the efficacy and specificity of KRAS inhibitors in models with different KRAS mutations. The HUB PDO platform is valuable for studying combinatorial treatments to overcome KRAS inhibitor resistance, highlighting the potential of PDOs in developing novel KRAS inhibitors. Merel Derksen, Mariana Silva, Gerben ten Hag, Nilofar Ehsani, Yasmine Abouleila, Vineeta Adegbenro, Annemarie Buijs, Mayke Doorn, Nikki van Dorp, Javier Frias Aldeguer, Foteini Gkogkou, Yara Hendriksen, Ricardo Korporaal, Ka Wai La, Joris Maas, Francisco Morales-Rodrigues, Daniele Mori, Sumeyra Mucuk, Cesar Oyarce, Nima Salehi, Anna Schepers, Marvin Statia, Roel Verkerk, Timo Voskuilen, Rene Overmeer, Farzin Pourfarzad, Sylvia F. Boj. A patient-derived organoid screening platform as a powerful tool to study efficacy of KRAS inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5706.

Genomic landscape of 36 tumors and/or PDOs derived from patients with mCRC. Top, microsatellite stability, tumor mutational load, tumor mutational burden, and whether the sequencing was performed on the PDOs or the tumor of origin; bottom, somatic driver mutations. MS, microsatellite status; MSI, microsatellite instability; MSS, microsatellite stability; Seq, sequencing; TML, tumor mutational load.