Intestinal Stem Cell Marker Research Articles

Dissecting the cellular responses of immune cells and Lgr5+ stem cells in the inflamed mouse colon with the RNAscope in situ hybridization technology

Abstract Due to its exposure to a harsh luminal environment, the intestinal epithelium has a remarkably fast turnover rate that is facilitated by a resident intestinal stem cell (ISC) population present at the base of the intestinal crypt. These ISCs, marked by the GPCR Lgr5, allow the intestinal epithelium to adapt to different types of damage, such as inflammation. Chronic intestinal inflammation is a hallmark of the inflammatory bowel diseases (IBD) yet the interplay between inflammatory immune cells and ISCs remains to be elucidated. Therefore, we utilized the single-molecule RNA in situ hybridization (ISH) technology RNAscope to visualize the expression of multiple immune and ISC markers within the inflamed intestinal tissue environment. To interrogate the expression pattern of inflammatory immune cell and ISC markers within the intestinal crypt, we performed the assay on colons from either control or TNBS-treated mice. We visualized the location of each intestinal cell population, including the resident Lgr5+ ISC population, within the crypt. The impact of inflammation on the Lgr5+ ISC population, as well as the Wnt/β-catenin pathway, was also examined. Using a multiplex assay, we assessed infiltration of regulatory T cells and Th17 cells in the inflamed region. Lastly, we examined the expression of several receptor-ligand pairs for cytokines and ISC markers. Taken together, these results demonstrate the ability of the RNAscope assay to visualize the ISCs within the morphological context of the intestinal crypt and in relationship to inflammatory immune cells. This work may help understand mechanisms behind the pathogenesis of IBD and other inflammatory diseases and also aid in the development of potential therapies.

Culture and characterization of mouse intestinal organoids

Background & Aim Organoids formed by self-organizing stem cells resemble their native counterparts in cellular content, multicellular architecture and functional features. As such, they are emerging as powerful tools in basic and translational research. Intestinal organoids possess a particularly high level of multicellular organization and a wide range of potential applications. However, these cultured intestinal tissue pieces from the intestinal epithelial could be maintained in culture for several days at maximum, and thus were not self-renewing. Lgr5+ cells isolated from intestine, colon, or stomach can form organoids in long-term culture. An essential component of these cultures is R-spondin, the ligand of Lgr5. Thus, when cultured under the appropriate 3D conditions, single Lgr5-expressing intestinal stem cells (ISCs) undergo cycles of self-renewal, differentiation and morphogenesis, and self-organize into crypt-villus domains that house cycling ISCs and differentiated intestinal cells. In this study, we performed isolation and characterization of ISCs from BALB/c-nude mouse. Methods, Results & Conclusion Crypts were isolated from the mouse small intestine by minor modification of the method described by Khalil. Freshly isolated mouse crypts from dissociated mouse ISCs colonies were embedded in Matrigel, cast into 40 mL droplets at the bottom of well in a 48 well plate. Following polymerization, the gels were overlaid with ISC expansion medium containing B27, N2, epidermal growth factor, noggin, and R-spondin 1. As a result, mouse ISCs had enteroids and spheroid morphologies. We also confirmed by quantitative real-time RT-PCR that expression of ISCs related genes (Lgr5, mucin-1, mucin-2, defensin-5, chromogranin A, and villin) was maintained at 8 passage or more. In this study, we observed that expression of specific ISC markers and consecutive self-renewing in the mouse intestinal organoids.

Characterization of LGR5 expression in poorly differentiated colorectal carcinoma with mismatch repair protein deficiency

BackgroundLeucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a promising intestinal stem cell and carcinoma stem cell marker. We examined the relationship between mismatch repair (MMR) protein deficiency and LGR5 expression in poorly differentiated (PD) colorectal carcinoma (CRC).MethodsIn 29 cases of PD-CRC, deficiencies in MMR proteins (MLH1, PMS2, MSH2, MSH6) and β-catenin expression were identified by immunohistochemistry (IHC). LGR5 expression was examined by the RNAscope assay in tissue microarrays.ResultsLGR5 H-scores in MMR-deficient (MMR-D) cases were significantly lower than those in MMR-proficient (MMR-P) cases (P = 0.0033). Nuclear β-catenin IHC scores in MMR-D cases were significantly lower than those in MMR-P cases (P = 0.0024). In all cases, there was a positive correlation between LGR5 H-score and nuclear β-catenin IHC score (r = 0.6796, P < 0.001). Even in MMR-D and MMR-P cases, there was a positive correlation between LGR5 H-score and nuclear β-catenin IHC score (r = 0.7180, P < 0.0085 and r = 0.6574, P < 0.003, respectively). MMR-D CRC cases showed low expression of LGR5, which may be due to low activation of the Wnt/β-catenin signaling pathway.ConclusionsOur results reveal the relationship between LGR5 expression and MMR protein profiles in PD-CRC. A further study is warranted to confirm these findings.

Expression Profile and Prognostic Significance of EPHB3 in Colorectal Cancer.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) is expressed in cells at the base of intestinal crypts, acting as a cellular guide in the maintenance of intestinal crypt architecture. We aimed to investigate the expression profile of EPHB3 in colorectal precancerous lesions and colorectal cancers (CRCs), and assess its prognostic value. EPHB3 expression was higher in CRCs than in normal mucosa and was associated with the intestinal stem cell markers EPHB2, OLFM4, LRIG1, and a proposed cancer stem cell marker, CD44. Enhanced EPHB3 expression significantly declined during the transformation from adenoma to carcinoma and as the tumor invaded into deeper tissue layers. Namely, a substantial reduction of EPHB3 expression was observed in the budding cancer cells at the invasive tumor fronts, which was more extensive than E-cadherin downregulation. In an azoxymethane/dextran sulfate sodium-induced, colitis-associated, CRC model, EPHB3 expression increased along with tumor development. In a large cohort of CRC patients, EPHB3 positivity was observed in 24% of 610 CRCs and was negatively correlated with tumor differentiation, lympho-vascular invasion, and tumor, node, and metastasis stages. EPHB3 was positively associated with microsatellite instability but was associated with neither CpG island methylation, nor with KRAS and BRAF mutations. Notably, EPHB3 positivity was associated with better clinical outcomes, although it was not an independent prognostic marker. Overexpression of EPHB3 in the colon cancer cell line, DLD1, led to decreased cell growth and migration and reduced mitogen-activated protein kinase signaling. Taken together, our data demonstrate the suppressive role of EPHB3 in CRC progression.

T‐cell Protein Tyrosine Phosphatase is a Key Regulator of Paneth Cell Function

Loss‐of‐function single nucleotide polymorphisms (SNPs) in the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene, are associated with increased susceptibility to Inflammatory Bowel Disease (IBD). PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) which is a negative regulator of intracellular signaling pathways including JAK‐STAT. We previously presented that Paneth and goblet cell numbers are reduced in Ptpn2 knockout (KO) mice, consistent with altered differentiation of these intestinal epithelial cell (IEC) types in IBD. Alterations in IEC populations are detrimental for intestinal homeostasis and increase susceptibility to microbial infection.GoalTo identify whether Ptpn2‐KO mice display altered IEC differentiation and Paneth cell antimicrobial peptide (AMP) production.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old constitutive Ptpn2‐KO mice and control littermates, and from adult tamoxifen‐inducible intestinal epithelial‐specific Ptpn2ΔIEC mice (Vil‐Cre/Ptpn2), were harvested and processed for imaging, western blot, flow cytometry and qPCR.ResultsConstitutive Ptpn2‐KO mice display reduced numbers of Paneth cells and loss of the AMP lysozyme (p=0.0158; n=5). This was confirmed by Western blots of isolated IECs (~67% reduction; n=4). Expression of Paneth cell markers and differentiation factors (Mmp7, Defa5 and Sox9) were not altered in KO mice (n=10). Interestingly, Reg3‐γ, which like lysozyme also targets gram‐(+) bacteria and is secreted by Paneth cells and enterocytes, was increased in KO mice vs. control littermates (p=0.0066; n=10). Next, we identified by immunofluorescence that loss of Ptpn2 only in intestinal epithelium (Ptpn2ΔIEC) dramatically reduced colocalization of lysozyme and another Paneth cell marker, UEA‐1 (n=6). Decreased lysozyme expression in Ptpn2ΔIEC ileum was confirmed by western blot (p=0.0315; n=6). Since Paneth cells reside in the stem cell compartment, we investigated whether Ptpn2 deficiency affects the intestinal stem cells. Gene expression of Lgr5, an intestinal stem cell marker, Ephb3, a marker of the stem cell compartment, and Ascl2, a gene that controls stem cell renewal in the crypts, was unchanged in Ptpn2‐KO mice (n=10). In addition, expression of early IEC differentiation factors, Hes1 and Math1, were not altered (n=10). Il‐22 and IFN‐γ are known to stimulate secretion of AMPs by Paneth cells. Interestingly, flow cytometry showed increased abundance of Il‐22+ T cells (p=0.0187; n=5) and IFN‐γ+ T cells (p=0.0277; n=5) in the Ileum of constitutive KO mice, indicating that the defect in AMP production is likely not due to a lack of immune cell‐derived stimuli.ConclusionPtpn2‐deficiency decreases Paneth cell number and has opposing effects on their production of specific AMPs. These effects on Paneth cell number and function may contribute to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2‐deficient mice.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM) and the Crohn’s and Colitis Foundation (DFM)

Salmonella infection induced intestinal crypt hyperplasia through Wnt/β-catenin pathway in chicken

S. Pullorum is a causative agent of enteric disease of poultry with serious diarrhea. However, the detailed mechanism behind its injury to intestinal mucosa barrier, especially for intestinal stem cells, is unclear. In this study, S. Pullorum were orally administrated to 3 days old chicken to investigate the pathogenesis of S. Pullorum on intestinal mucosal barrier, especially on the proliferation of epithelial cells. We found that S. Pullorum could colonize in the cecum and invade into the liver through intestinal mucosa damage, which caused obvious pathological changes in liver and intestine and even leaded to death, as well as significant reduction of body weight. We also found that S. Pullorum infection enhanced the mRNA expression of IL-1β and IL-6 through TLR4/MyD88 pathway, which was also further verified by the increased lipopolysaccharide (LPS) levels in serum. Furthermore, S. Pullorum increased the depth of crypt and density of PCNA+ cells significantly through the over-activation of Wnt/β-catenin signaling pathway. The expression of intestinal stem cells markers Lgr5 and Bmi1 was also increased after S. Pullorum infection to support the crypt hyperplasia. In addition, we verified that S. Pullorum infection enhanced the mRNA expression of IL-1β, TLR4, Lgr5 and Bmi1. Our study indicated that S. Pullorum infection damaged the intestinal mucosa barrier to induce diarrhea, affected the abnormal proliferation of intestinal stem cells by over-activation of Wnt/β-catenin pathway in chicken.

A75 ON THE TRAIL OF ALTERNATIVE LIGANDS AND SIGNALING PATHWAYS FOR LGR5

Abstract Background Leucine-rich G protein coupled receptor-5 (LGR5) is a GPCR originally identified as a marker for intestinal stem cells but is now associated to stemness in numerous other tissues. R-spondins (RSPO) are the only reported ligands of LGR5, which upon receptor binding, potentiates the canonical Wnt/b-catenin pathway. Surprisingly, despite the presence of classical GPCR features such as conserved DRY and NPXXY motifs RSPO binding to LGR5 does not induce classical GPCR behaviors such as coupling to heterotrimeric G-proteins or engagement of b-arrestins. For this reason, LGR5 is still considered to be an orphan receptor. Aims Aberrant expression of LGR5 is a common feature of many cancers, including gastrointestinal cancers, but it is still unclear why LGR5 is pro-tumorigenic in some cancers, while anti-tumorigenic in others. One potential explanation is that LGR5 does not only signals through the Wnt/b-catenin pathway, but also to alternative pathways. Thus, the goals of this study were 1), to elucidate LGR5 signaling networks and 2), to identify new LGR5 ligands and activity modulators. We suggested that the intestinal lumen content could be a source of bioactive molecules that could act as alternative LGR5 ligands Methods All experiments were performed on HEK293 cells expressing recombinant LGR5 (HEK293/LGR5) and compared to control cells expressing empty vector. Characterization of LGR5 signaling network was initiated with LC MS/MS spectrometric analyses using cells stimulated with Wnt3a and RSPO1. To identify new LGR5 modulators, commercially available bacteria-derived metabolites (BDM) were tested on HEK293/LGR5 cells stimulated with Wnt3a and RSPO1 and assessed using the TOP-Flash luciferase system. Finally, a mouse intestinal lumen extract (ILE) was investigated in whole-cell impedance assay to detect if it contained native BDM or potential host-derived LGR5 ligands. Results Mass spectrometric analyses revealed distinct protein expression and phosphorylation profiles in HEK293/LGR5 cells stimulated with Wnt3a and RSPO1. The TOP/Flash luciferase assays showed that the flavonoid derivative 3,4-dihydroxyphenylacetic acid (DOPAC) and lipopolysaccharide (LPS) acted as negative regulators of LGR5, while butyrate had no effect. Interestingly, stimulation of HEK293/LGR5 with ILE induced a Gαq-like response in the impedance assay, while control cells did not respond. Conclusions Stimulation of LGR5 with Wnt and RSPO triggered a complex signaling network. In agreement with our hypothesis, some BDM such as DOPAC and LPS were indeed activity modulators of LGR5. Moreover, a still unidentified compound present in the ILE triggered a selective GPCR-like response in HEK293/LGR5 cells. Future works aim at identifying this putative LGR5 ligand(s) and establish the LGR5 interactome using proximity labeling and mass spectrometry. Funding Agencies NSERC, Université de Sherbrooke, FRQS

Haploinsufficiency of Casitas B-Lineage Lymphoma Augments the Progression of Colon Cancer in the Background of Adenomatous Polyposis Coli Inactivation

Casitas B-lineage lymphoma (c-Cbl) is a recently identified ubiquitin ligase of nuclear β-catenin and a suppressor of colorectal cancer (CRC) growth in cell culture and mouse tumor xenografts. We hypothesized that reduction in c-Cbl in colonic epithelium is likely to increase the levels of nuclear β-catenin in the intestinal crypt, augmenting CRC tumorigenesis in an adenomatous polyposis coli (APCΔ14/+) mouse model. Haploinsufficient c-Cbl mice (APCΔ14/+ c-Cbl+/-) displayed a significant (threefold) increase in atypical hyperplasia and adenocarcinomas in the small and large intestines; however, no differences were noted in the adenoma frequency. In contrast to the APCΔ14/+ c-Cbl+/+ mice, APCΔ14/+ c-Cbl+/- crypts showed nuclear β-catenin throughout the length of the crypts and up-regulation of Axin2, a canonical Wnt target gene, and SRY-box transcription factor 9, a marker of intestinal stem cells. In contrast, haploinsufficiency of c-Cbl+/- alone was insufficient to induce tumorigenesis regardless of an increase in the number of intestinal epithelial cells with nuclear β-catenin and SRY-box transcription factor 9 in APC+/+ c-Cbl+/- mice. This study demonstrates that haploinsufficiency of c-Cbl results in Wnt hyperactivation in intestinal crypts and accelerates CRC progression to adenocarcinoma in the milieu of APCΔ14/+, a phenomenon not found with wild-type APC. While emphasizing the role of APC as a gatekeeper in CRC, this study also demonstrates that combined partial loss of c-Cbl and inactivation of APC significantly contribute to CRC tumorigenesis.

MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium

Intestinal stem cells (ISCs) fuel the lifelong self‐renewal of the intestinal tract and are paramount for epithelial repair. In this context, the Wnt pathway component LGR5 is the most consensual ISC marker to date. Still, the effort to better understand ISC identity and regulation remains a challenge. We have generated a Mex3a knockout mouse model and show that this RNA‐binding protein is crucial for the maintenance of the Lgr5+ ISC pool, as its absence disrupts epithelial turnover during postnatal development and stereotypical organoid maturation ex vivo. Transcriptomic profiling of intestinal crypts reveals that Mex3a deletion induces the peroxisome proliferator‐activated receptor (PPAR) pathway, along with a decrease in Wnt signalling and loss of the Lgr5+ stem cell signature. Furthermore, we identify PPARγ activity as a molecular intermediate of MEX3A‐mediated regulation. We also show that high PPARγ signalling impairs Lgr5+ ISC function, thus uncovering a new layer of post‐transcriptional regulation that critically contributes to intestinal homeostasis.

Single-Cell Studies of Intestinal Stem Cell Heterogeneity During Homeostasis and Regeneration.

Single-cell RNA-sequencing (scRNA-seq) provides a unique opportunity to study heterogeneous cell populations within tissues, including the intestinal epithelium, to gain detailed molecular insights into their biology. Many new putative markers of intestinal stem cells and their progeny have been described using single-cell transcriptomics, which has contributed to the identification of novel subpopulations of mature cell types and insight into their developmental trajectories. This approach has revealed tremendous cellular heterogeneity within the intestinal epithelium that is concordant with its diverse and multifaceted functions. We discuss the function of these subpopulations during tissue homeostasis, as well as putative subpopulations with inducible regenerative potential following tissue injury.

The role of tumor budding in colorectal adenocarcinoma: Possible involvement of the intestinal cancer stem cell marker Lgr5.

Tumor budding (TB) is a promising prognostic factor in colorectal cancer (CRC) that is independent of tumor-node-metastasis (TNM) staging. Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a stem cell marker and a member of the canonical Wnt-signaling cascade. It is involved in colorectal carcinogenesis. However, its role in CRC progression and TB needs to be clarified. TB was assessed in both H and E and CK immunostained sections of 92 CRC cases. Associations between TB grade and different clinicopathological parameters were evaluated. Lgr5 expression in CRC cases and its association with TB grade and other clinicopathological features was also evaluated. H and E stained sections revealed low- and high-grade budding in 55 (59.8%) and 37 (40.2%) tumors, respectively, whereas Cytokeratin Immunohistochemistry (CK-IHC) showed low- and high-grade budding in 31 (33.7%) and 61 (66.3%) tumors, respectively. TB grade (in H and E and CK stained sections) was significantly associated with adverse pathological prognostic variables including vascular invasion (P = 0.03 and 0.001), lymph node metastasis (P = 0.001 and 0,001), advanced Dukes (P = 0.000 and 0.000), and TNM (P = 0.001 and 0.000) stages and inversely associated with Tumor infiltrating lymphocytes (TILS) (P = 0.02 and 0.0001) which is known to be a good prognostic indicator. Lgr5 protein was positively expressed in 52.2% (48/92) of the CRCs. Immunoreactivity of Lgr5 was significantly associated with histological grade (P = 0.01), lymph node metastasis (P = 0.002), vascular invasion (P = 0.02), TNM stage (P = 0.000), Dukes stage (P = 0.000), and TILS (P = 0.03). Furthermore, Lgr5 was found to be significantly associated with TB estimated in both H and E and CK stained tumors (P = 0.003 and 0.001 respectively). This study supported the relevance of TB in the assessment of CRC aggressiveness. It also revealed that Lgr5 expression is related to morphologic features in the invasive front of CRC. Lgr5 could have an important role in forming a morphologic feature at the invasive front associated with the aggressiveness of the tumor.

Single-Molecule RNA FISH in Whole-Mount Organoids.

Single-molecule RNA fluorescent in situ hybridization (smFISH) enables the detection and quantification of single RNA molecules. Three-dimensional organoid cultures have emerged as versatile in vitro primary culture models that recapitulate many physiological features of their tissue of origin. Here we describe a protocol to visualize single RNA molecules in organoid cultures. Our method accommodates both a whole-mount staining workflow which requires spinning disk confocal microscopy, and a cryosectioning workflow which is compatible with widefield microscopy. Organoid smFISH enables to address various biological problems that range from the identification of cell types (e.g.,via the intestinal stem cell marker Lgr5) to the quantification of RNA localization in an epithelium.

NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor

The intestinal stem cell (ISC) marker LGR5 is a receptor for R‐spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self‐renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post‐translational regulation of LGR5. Here, we show that the HECT‐domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high‐grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/β‐catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post‐translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression.

Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

Background & AimsClaudin-7 (Cldn7) is a tight junction (TJ) membrane protein located at the apical TJ and basolateral side of intestinal epithelial cells. Deletion of Cldn7 by gene targeting leads to the inflammatory bowel disease–like phenotype in mice, which includes weight loss, diarrhea, mucosa ulceration, and severe intestinal epithelial damage. In this study, we test our hypothesis that Cldn7 plays a critical role in regulating intestinal crypt stem cell functions.MethodsGene expression microarray, quantitative reverse-transcription polymerase chain reaction, in situ hybridization, histologic examinations, immunoblotting, 3-dimensional organoid culture, and various treatments to rescue Cldn7-deficient organoid defects were conducted using global Cldn7 knockout mice and inducible, conditional Cldn7 knockout mice.ResultsGene deletion of Cldn7 in intestines showed significant alteration of expression profiles with striking down-regulation of intestinal crypt stem cell markers such as Olfm4, dislocated proliferative cells, and disrupted epithelial cell differentiation. In addition, the isolated Cldn7-deficient crypts where the stem cells reside were either unable to survive at all or formed defective spheroids, highlighting the functional impairment of crypt stem cells in the absence of Cldn7. Remarkably, the Cldn7-expressing organoids with buddings underwent rapid cell degeneration within days after turning off Cldn7 expression in the culture. We identified that activation of Wnt/β-catenin signaling rescued the organoid defects caused by Cldn7 deletion.ConclusionsIn this study, we show that Cldn7 is indispensable in controlling Wnt/β-catenin signaling–dependent intestinal epithelial stem cell survival, self-renewal, and cell differentiation. This study could open a door to study roles of TJ proteins in stem cell regulations in other tissues and organs.

Abstract A42: Single-cell RNA-seq analysis of human pancreatic ductal adenocarcinoma identifies a novel cell type expressing the intestinal stem cell marker OLFM4

Abstract Pancreatic ductal adenomcarcinoma (PDAC) is one of the most lethal forms of cancer with a 5-year survival rate of 8%. To determine the composition of different cell types in PDAC, we performed single-cell RNA-sequencing (scRNA-seq) analysis of two resected patient tumors. Nine distinct cell types were identified: (1) ductal carcinoma cells expressing mutant KRAS, (2) normal pancreatic islet cells, (3) B-lymphocytes, (4) macrophages, (5) mast cells, (6) nerve cells, (7-8) two types of cancer-associated fibroblasts (CAFs), and (9) a previously unidentified epithelial type with distinctly high expression of OLFM4 and CRISP3. Histologic analysis of human PDAC and chronic pancreatitis (CP) tissue sections confirmed the presence of this distinct cell type and furthermore demonstrated that it corresponded to inflamed/injured ducts and early pancreatic intraepithelial neoplasms (PanINs) but that actual PDACs did not express OLFM4. CRISP3 was not always expressed in this new cell type, but the expression of OLFM4 was always detected in these histologic structures. This distinct pancreatic epithelial cell type was also observed in mouse models for chronic pancreatitis and in mutant-KRAS induced PanINs. We hypothesize that inflammation-induced OLFM4 expression helps drive PanIN formation, and that its expression could be exploited for early PDAC detection. We are currently performing functional studies using organoids to address the role that OLFM4 might play in generation of this distinct cell type. Citation Format: Manisha Rao, Alice Nemajerova, Jinyu Li, Mei Gao, Ki Oh, Richard Moffit, Joseph Kim, George Georgakis, Aaron Sasson, Agnieszka Bialkowkska, Scott Powers. Single-cell RNA-seq analysis of human pancreatic ductal adenocarcinoma identifies a novel cell type expressing the intestinal stem cell marker OLFM4 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A42.

640P - Expression profile of EPHB3 and its prognostic significance in colorectal cancer progression (Running head: Prognostic value of EPHB3 in colorectal cancers)

BackgroundA tumour suppressive role for EPHB receptors in various malignancies has been described, however their expression profile and prognostic significance in human colorectal cancers (CRCs) remain unclear. In this study, we aimed to investigate the expression profile of EPHB3 during CRC progression and determined its prognostic impact in a large cohort of CRC samples. MethodsWe examined the EPHB3 mRNA levels by real time PCR with fresh frozen CRC samples and evaluated protein expression by immunohistochemistry with tissue microarrays containing various benign tumours and 610 CRC samples. AOM-DSS induced colitis model was used to investigate the EPHB3 expression profile during the colitis-associated carcinogenesis. ResultsEPHB3 expression was upregulated in CRCs than in normal colonic mucosa, and showed positive correlations with intestinal stem cell (ISC) markers (EPHB2, OLFM4, LRIG1) and CD44, a candidate cancer stem cell marker. EPHB3 positivity was observed in 24% of 610 CRCs and showed negative correlations with differentiation, lympho-vascular invasions and TNM stages. EPHB3 expression significantly declined during adenoma-carcinoma transition and invasion into deeper layers. In particular, a substantial reduction of EPHB3 was observed in the budding cancer cells at the invasive fronts. In AOM-DSS induced colitis-associated colon cancer model, EPHB3 expression increased along with tumor development. Notably, EPHB3 was positively associated with microsatellite instability (MSI) phenotype but was not associated with CpG island methylator phenotype, KRAS and BRAF mutations. Furthermore, EPHB3 positivity was a prognostic marker for better clinical outcomes in CRC patients. ConclusionsEPHB3 was upregulated in CRCs and showed positive correlations with candidate cancer stem cell marker CD44. EPHB3 expression decreased during adenoma-carcinoma transition and particularly in the budding cancer cells at the invasive fronts. EPHB3 positivity was associated with MSI phenotype and demonstrated to be a good prognostic marker in CRCs. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

638P - Development of a living organoid biobank derived from colorectal cancer patients: Towards personalized medicine

BackgroundOrganoids are 3D in vitroprimary culture of great interest for translational research representing an efficient and reproducible cancer model. The aim of this project is to generate a biobank of colorectal cancer (CRC) patients derived organoids (PDOs) that could be used to analyze molecular characteristics and to test different treatments as well as to study the underlying molecular causes of cancer and treatment resistance. MethodsPrimary or metastatic CRC tissues have been obtained from patients who underwent surgery. Tissue has been washed and incubated with antibiotics. After mechanical and enzymatic digestion, free cells have been seeded in Matrigel with proper medium. Development of organoids has been observed the day after seeding. Organoids have been passaged, expanded and stored in liquid nitrogen to constitute a biobank. For morphological characterization, after reaching an appropriate volume, organoids have been fixed in 4% paraformaldehyde, stained for hematoxylin and eosin (H&E) and immunohistochemistry (IHC) for ki67, CDX2, CK20, MUC2/5 was performed. Immunofluorescence (IF) for DAPI, Lgr5 and CDH1 has been performed.For genomic characterization, DNA has been extracted with phenol-chlorophorm and sequenced with an internal customized panel. A comparison statistical test with matched tissue has been performed. ResultsThe success rate in CRC-PDOs establishment is around 80%. PDOs staining with H&E shows a morphology comparable to the original tissue. IHC shows ki67 staining, nuclear positivity for CDX2, cytoplasmic CK20 and MUC2/5. IF evidences the positivity for the intestinal stem cells marker Lgr5 and CDH1 thus confirms that PDOs faithfully recapitulate original tissue morphology and cell composition.Between 80 and 120ng of genomic DNA has been sequenced. Spectrum of PDOs mutations and polimorphisms displays a good concordance with that of matched patients with an R of linear concordance of 0.9. ConclusionsWe have developed a robust protocol for efficient development of CRC PDOs. Organoids recapitulate morphological and genomic features of the original patient. They work as a solid in vitromodel, suitable for functional studies and drug screening, helping in promoting precision medicine. Legal entity responsible for the studyThe authors. FundingGrants from the Instituto de Salud Carlos III (PI15/02180 to AC and PI18/01508 to TF). FP is supported by the ESMO 2018 fellowship programme. VG was supported by the ESMO 2014 fellowship programme and by Rio Ortega contract CM18/00241 from the Carlos III Health Institute; TF is supported by Joan Rodes contract 17/00026 from the Carlos III Health Institute. NT was supported by Rio Ortega contract CM15/00246 from the Carlos III Health Institute; DR was supported by Joan Rodes contract 16/00040 from the Carlos III Health Institute. MFGB is supported by a Santiago Grisolia fellowship. DisclosureA. Cervantes: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Merck Serono; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Beigene; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Servier; Advisory / Consultancy, Research grant / Funding (institution): Lilly; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): Takeda; Advisory / Consultancy, Research grant / Funding (institution): Astellas; Advisory / Consultancy: Pierre Fabre; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Fibrogen; Research grant / Funding (institution): Amcure; Research grant / Funding (institution): Sierra Oncology; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Medimmune; Research grant / Funding (institution): BMS; Research grant / Funding (institution): MSD; Speaker Bureau / Expert testimony: Amgen; Speaker Bureau / Expert testimony: Foundation Medicine. All other authors have declared no conflicts of interest.

Author Correction: The Expression Analysis of Intestinal Cancer Stem Cell Marker Lgr5 in Colorectal Cancer Patients and the Correlation with Histopathological Markers

The original version of this article unfortunately contained a mistake. In the author group section, the correct name of the first author is "Shirin Salehzadeh." The authors apologize for this oversight and for any confusion it may have caused.

The Expression Analysis of Intestinal Cancer Stem Cell Marker Lgr5 in Colorectal Cancer Patients and the Correlation with Histopathological Markers.

Cancer stem cells (CSCs) have frequently been utilized in the cell characterization and identified responsible for tumor development, metastasis, recurrence, and chemoresistance. CSC surface markers function in cancer cell signaling and are indicated as potential biomarkers for cancer diagnosis and prognosis. As well, dysregulation of cancer-related signaling pathways could promote CSC development and progression. Our aim was to evaluate the expression of colorectal CSC markers and their correlation with cancer proliferation and angiogenesis. In this case-control study, total RNA was extracted from a total of 74 colorectal tumors and 74 adjacent normal tissue biopsies. Then, using a quantitative real-time PCR, the relative expression levels of Lgr5 and Lrig1 were measured in all malignant and healthy samples. Also, immunohistochemical (IHC) staining of tumor tissues was performed for Ki-67 (proliferation) and CD34 (angiogenesis) markers, and the immunoexpression staining scores were obtained. The diagnostic value of the genes was evaluated using receiver operating characteristic (ROC) curve. Possible correlation between CSC markers and immunohistochemical markers in CRC was analyzed by Pearson's correlation test and linear regression. The expression level of Lgr5 in tumor samples showed a significant increase compared with normal samples (p < 0.001) with a fold change of 2.54 (± 0.182). However, there was no significant difference in the relative expression of Lrig1 gene in tissue samples of healthy subjects and patients. The analysis of the ROC showed an AUC of 0.92 for Lgr5 and sensitivity 80% and specificity 96%. Further analysis revealed a significant correlation between mRNA levels of Lgr5 and immunoexpression of Ki-67 (r2 = 0.680, p < 0.001). The high expression levels of Lgr5 found in tumor tissues were correlated with histological parameters, indicating a significant role in CRC development and diagnosis.

Genome-wide mapping of DNA-binding sites identifies stemness-related genes as directly repressed targets of SNAIL1 in colorectal cancer cells

At the molecular level, epithelial-to-mesenchymal transition (EMT) necessitates extensive transcriptional reprogramming which is orchestrated by a small group of gene-regulatory factors that include the zinc-finger DNA-binding protein SNAIL1. Although SNAIL1 is a well-known master regulator of EMT, knowledge of its immediate target genes is incomplete. Here, we used ChIP-seq to identify genes directly regulated by SNAIL1 in colorectal adenocarcinoma cells. When comparing the genomic distribution of SNAIL1 to that of the intestinal stem cell (ISC) transcription factors ASCL2 and TCF7L2, we observed a significant overlap. Furthermore, SNAIL1 ChIP-seq peaks are associated with a substantial fraction of ISC signature genes. In two colorectal cancer cell lines, we verified that SNAIL1 decreases ISC marker expression. Likewise, SNAIL1 directly represses the proto-oncogene MYB, and the long noncoding RNA (lncRNA) WiNTRLINC1, a recently described regulator of ASCL2. SNAIL1 targets multiple regulatory elements at the MYB and WiNTRLINC1 loci, and displaces ASCL2 and TCF7L2 from their binding regions at a MYB downstream regulatory element. Correlation analyses and expression profiling showed antiparallel expression of SNAIL1 and MYB in colorectal and breast cancer cell lines and tumor transcriptomes, suggesting that SNAIL1 controls MYB expression in different tissues. MYB loss-of-function attenuated proliferation and impaired clonogenicity in two- and three-dimensional cell cultures. Therefore, SNAIL1-mediated downregulation of MYB and ISC markers like WiNTRLINC1 likely contributes to the decrease in proliferation known to be associated with EMT, while simultaneously abrogating stemness features of colorectal cancer cells. Apparently, the relationship between EMT and stemness varies in different tumor entities.