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Colorectal cancer (CRC) is the third leading cause of cancer and cancer-related death. A large fraction of the CRC patients diagnosed with de novo metastatic disease do not benefit from the standard of care but still experience substantial side effects. Therefore, there is the urgency for a new model to predict clinical response. Adult epithelial stem cell (ASC) -derived organoids are proving to be a major breakthrough in pre-clinical models. ASC-derived organoids can be developed from healthy as well as diseased tissue, including cancer lesions and therefore are often referred to as patient-derived organoids (PDOs or HUB Organoids࣪). These model is established directly from patient tissue, represent the tissue of origin and faithfully recapitulate patient disease in vitro and can be propagated for drug testing in a matter of weeks. PDOs bridges the gap between the lab and the clinic and effectively bring a “patient in the lab.” In this study we aimed to validate the predictive value of PDOs in the stratification of metastatic CRC (mCRC) patients for treatment with chemotherapeutic agents. PDOs from mCRC tissues were established following our optimized procedures and drug screening was performed. Clinical response was compared with PDO drug response and the best predicting drug response parameters (growth rate (GR), GRmax, GR50 and area under the curve) were identified. The patient response was evaluated by the percentage of change in size of the target lesions on response scans (% size change), best Response Evaluation Criteria in Solid Tumors (RECIST) response and progression-free survival (PFS). Importantly, we validated the predictive value of organoids towards fluorouracil (5-FU). We are validating these results in a large trial. Current efforts to further expand our PDO and drug sensitivity biobank will enable the implementation of the personalized HUB Organoid Technology to accurate and fast predict of the treatment response to improve clinical outcome of mCRC patients. Citation Format: Carla S. Verissimo, Lidwien Smabers, Emerens Wensink, Esmee Koedoot, Maarten Huismans, Celia Higuera Barón, Ricardo Korporaal, Emma Teal, Katerina-Chara Pitsa, Edwin van Oosten, Roshni Nair, Liselot Valkenburg, Geert Cirkel, Anneta Brousali, Jorieke Salij, René Overmeer, Manon Braat, Sjoerd Elias, Robert Vries, Onno Kranenburg, Miriam Koopman, Sylvia F. Boj, Jeanine Roodhart. Patient derived organoids predict clinical response: A patient in the lab [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 4112.

Organoid cultures generated from gastrointestinal tissues have been an invaluable advancement for in vitro studies of physiological function and disease. Here we present a comprehensive protocol for the establishment and culture of human- and mouse-derived 3-dimensional gastric organoids transferred to 2-dimensional gastric epithelial cell monolayers. We introduce two methods that include the establishment of monolayers from: (1) intact organoids, and (2) single cells dissociated from intact organoids.

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a funcitonal role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-deficient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (55 years) or young (14-20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile anlayzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastic epithelium CD44v9 emerges within a regenerative cell lineage that coincides with macrophage inflitration within the injured mucosa. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.