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In colorectal cancers, the tumor microenvironment plays a key role in prognosis and therapy efficacy. Patient-derived tumor organoids (PDTO) show enormous potential for preclinical testing; however, cultured tumor cells lose important characteristics, including the consensus molecular subtypes (CMS). To better reflect the cellular heterogeneity, we established the colorectal cancer organoid-stroma biobank of matched PDTOs and cancer-associated fibroblasts (CAF) from 30 patients. Context-specific phenotyping showed that xenotransplantation or coculture with CAFs improves the transcriptomic fidelity and instructs subtype-specific stromal gene expression. Furthermore, functional profiling in coculture exposed CMS4-specific therapeutic resistance to gefitinib and SN-38 and prognostic expression signatures. Chemogenomic library screening identified patient- and therapy-dependent mechanisms of stromal resistance including MET as a common target. Our results demonstrate that colorectal cancer phenotypes are encrypted in the cancer epithelium in a plastic fashion that strongly depends on the context. Consequently, CAFs are essential for a faithful representation of molecular subtypes and therapy responses ex vivo., Significance: Systematic characterization of the organoid-stroma biobank provides a resource for context dependency in colorectal cancer. We demonstrate a colorectal cancer subtype memory of PDTOs that is independent of specific driver mutations. Our data underscore the importance of functional profiling in cocultures for improved preclinical testing and identification of stromal resistance mechanisms. This article is featured in Selected Articles from This Issue, p. 2109., (©2023 The Authors; Published by the American Association for Cancer Research.)

Tumor progression is recognized as a result of an evolving cross-talk between tumor cells and their surrounding nontransformed stroma. Although Wnt signaling has been intensively studied in colorectal cancer, it remains unclear whether activity in the tumor-associated stroma contributes to malignancy. To specifically interfere with stromal signals, we generated Wnt-independent tumor organoids that secrete the Wnt antagonist Sfrp1. Subcutaneous transplantation into immunocompetent as well as immunodeficient mice resulted in a strong reduction of tumor growth. Histologic and transcriptomic analyses revealed that Sfrp1 induced an epithelial-mesenchymal transition (EMT) phenotype in tumor cells without affecting tumor-intrinsic Wnt signaling, suggesting involvement of nonimmune stromal cells. Blockage of canonical signaling using Sfrp1, Dkk1, or fibroblast-specific genetic ablation of β-catenin strongly decreased the number of cancer-associated myofibroblasts (myCAF). Wnt activity in CAFs was linked with distinct subtypes, where low and high levels induced an inflammatory-like CAF (iCAF) subtype or contractile myCAFs, respectively. Coculture of tumor organoids with iCAFs resulted in significant upregulation of EMT markers, while myCAFs reverted this phenotype. In summary, we show that tumor growth and malignancy are differentially regulated via distinct fibroblast subtypes under the influence of juxtacrine Wnt signals. SIGNIFICANCE: This study provides evidence for Wnt-induced functional diversity of colorectal cancer-associated fibroblasts, representing a non-cell autonomous mechanism for colon cancer progression. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5569/F1.large.jpg., (©2020 American Association for Cancer Research.)