Induced organoids derived from patients with ulcerative colitis recapitulate colitic reactivity.

The pathogenesis of ulcerative colitis (UC), a major type of inflammatory bowel disease, remains unknown. No model exists that adequately recapitulates the complexity of clinical UC. Here, we take advantage of induced pluripotent stem cells (iPSCs) to develop an induced human UC-derived organoid (iHUCO) model and compared it with the induced human normal organoid model (iHNO). Notably, iHUCOs recapitulated histological and functional features of primary colitic tissues, including the absence of acidic mucus secretion and aberrant adherens junctions in the epithelial barrier both in vitro and in vivo. We demonstrate that the CXCL8/CXCR1 axis was overexpressed in iHUCO but not in iHNO. As proof-of-principle, we show that inhibition of CXCL8 receptor by the small-molecule non-competitive inhibitor repertaxin attenuated the progression of UC phenotypes in vitro and in vivo. This patient-derived organoid model, containing both epithelial and stromal compartments, will generate new insights into the underlying pathogenesis of UC while offering opportunities to tailor interventions to the individual patient.

Cancer-predicting transcriptomic and epigenetic signatures revealed for ulcerative colitis in patient-derived epithelial organoids.

Ulcerative colitis (UC) is a prevalent form of inflammatory bowel disease (IBD) whose pathogenic mechanisms remain unclear. Elucidating these mechanisms is important to reduce UC symptoms and to prevent UC progression into colitis-associated colon cancer (CAC). Our goal was to develop and validate faithful, human-derived, UC models and analyze them at histologic, transcriptomic and epigenetic levels to allow mechanistic studies of UC and CAC pathogenesis. We generated patient-derived primary-organoid cultures from UC and non-IBD colonic epithelium. We phenotyped them histologically and used next-generation-sequencing approaches to profile whole transcriptomes and epigenomes of organoids and primary tissues. Tissue organization and expression of mucin 2 (MUC2) and lysozyme (LYZ) demonstrated histologic faithfulness of organoids to healthy and diseased colonic epithelium. Transcriptomic analyses showed increased expression of inflammatory pathways in UC patient-derived organoids and tissues. Profiling for active enhancers using the H3K27ac histone modification revealed UC-derived organoid enrichment for pathways indicative of gastrointestinal cancer, including S100 calcium-binding protein P (S100P), and revealed novel markers for GI cancer, including both LYZ and neuropeptide S receptor 1 (NPSR1). Immunolocalization showed increased levels of LYZ, S100P, and NPSR1 proteins in UC and CAC. In conclusion, primary colonic organoid cultures from UC and non-IBD patients can be established that faithfully represent diseased or normal colonic states. These models reveal precancerous molecular pathways that are already activated in UC. The findings demonstrate the suitability of primary organoids for dissecting UC and CAC pathogenic mechanisms and suggest new targets for therapeutic intervention.

Stromal miR-20a controls paracrine CXCL8 secretion in colitis and colon cancer.

Inflammatory bowel disease (IBD) affects one million people in the US. Ulcerative colitis (UC) is a subtype of IBD that can lead to colitis-associated cancer (CAC). In UC, the rate of CAC is 3-5-fold greater than the rate of sporadic colorectal cancer (CRC). The pathogenesis of UC and CAC are due to aberrant interactions between host immune system and microenvironment, but precise mechanisms are still unknown. In colitis and CAC, microenvironmental fibroblasts exhibit an activated, inflammatory phenotype that contributes to tumorigenesis accompanied by excessive secretion of the chemokine CXCL8. However, mechanisms regulating CXCL8 secretion are unclear. Since it is known that miRNAs regulate chemokines such as CXCL8, we queried a microRNA library for mimics affecting CXCL8 secretion. Among the identified microRNAs, miR-20a/b was further investigated as its stromal expression levels inversely correlated with the amounts of CXCL8 secreted and predicted fibroblast tumor-promoting activity. Indeed, miR-20a directly bound to the 3'UTR of CXCL8 mRNA and regulated its expression by translational repression. In vivo co-inoculation studies with CRC stem cells demonstrated that fibroblasts characterized by high miR-20a expression had reduced tumor-promoting activities. These studies reveal that in stromal fibroblasts, miR-20a modulates CXCL8 function, therefore influencing tumor latency.

Colon Cancer: Inflammation-Associated Cancer.

Colitis-associated cancer is a relatively rare form of cancer with an unclear pathogenesis. Colitis-associated cancer serves as a prototype of inflammation-associated cancers. Advanced colonoscopic techniques are considered standard of care for surveillance in patients with long-standing colitis, especially those with other risk factors, including sclerosing cholangitis and a family history of colorectal cancer. When colitis-associated cancer is diagnosed, the standard operation involves total proctocolectomy. Restorative procedures and surveillance after colectomy require special considerations. In these contexts, new 3-dimensional human models may be used to usher in personalized medicine.

Inhibition of PI3K/Akt/mTOR signaling in PI3KR2-overexpressing colon cancer stem cells reduces tumor growth due to apoptosis.

In sporadic colon cancer, colon cancer stem cells (CCSCs) initiate tumorigenesis and may contribute to late disease recurrences and metastases. We previously showed that aldehyde dehydrogenase (ALDH) activity (as indicated by the ALDEFLUOR® assay) is an effective marker for highly enriching CCSCs for further evaluation. Here, we used comparative transcriptome and proteome approaches to identify signaling pathways overrepresented in the CCSC population. We found overexpression of several components of the phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway, including PI3KR2, a regulatory subunit of PI3K. LY294002, a PI3K inhibitor, defined the contribution of the PI3K/Akt/mTOR signaling pathway in CCSCs. LY294002-treated CCSCs showed decreases in proliferation, sphere formation and self-renewal, in phosphorylation-dependent activation of Akt, and in expression of cyclin D1. Inhibition of PI3K in vivo reduced tumorigenicity, increased detection of cleaved caspase 3, an indicator of apoptosis, and elevated expression of the inflammatory chemokine, CXCL8. Collectively, these results indicate that PI3K/Akt/mTOR signaling controls CCSC proliferation and CCSC survival, and suggests that it would be useful to develop therapeutic agents that target this signaling pathway.