A Practical Guide to Developing and Troubleshooting Patient-Derived “Mini-Gut” Colorectal Organoids for Clinical Research

Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) have been approved as monotherapy and combination therapy for BRCA-mutated breast, ovarian, and prostate cancer and it is proposed that they may be effective for other BRCA-mutated cancer as well. However, recent clinical trials show inconsistent efficacy of PARPi on BRCA-mutated cancers, partially due to imperfect biomarker definition and unknown resistance mechanism. BRCA status alone may not be a good indicator for PARPi responsiveness as many proteins are involved in the homologous recombination (HR) pathway, which is why this experiment raises the question of whether time plays a role in tumor drug sensitivity. Patient-derived tumor organoids (PDTOs) can be used to study the pharmacogenomics between PARPis and HR deficient cancer. PDTOs are emerging ex vivo cancer models used for precision medicine because they harbor the mutational landscape of primary tumors. The common pipeline for organoid preclinical research includes confirming mutation profile, performing drug screening, and proposing clinically relevant candidates. The goal of this experiment was to model if the responses of patient-derived colorectal cancer organoids with BRCA gene deletion and TP53, SMAD4, and KRAS gene mutations to the PARPi talazoparib and olaparib differ depending on time.Methods: Patient sample: Colorectal: rectal cancer with BRCA1 gene deletion. Mutations in ⦁ KRAS p.Gly12Val ⦁ TP53 p.Arg175His ⦁ SMAD4 p.Arg361Cys + control: Puromycin 2 ug/mL - control: DMSO PARPi: talazoparib and olaparib In a 96 well plate, 1500 cells per well were plated in 10uL of a media/matrigel mixture and 100 uL of additional media were added to each well. The plate was put in the incubator at 37 C for 48 hours. The PARPi talazoparib and olaparib were then added after a serial dilution at max 50 uM concentration. The plates with the drugs (additional 100uL media with 2X drugs) were put in the incubator at 37 C for 120 hours. The plate for timepoint 1 was then read using the viability assay called Cell Titer Glo 3D. The next timepoint was taken after 168 hours using the same method. In addition, cell viability was measured by high content imaging which is being developed at EIPM.Results: Patient-Derived organoids were sensitive to talazoparib at hour 120 and hour 168 (IC50 1.6 and 1.9 respectively). Patient-derived organoids were not sensitive to olaparib at any time point tested and did not yield a meaningful IC50.Conclusion: The sensitivity of patient-derived colorectal cancer organoids with BRCA gene deletion and TP53, SMAD4, and KRAS gene mutations to the PARPi talazoparib and olaparib did not significantly differ depending on time. However, organoids were more sensitive to talazoparib than to olaparib. These results can guide which PARPi should be used in similar ex vivo experiments to yield meaningful results to eventually lead to successful clinical treatments for patients. Citation Format: Maria Mastropaolo, Helen Kuo, John Nguyen, Florencia Madorsky Rowdo, Jared Capuano, Jenna Moyer, M. Laura Martin, Julianne Hall, Olivier Elemento. Patient-Derived Organoids model time-dependent sensitivities to PARP inhibitors in patients with metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B55.

Introduction: Colorectal cancer is the second cause of cancer related deaths worldwide, with the poor survival outcomes attributable to the presence of metastases. To tackle this problem, (phospho)proteomics analysis enables valuable insights into changes of protein expression and signalling in cancer that can be perturbed by drugs. To study mechanisms driving metastasis and perform subsequent drug testing, patient derived organoids (PDOs) are in development as preclinical models. PDOs are obtained by 3D culture of tumour tissue ex-vivo, which enables them to retain the heterogeneity and architecture of the tumor source. To identify putative drug targets for colon cancer metastasis, we performed comparative (phospho)proteomics analysis of metastatic colon tissues and their matched PDOs. Methods: (Phospho)proteomics profiling was performed on PDOs generated from 10 patients with colon metastases to the liver, 10 matched tumour tissues and 4 normal colon mucosa. Unsupervised analysis of the (phospho)proteomic landscape was used to identify differentially expressed genes and pathways in colon metastases and their matched PDOs, compared to normal colon mucosa. We focused on phosphosites, proteins and pathways showing consistently altered expression in tumour tissues and PDOs. Kinase-substrate enrichment analysis was used to identify aberrantly activated kinases, based on the abundance of phosphorylation on the substrates. Putative drugs were selected using the consensus expression response to multiple small molecule drugs across cell lines and conditions from the Library of Integrated Network-Based Cellular Signatures (LINCS) database. Drug treatment of drug candidates was performed using the MTT Cell Proliferation Assay. Results: Using the approach described above we identified 103 differentially expressed proteins and 236 phosphosites between tumour tissues and normals, which were also detected in PDOs, with overall high correlation of t statistics (0.6 Spearman’s rho) between tumor tissues and PDOs. MYC-targets, G2M checkpoints and E2F-targets were amongst the top positively enriched pathways in common, and the LINCS analysis identified multi-kinase inhibitor Nintedanib and NFKB pathway-targeting KIN0-260 as putative drugs for upregulated proteins. The kinase CSNK2A1 was overactivated in both groups, pointing towards the use of CK2 inhibitors for drug testing. Treatment at 5 days with Nintedanib and KIN001-260 on the PDOs resulted in significant reduction of cell viability compared with 5-FU. Conclusion: Our study provides evidence that PDOs recapitulate relevant tumor features at the proteomic and phosphoproteomic levels, supporting the utility of this ex-vivo model as a tool for drug sensitivity testing for metastatic colon cancer. Citation Format: Gina Faye Boot, Federica Panebianco, John Gallon, Charlotte Kiu Yan Ng, Salvatore Piscuoglio. (Phospho)proteomics profiling of patient-derived colon cancer organoids for the discovery of putative drug targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3074.

Patient-derived functional organoids as a personalized approach for drug screening against hepatobiliary cancers.

Advanced pancreatic cancer has a dismal prognosis and current treatment options (FOLFIRINOX, Gemcitabine/nab-paclitaxel [GnP]) are associated with toxicity. Although some patients achieve partial responses, most progress rapidly and become chemorefractory. While RNA subtypes, genomic alterations, and protein biomarkers have prognostic value, predictive biomarkers to guide therapy are needed. Patient-derived organoids (PDOs) are an increasingly popular model for predicting patient responses to standard-of-care therapy and investigating personalized therapy options. We present a novel biobank of 42 PDOs and drug profiling data with 5 standard of care agents and 3 kinase inhibitors. Tissue was processed from n=103 biopsies from 97 patients with a confirmed pathologic diagnosis of advanced (Stage III-IV) pancreatic ductal adenocarcinoma who presented to a single Canadian tertiary care centre between 2017-2020. Matched WGS was available in all cases. Our PDO generation success was 42/103 (41%). We observed a trend towards decreased establishment in tumors that were KRAS WT, TP53 WT, or had higher HRDetect scores. Conversely, polyploidy, SMAD4 WT, and major imbalances in mutant KRAS were associated with successful PDO establishment. These associations were not statistically significant after multiple comparisons correction, but suggest selection for success with more aggressive tumors. Drug profiling was performed on all 42 PDOs with the individual agents of FOLFIRINOX (5-FU, irinotecan, oxaliplatin), GnP (gemcitabine, paclitaxel), and three targeted agents (afatinib, trametinib, and talazoparib). Combination testing was also performed for gemcitabine + paclitaxel. Drug responses were measured through both viability and growth rate (GRMetrics). We found that GRMetrics minimized effects from different PDO growth rates. Matched clinical data were available for 23 patients who received FOLFIRINOX, 11 patients who received GnP, and one patient who received gemcitabine monotherapy. Similar to previous studies, we found that in vitro PDO responses to 5-FU, irinotecan, and GnP were correlated with patient responses based on RECIST criteria. Interestingly, and similar to previous reports in colorectal cancer PDOs, we found that oxaliplatin responses were not predictive of RECIST response. As expected, PDOs were resistant to afatinib (EGFRi), which reflects negative clinical trials, and may also be masked by use of EGF in growth media. A range of responses to trametinib (MEKi) were seen but were not correlated with KRAS allelic dosage. A similar range of response was seen to talazoparib (PARPi), but did not correlate with oxaliplatin response or HRDetect scores. In summary, we have established a novel biobank of PDOs from advanced pancreatic cancer patients. Notably, PDOs were less likely to establish from tumors that were KRAS WT or HR-deficient, even though these patients are likely to benefit from targeted approaches. Further investigation is required to develop PDO use in clinical drug prediction and drug discovery. Citation Format: Irene Y. Xie, Laura Tamblyn, Karen Ng, Eugenia Flores-Figueroa, Julie M. Wilson, Gun Ho Jang, Amy X. Zhang, Stephanie Ramotar, Anna Dodd, Nikolina Radulovich, Jennifer J. Knox, Grainne M. O'Kane, Steven Gallinger, Faiyaz Notta. Establishment of a novel living biobank of patient-derived pancreatic cancer organoids with genomic and drug response characterization [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-077.

Although right‐sided colorectal cancer (CRC) shows a worse prognosis than left‐sided CRC, the underlying mechanism remains unclear. We established patient‐derived organoids (PDOs) from left‐ and right‐sided CRCs and directly compared cell proliferation and invasion capability between them. We then analyzed the expression of numerous genes in signal transduction pathways to clarify the mechanism of the differential prognosis. Cell proliferation activity and invasion capability in right‐sided cancer PDOs were significantly higher than in left‐sided cancer PDOs and normal PDOs, as revealed by Cell Titer Glo and transwell assays, respectively. We then used quantitative RT‐PCR to compare 184 genes in 30 pathways among right‐sided and left‐sided cancer and normal PDOs and found that the TIMP1 mRNA level was highest in right‐sided PDOs. TIMP1 protein levels were upregulated in right‐sided PDOs compared with normal PDOs but was downregulated in left‐sided PDOs. TIMP1 knockdown with shRNA significantly decreased cell proliferation activity and invasion capability in right‐sided PDOs but not in left‐sided PDOs. Moreover, TIMP1 knockdown significantly decreased pFAK and pAkt expression levels in right‐sided PDOs but not in left‐sided PDOs. A database analysis of The Cancer Genome Atlas revealed that TIMP1 expression in right‐sided CRCs was significantly higher than in left‐sided CRCs. Kaplan–Meier survival analysis showed significantly shorter overall survival in high‐TIMP1 patients versus low‐TIMP1 patients with right‐sided CRCs but not left‐sided CRCs. Our data suggest that TIMP1 is overexpressed in right‐sided CRCs and promotes cell proliferation and invasion capability through the TIMP1/FAK/Akt pathway, leading to a poor prognosis. The TIMP1/FAK/Akt pathway can be a target for therapeutic agents in right‐sided CRCs.

BackgroundAlloplex Biotherapeutics has developed a novel autologous cellular therapy for cancer that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand a heterogeneous population of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). The 2-week manufacturing process from PBMCs consistently results 300-fold expansion of NK cells, CD8+ T cells, gamma/delta T cells, NKT cells and some CD4+ T cells, collectively called SUPLEXA therapeutic cells. SUPLEXA cells will be delivered back to cancer patients via intravenous administrations on a weekly schedule as an autologous adoptive cellular immunotherapy for cancer. In this study, we tested SUPLEXA cells developed from normal healthy volunteer PBMCs for their ability to infiltrate and kill patient-derived tumor organoids (PDO) as a pre-clinical assessment for potency against 2 different types of tumor organoids.MethodsTumor organoids derived from colorectal cancer (CRC) or non-small cell lung carcinoma (NSCLC) patients were labeled with cell-trace red dye and plated at equal density in a 96-well plate. After 3 days culture, SUPLEXA cells were thawed (82.8% viable), labeled with cell-trace violet dye, and added to PDO at 1:2 serial diluted numbers ranging from 2 million to 7,800 cells per well. Fluorescent images were captured at 24 hours after adding SUPLEXA cells to PDO models to measure PDO size, tumor infiltration, and PDO killing.ResultsAdding SUPLEXA cells to PDO from CRC and NSCLC resulted in significant infiltration and killing of organoids by 24 hours as shown by the fluorescent images and the organoid size plot for the CRC PDO model (figure 1). Significant reduction in PDO size was observed by adding 31,240 SUPLEXA cells. Similar results were observed with the NSCLC PDO model with significant reduction in PDO size by adding 15,600 SUPLEXA cells. Obvious organoid infiltration was observed in both PDO models and organoid fluorescence was significantly reduced by addition of SUPLEXA cells in both PDO models to suggest that SUPLEXA cells were able to reduce tumor burden (figure 2).Abstract 164 Figure 1CRC organoid infiltration and killing by SUPLEXA. A representative fluorescent image of CRC organoid killing with addition of increasing SUPLEXA cell numbers and a plot showing statistical analysis of 6 replicate wells for changes in CRC organoid size in relation to SUPLEXA cell number additionsAbstract 164 Figure 2Dose-dependent killing in CRC and NSCLC PDO models. CRC and NSCLC organoids were detected by total red fluorescence at 24 hours after adding the indicated numbers of SUPLEXA cells. Loss of red fluorescence after adding SUPLEXA is a measure of overall tumor cell killing/burden in organoids. Data is plotted as mean ± SEM for n=6 replicates per group.ConclusionsSUPLEXA cells infiltrated and killed tumor cells in patient-derived organoids within 24 hours of culture at low cell concentrations indicating potent tumor killing activity. The observed activity in both colorectal and lung cancer organoid models support broad anti-tumor killing activity by SUPLEXA. These results provide further evidence that PBMCs from cancer patients can be activated and expanded by our approach as a novel autologous cellular immunotherapy for cancer.

The EGFR-MAPK signaling pathway is frequently activated in cancers, particularly due to KRAS gain-of-function mutations. Certain types, such as pancreatic ductal adenocarcinoma (PDAC) with KRAS mutations and cholangiocarcinoma with FGFR2 fusions, show dependency on EGFR. Recent studies demonstrate promising outcomes in metastatic colorectal cancer by combining an irreversible KRASG12C inhibitor with an EGFR inhibitor. To explore therapeutic benefits across tumor types, we analyzed the expression of EGFR and co-receptors (ERBB) in TCGA samples and CCLE models with different KRAS mutations. We also verified the reliance on ERBB receptors using DepMap data. Patient-derived organoid (PDO) models from PDAC, colorectal cancer (CRC), and CRC liver metastases were utilized to develop orthotopic tumor models. These PDO lines were characterized through mutational profiling, cell signaling analysis, RNA-sequencing, and whole exome sequencing, providing insights into their molecular features. Higher EGFR expression levels at both protein and RNA levels were observed in specific CRC PDO models, consistent in in-vitro PDO models and in-vivo PDO-derived Xenograft (PDOX) tissues from subcutaneous implantation in immunocompromised mice. Additionally, the PDO models were evaluated for sensitivity to standard of care (SOC) drugs and EGFR inhibitors, revealing EGFR expression-independent and tumor type-dependent sensitivities (e.g., Afatinib IC50: PDAC (128nM+/-123) vs CRC (2776nM+/-3099)). Surface expression levels of ERBBs were measured in in-vivo PDOX tissues to assess the comparative use case for targeting the EGFR pathway via inhibition or antibody-drug conjugate (ADC) molecules. Furthermore, we generated and validated stable tumoroid models expressing firefly luciferase in PDOs derived from primary CRC, CRC metastasis to the liver, and primary PDAC. To further validate the potential for in-vivo models, orthotopic tumor models were established in immunocompromised mice for each PDO line, monitoring tumor burden using bioluminescence imaging. Correlation with MRI and ultrasound imaging was conducted for PDAC and liver tumor burden, respectively, with significant correlation observed. Overall, this study aims to enhance understanding of tumor biology and response to treatment through the development and validation of PDO-based orthotopic models, with future work involving validation of EGFR inhibitors targeting the pathway and ADCs based on ERBBs targeting tumor cells expressing these proteins. In vivo luciferase reactivation in our models will allow us to dissect the mechanisms of resistance to these agents using physiologically relevant tumor models and single-cell RNA-sequencing analysis. Citation Format: Krishna S. Tummala, Milind Chalishazar, Minilik Angagaw, Ali Savadipour, Qingyun Yan, Cui Long, Mark Buchanan, Panagiotis N. Lalagkas, Eric Muise, Lan Chen, Kuoyuan Cheng, Eunsil Park, Jennifer Piesvaux, Erica Leccese, Brian Long, Nicolas Solban. Orthotopic tumor models using luciferized patient-derived organoids for investigating EGFR-MAPK pathway inhibition in colorectal and pancreatic cancer [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 27.

Treatment with EGFR-targeted therapies, such as the EGFR monoclonal antibody cetuximab, has improved the outcome of patients with metastatic colorectal cancer (mCRC). However, EGFR inhibition is mainly cytostatic and does not lead to tumor eradication: subsets of drug-tolerant cancer cells often persist even after maximal response to therapy and foster residual disease, leading to relapse. Importantly, drug-tolerant cells are less prone to undergo apoptosis, and thus constitute a reservoir of treatment-refractory persisters that are difficult to extinguish. Here we aimed to investigate the mechanisms by which apoptosis escape mediates survival of cetuximab-tolerant mCRC cells as a means to find new therapeutic strategies for limiting residual disease and delaying disease recurrence. By performing an innovative functional assay named Dynamic BH3 Profiling in cetuximab-sensitive patient-derived organoids (PDOs), we found that antibody treatment increased the “apoptotic priming”, i.e. decreased the distance of cells from the apoptotic threshold without eliciting overt cell death. To start investigating the mechanisms of this increased apoptotic priming, protein levels of the major anti-apoptotic molecules (BCL2, BCL-XL, MCL1) and the pro-apoptotic molecule BIM were evaluated by Western blot in cetuximab-treated PDOs. BIM was consistently upregulated by cetuximab in all tested models. Increased BIM was sequestered by the anti-apoptotic BCL-XL, suggesting that EGFR blockade may render mCRC residual cells dependent on BCL-XL to elude cell death and, consequently, more susceptible to its inhibition. We therefore challenged a panel of 14 PDOs with different BH3 mimetics currently in preclinical or clinical development and found that only the combination of cetuximab with BH3 mimetics against BCL-XL strongly reduced cell viability and induced apoptosis. BCL-XL blockade enhanced cetuximab efficacy also in mCRC patient-derived xenografts, which displayed more durable tumor regression that was maintained also after treatment discontinuation. Notably, the combination of cetuximab with BCL-XL inhibitors was effective in vivo in all tested models except for one, which was characterized by low basal expression of BIM. Mechanistically, unleashed apoptosis by combination therapy was due to cetuximab-induced BIM upregulation concomitantly with its displacement from BCL-XL by the BH3 mimetic. Overall, this study offers new insights into the molecular mechanisms assisting residual disease after cetuximab treatment in mCRC, providing hints to design rational strategies that may convert the cytostatic effect of cetuximab into a cytotoxic, fully apoptotic outcome. Basal expression of BIM could be exploited as a predictive biomarker to enrich for potential responders to the combination of cetuximab and BH3 mimetics. Citation Format: Simonetta Maria Leto, Irene Catalano, Martina Ferri, Valentina Vurchio, Francesca Cottino, Eugenia Rosalinda Zanella, Francesco Sassi, Giorgia Migliardi, Andrea Bertotti, Livio Trusolino. Exploiting patient-derived xenografts and organoids to tackle apoptotic dependencies in EGFR-inhibited colorectal tumours [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3697.

Introduction: The inability to predict treatment response results in unnecessary toxicity, decreased efficacy and survival. There is a clinical need for an effective biomarker to select patients for cancer treatment. A promising biomarker for treatment efficacy is response testing on patient-derived organoids (PDOs). For metastatic colorectal cancer (mCRC), larger prospective studies are needed to evaluate the predictive value of standardized PDO screens. Methods: OPTIC is an ongoing prospective multicenter trial (NL61668.041.17, started 2018) that will evaluate the value of tumor PDOs in therapy response prediction. We establish PDOs for mCRC patients from newly obtained biopsies, prior to the start of treatment. We compare PDO sensitivity (area under drug response curve) with patient response measured by changes in biopsied metastatic lesion size on CT scans. Secondary outcome measures are RECIST response and progression-free survival. We evaluate the feasibility of using PDOs as a biomarker by examining drug screen availability in a clinically relevant timeframe of 4 weeks. From 2020 onwards, we incorporated whole genome sequencing (WGS) on fresh-frozen biopsies to correlate drug response in vitro and in vivo, to genetic tumor characteristics. Additionally, PDO screens with experimental drugs based on genetic alterations identified by WGS could be performed. Trial inclusion will continue until 85 PDOs from clinically evaluable patients for standard of care (SOC) treatment are established, with expected completion in Q1 2024. Results: Currently, 206 patients were included in 5 hospitals and 180 biopsies were taken. PDO establishment success gradually increased from 22% in 2018-2020 to 75% in 2023, yielding an overall success rate of 50% (n=84 PDOs and 13 in culture). Critical factors in optimizing culture success were replacement of several growth factors in the culture medium, reducing time to processing and more frequent handling of biopsies. We have established PDOs for comparing with patient response for the following treatment categories: 5-fluorouracil (n=4), CAPOX/FOLFOX (n=23), irinotecan/FOLFIRI (n=11), FOLFOXIRI (n=12), trifluridine/tipiracil (n=14), panitumumab (n=14), encorafenib-cetuximab (n=2), non-SOC drugs (n=4). We have successfully screened the first 8 PDOs in consistent technical triplicates and biological duplicates (R<0.88, p<0.02). WGS succeeded in 76% (n=75), primarily failing when tumor percentage was <20%. Conclusion: We improved PDO establishment success to 75% which is essential to facilitate the implementation of PDOs in clinical treatment. OPTIC will enhance the clinical application of PDOs by defining thresholds for PDO sensitivity and analyzing the diagnostic power for different treatments. To enable personalized treatment in clinical practice, PDO screening should guide mCRC treatment and result in enhanced chance of response and reduced over- and mistreatment. Citation Format: Lidwien P. Smabers, Emerens Wensink, Carla S. Verissimo, Mayke Doorn, Tao Yang, Timo Voskuilen, Yasmine Abouleila, Maarten A. Huismans, Liselot Valkenburg-van Iersel, Geert A. Cirkel, Elske C. Gootjes, Frank J. Jeurissen, Guus M. Bol, Hilde H. Nienhuis, Manon M. Braat, Bas Penning de Vries, Sjoerd G. Elias, Edwin Cuppen, Robert G. Vries, Onno Kranenburg, Miriam Koopman, Sylvia F. Boj, Jeanine M. Roodhart. Organoids to predict treatment response in metastatic colorectal cancer (OPTIC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5174.

Colorectal cancer (CRC) is a substantial global health concern due to its limited treatment options, especially for oxaliplatin (L-OHP) regimen resistance. This study used organoid-based screening methodologies to evaluate drug responses in CRC while validating the approach with patient-derived CRC organoids and investigating potential biomarkers. Patient-derived organoids were created from CRC surgical specimens, and drug screening were performed. Selected organoids with high and low L-OHP sensitivity underwent next-generation sequencing (NGS), and in vivo experiments using xenotransplantation were used to validate in vitro results. Moreover, the clinical application of homologous recombination deficiency (HRD) as a biomarker was investigated. Organoid drug screening revealed differences in L-OHP sensitivity among 34 patient-derived CRC organoids, and NGS deemed HRD as a potential biomarker. In vivo experiments validated the correlation between HRD status and L-OHP sensitivity, and clinical data suggested the potential of HRD as a biomarker for recurrence-free survival in patients treated with L-OHP. Additionally, HRD exhibited potential as a biomarker for other platinum agents and poly (ADP-ribose) polymerase inhibitors in CRC. The study underscores HRD as a potential biomarker for predicting L-OHP sensitivity, expanding its application to other drugs in CRC. Organoid screening is reliable, providing insights into the intricate association between genetic features and treatment responses.

The development of patient-derived organoid (PDO) technologies has greatly expanded the toolbox for drug discovery and personalized drug screening for several cancer types. While PDO models more closely represent the molecular characteristics and heterogeneity of patient tumors than traditional immortalized cancer cell lines, they are inherently limited in their ability to reflect the tumor microenvironment in vitro as they comprise exclusively of epithelial cells. The lack of stromal cells in PDO models, such as cancer-associated fibroblasts (CAFs), poses a major problem as these tumor microenvironmental components contribute to the various hallmarks of cancer and response to therapy. Particularly for colorectal cancer, CAFs comprise the majority of the tumor microenvironment and play important roles in cancer progression and drug resistance. In this study, we addressed this problem by establishing in vitro conditions that robustly enable the co-culture of CRC PDO with patient-derived CAFs. We report the development of an engineered tumor microenvironment consisting of CRC PDO encapsulated within a well-defined three-dimensional (3D) hyaluronan-gelatin hydrogel and co-cultured with patient-derived CAFs. Basement membrane extracts conventionally used for PDO culture exhibit batch-to-batch variability. Considering that the CRC extracellular matrix is high in hyaluronan and collagen I, and that hyaluronan-based matrices have been shown to be conducive for the culture of various human cancers, we hypothesized that hyaluronan-gelatin hydrogels may serve as a suitable alternative 3D matrix to support the co-culture of CRC PDO and CAFs. Through RNA- and whole-exome sequencing, we first show that these hydrogels are capable of maintaining key molecular characteristics of the original patient tumors in CRC PDO but not support the culture of CAFs. Further, based on our findings that CRC PDO culture medium poorly supports CAF viability, we developed a co-culture strategy that maintains the viability of both CRC PDO and CAFs. We found that in the absence of growth factors added to the co-culture, CAFs were able to maintain the proliferation of the cultured CRC PDO in the hydrogels and restore distinct biological pathways absent in the PDO culture alone but present in patient tissues. Lastly, we demonstrate that these CRC PDO-CAFs models are suitable for evaluating standard-of-care drugs, making them potentially very useful for realizing personalized cancer medicine.

Loss-of-function (LOF) mutations in genes encoding subunits of SWI/SNF chromatin remodeling complexes are found in approximately 25% of human cancers. Colorectal cancers (CRC), the second leading cause of cancer related deaths, are particularly vulnerable to SWI/SNF driver mutations, harboring subunit alterations in up to 55% of patient tumors. ARID1A, a BAF subcomplex specific subunit, is the most frequently mutated subunit in human cancers (12%) and CRCs (10-12%). Additionally, ARID1A was recently identified as a leading genetic alteration in inflammatory bowel diseases such as Crohn’s and Ulcerative Colitis, conditions which are at higher risk of developing CRC. These data suggest an important role in ARID1A regulation of colonic tissue homeostasis and the potential for ARID1A mutation as an initiating or early driving event in CRC, which has not yet been modeled in human colon tissues. To examine the role of ARID1A in colon cancer development, we took a bottom-up strategy using CRISPR edited patient-derived colon organoids to model ARID1A LOF mutations in combination with classic CRC mutations APC and TP53. We evaluated the functional consequences of ARID1A mutation on cell growth using colony formation and proliferation assays. To systematically investigate the phenotypic contribution of ARID1A loss in these cell contexts, we employed transcriptomic and epigenomic profiling with RNAseq and ATACseq. We found that triple knock-out (TKO) of ARID1A, APC and TP53 confers a growth advantage in colon organoids, and induces dramatic morphological changes such as loss of symmetry and the breakdown of cystic architectures. At the gene expression level, ARID1A mutations led to shared and cell context specific transcriptional changes enriched for pathways such as G2M checkpoint, KRAS Signaling, and STAT5 signaling. Mechanistically, we show that ARID1A dependent chromatin accessibility changes at putative enhancers are linked to promoters of differentially expressed genes. These data suggest that ARID1A can regulate multiple cancer hallmark pathways depending on the cellular context and indicate a pleiotropic tumor suppressive function requiring further investigation in clinically relevant genetic backgrounds. Citation Format: Luke T. Deary, Nicholas Sugiarto, Cameron Pigeon, Sara W. Mayo, Matthew Z. Wilson, Xiaofeng Wang. Modeling the role of ARID1A in colon cancer using patient-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB044.

BackgroundTyrosine kinase inhibitors (TKIs) are crucial in the targeted treatment of advanced colorectal cancer (CRC). Anlotinib, a multi-target TKI, has previously been demonstrated to offer therapeutic benefits in previous studies. Circular RNAs (circRNAs) have been implicated in CRC progression and their unique structural stability serves as promising biomarkers. The detailed molecular mechanisms and specific biomarkers related to circRNAs in the era of targeted therapies, however, remain obscure.MethodsThe whole transcriptome RNA sequencing and function experiments were conducted to identify candidate anlotinib-regulated circRNAs, whose mechanism was confirmed by molecular biology experiments. CircHAS2 was profiled in a library of patient-derived CRC organoids (n = 22) and patient-derived CRC tumors in mice. Furthermore, a prospective phase II clinical study of 14 advanced CRC patients with anlotinib-based therapy was commenced to verify drug sensitivity (ClinicalTrials.gov identifier: NCT05262335).ResultsAnlotinib inhibits tumor growth in vitro and in vivo by downregulating circHAS2. CircHAS2 modulates CCNE2 activation by acting as a sponge for miR-1244, and binding to USP10 to facilitate p53 nuclear export as well as degradation. In parallel, circHAS2 serves as a potent biomarker predictive of anlotinib sensitivity, both in patient-derived organoids and xenograft models. Moreover, the efficacy of anlotinib inclusion into the treatment regimen yields meaningful clinical responses in patients with high levels of circHAS2. Our findings offer a promising targeted strategy for approximately 52.9% of advanced CRC patients who have high circHAS2 levels.ConclusionsCircHAS2 promotes cell proliferation via the miR-1244/CCNE2 and USP10/p53/CCNE2 bidirectional axes. Patient-derived organoids and xenograft models are employed to validate the sensitivity to anlotinib. Furthermore, our preliminary Phase II clinical study, involving advanced CRC patients treated with anlotinib, confirmed circHAS2 as a potential sensitivity marker.

e15524 Background: Biomarker discovery and development are essential for stratifying cancer patients in order to improve treatment outcomes. In colorectal cancer (CRC), mutations in the TGF-β/BMP pathway, especially in the SMAD4 gene have been correlated with decreased overall survival and are suspected to modulate drug sensitivity on the cellular level, hence SMAD4 mutations are worthwile targets for novel targeted therapy aproaches. Methods: In the present study, we uncover the mechanistic role of a loss-of-function mutation in SMAD4 in syngeneic patient-derived organoids (PDOs). CRISPR-engineered SMAD4R361H PDOs were subjected to a comparative drug screening, RNA-Sequencing and multiplex protein profiling analysis (DigiWest®). We have confirmed the response towards MEK inhibition of the initial model in an additional set of 62 PDOs with known mutational status. Results: We show that acquisition of SMAD4 loss-of-function mutations renders PDOs sensitive to MEK-inhibitors. Further, an activation of the TGF-β/BMP signaling pathway, specifically of the BMP branch was observed in SMAD4wt PDOs; indicating that BMP signaling is likely responsible for the resistance towards MEK inhibition. It is plausible that functional loss of SMAD4 and thus loss of BMP signaling renders SMAD4 mutated tumors more sensitive to MEK-inhibitors. By looking at additional genes involved in TGF-β/BMP signaling that are frequently mutated in CRC, we identified the novel gene mutational SFAB-signature ( SMAD4, FBXW7, ARID1A, or BMPR2), when at least one pathogenic mutation is present in these genes. The frequency of SFAB in CRC patient cohort (TCGA, n = 594) was comparable to the frequency of SFAB in our PDOs. For PDOs with SFAB-signature, we found up to 95% and 70% significant positive prediction for cobimetinib and selumetinib, respectively and also up to 70% positive prediction for trametinib. Thus, the SFAB-signature predicts response to MEK inhibition in PDOs with a very high confidence. We further investigated whether the RAS status of CRC PDOs does predict sensitivity to MEK inhibition. The RAS status alone and in combination with SFAB-signature failed to yield better prediction sensitivity to MEK-inhibitors. Conclusions: The present study is a significant step forward to more personalized treatment regimens for CRC patients by early inclusion of MEK-inhibitors. The SFAB-signature should be put to clinical testing as a RAS-independent biomarker for stratification of patients providing a valuable alternative treatment option against CRC, thus ensuring that all patients receive effective and specific therapies as early as possible.

535 Background: The bispecific antibody CEA-TCB binds Carcino-Embryonic Antigen (CEA) on cancer cells and CD3 on T cells. This triggers T cell killing of colorectal cancer cell lines expressing moderate to high levels of CEA at the cell surface (Bacac, Clin Cancer Res 2016). Patient derived organoids (PDOs) may more accurately represent patient tumors than established cell lines. Yet, determinants of CEA-TCB resistance have not been studied in PDOs. Methods: PDOs were established from biopsies of eight multidrug-resistant metastatic CRCs, GFP labelled and adapted to 2D culture. Allogenic CD8 T cells and CEA-TCB or a non-targeting control antibody were added and cancer cell killing and growth were monitored for 10 days. CEA expression of PDOs was determined by FACS. Results: CRC PDOs could be categorized into three groups based on CEA cell-surface expression: CEAhigh (n = 3), CEAlow (n = 2), and CEA heterogeneous PDOs (n = 3) that stably maintained populations of both CEAhigh and CEAlow cells, which has not previously been described in CRC cell lines. Heterogeneity of cell-surface CEA expression is common in CRC cells in patients, supporting that PDOs may better represent these tumors than established cell lines. CEAhigh cells were sensitive whereas CEAlow cells showed resistance to CEA-TCB. All PDOs with heterogeneous CEA expression were resistant to CEA-TCB, suggesting that CEA-negative cells maintain cancer cell growth. Culture of FACS sorted CEAhigh and CEAlow cells from PDOs with heterogeneous CEA expression demonstrated high plasticity of CEA expression which may contribute to rapid resistance acquisition through CEA antigen loss. Conclusions: These results suggest that cell-surface CEA expression is a major determinant of CEA-TCB sensitivity and resistance in PDOs. In addition, we identified heterogeneous CEA expression in several PDOs and demonstrated that this could confer CEA-TCB resistance in vitro. These PDO models are likely to provide insights into the mechanism of CEA loss and may inform therapeutic opportunities to counter CEA-TCB resistance. RNA-sequencing and functional experiments are ongoing to investigate this and will be presented.