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.

Organoid Models of Colorectal Pathology: Do They Hold the Key to Personalized Medicine? A Systematic Review.

BACKGROUND: Colorectal cancer and IBD account for a large portion of the practice of colorectal surgery. Historical research models have provided insights into the underlying causes of these diseases but come with many limitations. OBJECTIVE: The aim of this study was to systematically review the literature regarding the advantage of organoid models in modeling benign and malignant colorectal pathology. DATA SOURCES: Sources included PubMed, Ovid-Medline, and Ovid Embase STUDY SELECTION:: Two reviewers completed a systematic review of the literature between January 2006 and January of 2020 for studies related to colon and intestinal organoids. Reviews, commentaries, protocols, and studies not performed in humans or mice were excluded. RESULTS: A total of 73 articles were included. Organoid models of colorectal disease have been rising in popularity to further elucidate the genetic, transcriptomic, and treatment response of these diseases at the individual level. Increasingly complex models utilizing coculture techniques are being rapidly developed that allow in vitro recapitulation of the disease microenvironment. LIMITATIONS: This review is only qualitative, and the lack of well utilized nomenclature in the organoid community may have resulted in the exclusion of articles. CONCLUSIONS: Historical disease models including cell lines, patient-derived tumor xenografts, and animal models have created a strong foundation for our understanding of colorectal pathology. Recent advances in 3-dimensional cell cultures, in the form of patient-derived epithelial organoids and induced human intestinal organoids have opened a new avenue for high-resolution analysis of pathology at the level of an individual patient. Recent research has shown the potential of organoids as a tool for personalized medicine with their ability to retain patient characteristics, including treatment response.

Disrupting Inflammation-Associated CXCL8-CXCR1 Signaling Inhibits Tumorigenicity Initiated by Sporadic- and Colitis-Colon Cancer Stem Cells.

Dysfunctional inflammatory pathways are associated with an increased risk of cancer, including colorectal cancer. We have previously identified and enriched for a self-renewing, colon cancer stem cell (CCSC) subpopulation in primary sporadic colorectal cancers (CRC) and a related subpopulation in ulcerative colitis (UC) patients defined by the stem cell marker, aldehyde dehydrogenase (ALDH). Subsequent work demonstrated that CCSC-initiated tumors are dependent on the inflammatory chemokine, CXCL8, a known inducer of tumor proliferation, angiogenesis and invasion. Here, we use RNA interference to target CXCL8 and its receptor, CXCR1, to establish the existence of a functional signaling pathway promoting tumor growth initiated by sporadic and colitis CCSCs. Knocking down either CXCL8 or CXCR1 had a dramatic effect on inhibiting both in vitro proliferation and angiogenesis. Likewise, tumorigenicity was significantly inhibited due to reduced levels of proliferation and angiogenesis. Decreased expression of cycle cell regulators cyclins D1 and B1 along with increased p21 levels suggested that the reduction in tumor growth is due to dysregulation of cell cycle progression. Therapeutically targeting the CXCL8-CXCR1 signaling pathway has the potential to block sustained tumorigenesis by inhibiting both CCSC- and pCCSC-induced proliferation and angiogenesis.

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.

Optimum 3D Matrix Stiffness for Maintenance of Cancer Stem Cells Is Dependent on Tissue Origin of Cancer Cells.

INTRODUCTION: The growth and expression of cancer stem cells (CSCs) depend on many factors in the tumor microenvironment. The objective of this work was to investigate the effect of cancer cells' tissue origin on the optimum matrix stiffness for CSC growth and marker expression in a model polyethylene glycol diacrylate (PEGDA) hydrogel without the interference of other factors in the microenvironment. METHODS: Human MCF7 and MDA-MB-231 breast carcinoma, HCT116 colorectal and AGS gastric carcinoma, and U2OS osteosarcoma cells were used. The cells were encapsulated in PEGDA gels with compressive moduli in the 2-70 kPa range and optimized cell seeding density of 0.6x106 cells/mL. Micropatterning was used to optimize the growth of encapsulated cells with respect to average tumorsphere size. The CSC sub-population of the encapsulated cells was characterized by cell number, tumorsphere size and number density, and mRNA expression of CSC markers. RESULTS: The optimum matrix stiffness for growth and marker expression of CSC sub-population of cancer cells was 5 kPa for breast MCF7 and MDA231, 25 kPa for colorectal HCT116 and gastric AGS, and 50 kPa for bone U2OS cells. Conjugation of a CD44 binding peptide to the gel stopped tumorsphere formation by cancer cells from different tissue origin. The expression of YAP/TAZ transcription factors by the encapsulated cancer cells was highest at the optimum stiffness indicating a link between the Hippo transducers and CSC growth. The optimum average tumorsphere size for CSC growth and marker expression was 50 µm. CONCLUSION: The marker expression results suggest that the CSC sub-population of cancer cells resides within a niche with optimum stiffness which depends on the cancer cells' tissue origin.

Experimental and computational investigation of the effect of hydrophobicity on aggregation and osteoinductive potential of BMP-2-derived peptide in a hydrogel matrix.

An attractive approach to reduce the undesired side effects of bone morphogenetic proteins (BMPs) in regenerative medicine is to use osteoinductive peptide sequences derived from BMPs. Although the structure and function of BMPs have been studied extensively, there is limited data on structure and activity of BMP-derived peptides immobilized in hydrogels. The objective of this work was to investigate the effect of concentration and hydrophobicity of the BMP-2 peptide, corresponding to residues 73-92 of the knuckle epitope of BMP-2 protein, on peptide aggregation and osteogenic differentiation of human mesenchymal stem cells encapsulated in a polyethylene glycol (PEG) hydrogel. The peptide hydrophobicity was varied by capping PEG chain ends with short lactide segments. The BMP-2 peptide with a positive index of hydrophobicity had a critical micelle concentration (CMC) and formed aggregates in aqueous solution. Based on simulation results, there was a slight increase in the concentration of free peptide in solution with 1000-fold increase in peptide concentration. The dose-osteogenic response curve of the BMP-2 peptide was in the 0.0005-0.005 mM range, and osteoinductive potential of the BMP-2 peptide was significantly less than that of BMP-2 protein even at 1000-fold higher concentrations, which was attributed to peptide aggregation. Further, the peptide or PEG-peptide aggregates had significantly higher interaction energy with the cell membrane compared with the free peptide, which led to a higher nonspecific interaction with the cell membrane and loss of osteoinductive potential. Conjugation of the BMP-2 peptide to PEG increased CMC and osteoinductive potential of the peptide whereas conjugation to lactide-capped PEG reduced CMC and osteoinductive potential of the peptide. Experimental and simulation results revealed that osteoinductive potential of the BMP-2 peptide is correlated with its CMC and the free peptide concentration in aqueous medium and not the total concentration.

Nanostructure formation and transition from surface to bulk degradation in polyethylene glycol gels chain-extended with short hydroxy acid segments.

Degradable, in situ gelling, inert hydrogels with tunable properties are very attractive as a matrix for cell encapsulation and delivery to the site of regeneration. Cell delivery is generally limited by the toxicity of gelation and degradation reactions. The objective of this work was to investigate by simulation and experimental measurement gelation kinetics and degradation rate of star acrylated polyethylene glycol (PEG) macromonomers chain-extended with short hydroxy acid (HA) segments (SPEXA) as a function of HA monomer type and number of HA repeat units. HA monomers included least hydrophobic glycolide (G), lactide (L), p-dioxanone (D), and most hydrophobic ε-caprolactone (C). Chain extension of PEG with short HA segments resulted in micelle formation for all HA types. There was a significant decrease in gelation time of SPEXA precursor solutions with HA chain-extension for all HA types due to micelle formation, consistent with the simulated increase in acrylate-acrylate (Ac-Ac) and Ac-initiator integration numbers. The hydrolysis rate of SPEXA hydrogels was strongly dependent on HA type and number of HA repeat units. SPEXA gels chain-extended with the least hydrophobic glycolide completely degraded within days, lactide within weeks, and p-dioxanone and ε-caprolactone degraded within months. The wide range of degradation rates observed for SPEXA gels can be explained by large differences in equilibrium water content of the micelles for different HA monomer types. A biphasic relationship between HA segment length and gel degradation rate was observed for all HA monomers, which was related to the transition from surface (controlled by HA segment length) to bulk (controlled by micelle equilibrium water content) hydrolysis within the micelle phase. To our knowledge, this is the first report on transition from surface to bulk degradation at the nanoscale in hydrogels.

Effect of CD44 binding peptide conjugated to an engineered inert matrix on maintenance of breast cancer stem cells and tumorsphere formation.

INTRODUCTION: As cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on cancer stem cells (CSCs) while keeping other factors unchanged. We have developed a synthetic inert 3D polyethylene glycol diacrylate (PEGDA) gel culture system as a unique tool to study the effect of microenvironmental factors on CSCs response. We have reported that CSCs formed in the inert PEGDA gel by encapsulation of breast cancer cells maintain their stemness within a certain range of gel stiffness. The objective was to investigate the effect of CD44 binding peptide (CD44BP) conjugated to the gel on the maintenance of breast CSCs. METHODS: 4T1 or MCF7 breast cancer cells were encapsulated in PEGDA gel with CD44BP conjugation. Control groups included dissolved CD44BP and the gel with mutant CD44BP conjugation. Tumorsphere size and density, and expression of CSC markers were determined after 9 days. For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined after 4 weeks. Effect of CD44BP conjugation on breast CSC maintenance was compared with integrin binding RGD peptide (IBP) and fibronectin-derived heparin binding peptide (FHBP). RESULTS: Conjugation of CD44BP to the gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP. CONCLUSION: CD44BP and IBP conjugated to the gel abolished tumorsphere formation by encapsulated 4T1 cells while FHBP enhanced tumorsphere formation compared to cells in the gel without peptide. The PEGDA hydrogel culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors.

Three-dimensional-engineered matrix to study cancer stem cells and tumorsphere formation: effect of matrix modulus.

Maintenance of cancer stem cells (CSCs) is regulated by the tumor microenvironment. Synthetic hydrogels provide the flexibility to design three-dimensional (3D) matrices to isolate and study individual factors in the tumor microenvironment. The objective of this work was to investigate the effect of matrix modulus on tumorsphere formation by breast cancer cells and maintenance of CSCs in an inert microenvironment without the interference of other factors. In that regard, 4T1 mouse breast cancer cells were encapsulated in inert polyethylene glycol diacrylate hydrogels and the effect of matrix modulus on tumorsphere formation and expression of CSC markers was investigated. The gel modulus had a strong effect on tumorsphere formation and the effect was bimodal. Tumorsphere formation and expression of CSC markers peaked after 8 days of culture. At day 8, as the matrix modulus was increased from 2.5 kPa to 5.3, 26.1, and 47.1 kPa, the average tumorsphere size changed from 37±6 µm to 57±6, 20±4, and 12±2 µm, respectively; cell number density in the gel changed from 0.8±0.1×105 cells/mL to 1.7±0.2×105, 0.4±0.1×105, and 0.2±0.1×105 cells/mL after initial encapsulation of 0.14×105 cells/mL; and the expression of CD44 breast CSC marker changed from 17±4-fold to 38±9-, 3±1-, and 2±1-fold increase compared with the initial level. Similar results were obtained with MCF7 human breast carcinoma cells. Mouse 4T1 and human MCF7 cells encapsulated in the gel with 5.3 kPa modulus formed the largest tumorspheres and highest density of tumorspheres, and had highest expression of breast CSC markers CD44 and ABCG2. The inert polyethylene glycol hydrogel can be used as a model-engineered 3D matrix to study the role of individual factors in the tumor microenvironment on tumorigenesis and maintenance of CSCs without the interference of other factors.