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CRISPR/CAS9 RIBONUCLEOPROTEINS AS A POWERFUL HIGH-THROUGHPUT SCREENING TOOL TO UNCOVER PATHWAY INTERACTIONS IN COLORECTAL CANCER

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Introduction

The Wnt signaling pathway and the p53 signaling pathway are key regulators in colorectal cancer (CRC) biology. For the development of new potential therapeutic approaches, a deeper understanding of the interactions between these pathways is required. We have developed a high-throughput arrayed phenotypic screening platform that combines multiplexed CRISPR/Cas9 ribonucleoprotein (RNP) knockouts with fluorophore pathway reporters, nuclear immunohistochemistry staining and fluorescence imaging. This allowed us to perform phenotypic analysis of knockout and pathway activation effects on a single-cell level to uncover new connections between Wnt and p53 signaling.

Aims & Methods

We established CRISPR/Cas9 RNP-based multiplexed knockouts both in colorectal cancer cell lines and patient-derived colorectal cancer organoids. We produce single guide RNA (sgRNA) by in vitro transcription. Utilizing RNPs for genome editing and mutating specific target genes allows for fast and precise editing and fewer off-target effects when compared with traditional plasmid or lentivirus-based CRISPR delivery methods. Furthermore, the small molecular size of an RNP editing complex provides the opportunity to easily multiplex several target gene knockouts or increase individual knockout efficiency by targeting the same gene with several sgRNAs simultaneously. We experimentally determined the ideal amount of individual sgRNAs to use in order to maximize knockout efficiency of a single target gene.
Using our optimized RNP knockout platform, we then conducted an arrayed phenotypic screen with 180 CRC-relevant target genes in 6 HCT116-derived adherent cell lines with different mutational backgrounds in the Wnt & p53 signaling pathways. After cell fixation and staining of nucleus and cell body, we used fluorescence microscopy images for cell segmentation in silico. This allowed us to monitor the transcriptional activity in p53 and Wnt/β-catenin signaling as well as the cell morphology on a single cell basis.

Results

We were able to show that gene knockout efficiency is highly dependent on the individual sgRNA used. Furthermore, we demonstrated that combining up to four sgRNAs that target the same gene in one knockout experiment significantly increases overall efficiency while not significantly affecting cell growth and viability when compared to using a single sgRNA. Using four target guides per gene, we succeeded in establishing high-efficiency CIRPSR/Cas9 RNP knockout protocols with >90% pooled knockout efficiency in adherent colorectal cancer cell lines using lipofection for complex delivery and >40% pooled knockout efficiency in patient-derived colorectal cancer organoids using electroporation.
In the arrayed phenotypic screen, we were able to detect pathway activation and deactivation using a fluorophore-based reporter system coupled to DNA transcription factor binding sites of Transcription Factor 7 Like 2 (TCF7L2) for Wnt signaling and a fluorescent immunohistochemistry staining for p53.

Conclusion

Our innovative approach combining CRISPR/Cas9 RNP technology with detailed imaging and phenotypic analysis has proven to be a powerful tool in dissecting the roles of the Wnt and p53 signaling pathways in colorectal cancer. This approach can be easily scaled to any other set of genes or signaling pathway of interest. Moving forward, we intend to verify target genes of interest discovered in our arrayed phenotypic screen in patient-derived colorectal cancer organoids.

CRISPR/CAS9 RIBONUCLEOPROTEINS AS A POWERFUL HIGH-THROUGHPUT SCREENING TOOL TO UNCOVER PATHWAY INTERACTIONS IN COLORECTAL CANCER

Carl Philipp Schilling 1, Kim Elisabeth Boonekamp 2, Michael Boutros 2

1 German Cancer Research Center (DKFZ), Heidelberg, Germany|||Heidelberg University, Heidelberg, Germany|||DKFZ-Hector Cancer Institute at the University Medical Center Mannheim, Heidelberg, Germany|||Freiburg University Medical Center, Freiburg im Breisgau

2 German Cancer Research Center (DKFZ), Heidelberg, Germany|||Heidelberg University, Heidelberg, Germany|||DKFZ-Hector Cancer Institute at the University Medical Center Mannheim, Heidelberg, Germany

Event

UEG Week Vienna 2024

Topics

Digestive Oncology Endoscopy Mechanisms & Personalised Medicine Colorectal

Submission format

Abstract

Session

Diagnose and treat to target in GI cancer: Latest news

Citation

United European Gastroenterology Journal 2024; 12 (Supplement 8)

Published

2024

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The role of nurses/technicians in performing endoscopic necrosectomy of peripancreatic collections

Ana Bertic 1

1 Clinical Hospital Center „Sisters of Charity“, Zagreb, Croatia

Event

UEG Week Vienna 2024

Topics

Nurses

Session

Abstracts II: Patient care and endoscopic techniques

Citation

United European Gastroenterology Journal 2024; 12 (Supplement 8)

Published

2024

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Endoscopy nursing education impact: The portuguese experience

Filomena Maia 1

1 Prelada`s Hospital, Oporto, Portugal

Event

UEG Week Vienna 2024

Topics

Nurses

Session

Abstracts I: Management and education

Citation

United European Gastroenterology Journal 2024; 12 (Supplement 8)

Published

2024

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PIGR-DEPENDENT INCREASE OF CIRCULATORY IGA TITRES EXACERBATES MURINE STEATOHEPATITIS

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Introduction

As the most abundant antibody in the gastrointestinal tract, IgA is an essential component of mucosal immunity due to generation of immune protection against invading pathogens. Interestingly, elevated systemic IgA titres are associated with steatotic liver diseases (SLD). Yet, the underlying mechanism responsible for systemic IgA accumulation is still not fully understood. Due to its control of IgA transport into the gut lumen, either from the liver via bile as well as across intestinal epithelium, the polymeric immunoglobulin receptor (pIgR) is one of the key regulators of IgA localization.

Aims & Methods

Here, our aim was to determine the contribution of pIgR-dependent IgA secretion to the development of steatohepatitis. First, we assessed systemic and faecal IgA titres in a cohort of biopsy-proven metabolic dysfunction-associated steatotic liver disease (MASLD) patients. Additionally, pIgR expression and IgA levels were assessed in different parts of the gut-liver axis in wild-type (Wt) mice fed chow or methionine- and choline-deficient (MCD) diet for 4 weeks to induce murine SLD. To study alterations in hepatic injury resulting from shifts in IgA titres, Wt and pIgR-deficient (pIgR^-/-) mice were fed MCD diet for 4 weeks. Moreover, IgA-deficient bone marrow was transplanted into Wt and pIgR^-/- mice which were then fed MCD diet, allowing us to further elucidate the specific effect of circulatory and intestinal IgA disturbances on murine steatohepatitis.

Results

MASLD patients had higher IgA titres in serum and stool in comparison to healthy individuals, which positively correlated with hepatic fibrosis. Similar changes in IgA concentrations were found in various murine MASLD models. Importantly, while increased faecal IgA levels associated with the induction of pIgR in murine colon epithelium, pIgR expression in the liver as well as in the distal small intestine was reduced during hepatic fibrosis in MCD-fed mice. Hence, altered site-specific pIgR regulation and aberrant hepatic IgA secretion might result in elevated systemic IgA deposition, thereby promoting SLD. In line, pIgR^-/- mice, which lack intestinal IgA and accumulate IgA in circulation, developed more hepatic inflammation and injury associated with increased neutrophil and macrophage infiltration compared to their Wt littermates after MCD diet. IgA-deficient bone marrow transplantation prevented aggravated hepatic injury and steatohepatitis in pIgR^-/- mice, indicating that pIgR-mediated systemic IgA accumulation exacerbates SLD.

Conclusion

Our research identified pIgR-dependent regulation of IgA secretion as an important mechanism contributing to the severity of murine steatohepatitis.

PIGR-DEPENDENT INCREASE OF CIRCULATORY IGA TITRES EXACERBATES MURINE STEATOHEPATITIS

Dragana Rajčić 1, Constanze Hoebinger 1, Sonja Lang 2, Münevver Demir 3, Tim Hendrikx 1

1 Medical University of Vienna, Vienna, Austria

2 University Hospital Cologne, Cologne, Germany

3 Charité Universitätsmedizin Berlin, Berlin, Germany

Event

UEG Week Vienna 2024

Topics

Hepatobiliary IBD Immunology Mechanisms & Personalised Medicine Pancreas

Submission format

Abstract

Session

Basic mechanisms of disease in liver, pancreas and intestine

Citation

United European Gastroenterology Journal 2024; 12 (Supplement 8)

Published

2024

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Liver nurses and allied health professions in Europe: The EASL taskforce

Malene O'Connell 1

1 None, None, Denmark

Event

UEG Week Berlin 2025

Topics

Nurses

Session

Innovative approaches in liver disease management

Citation

United European Gastroenterology Journal 2025; 13 (Supplement 8)

Published

2025

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Introduction

The number of individuals who will be treated for and cured from HCV infection world-wide is expected to exceed 1 million per year for the next decade. In those with compensated advanced chronic liver disease (cACLD), the risks of de-novo hepatocellular carcinoma (HCC) and hepatic decompensation are decreased but not completely abolished by HCV-cure. Thus, risk stratification is key to decrease resource utilization by individualizing post-treatment management. We evaluated whether the incidences of HCC and hepatic decompensation decrease with time after HCV-cure and whether the discriminatory ability of a one-time post-treatment assessment is maintained during long-term follow-up.

Aims & Methods

We retrospectively analyzed cACLD patients with paired liver stiffness measurement (LSM) & platelet count (PLT) before and after HCV-cure by interferon-free therapies from 7 European regions. Cumulative incidence curves were used to estimate the incidence of hepatic decompensation/HCC over time in the overall group and throughout previously defined risk strata: Baveno VII criteria for clinically significant portal hypertension after HCV-cure (CSPH excluded: FU-LSM <12kPa & FU-PLT >150G/L; CSPH ruled-in: FU-LSM ≥25kPa), and Semmler et al., J Hepatol 2022 (algorithm based on age, LSM, albumin and alcohol consumption) for HCC.

Results

2347 patients (mean age 60±12 years, 60% male, 21% obese, 21% diabetes) with a median LSM of 16.3kPa (IQR: 12.1-24.5, 25% ≥25kPa) before HCV-cure were followed for a median of 6.0 years after treatment. Interestingly, the incidence of first hepatic decompensation was strictly linear with 65 patients (2.8%) developing hepatic decompensation before eventually being diagnosed with HCC (incidence rate 0.57/100 patient years, incidence at 6 years: 3.1%). Similarly, incidence of HCC was also linear with 184 patients (7.8%) developing HCC (incidence rate 1.60/100 patient years, incidence at 6 years: 8.3%). Stratifying the risks for hepatic decompensation according to Baveno VII identified patients with a distinct prognosis during long-term follow-up (incidence at 6 years: 0.3% [CSPH excluded] vs. 2.1% [Gray-zone] vs. 15.6% [CSPH ruled-in]). Similarly, cumulative HCC incidence at 6 years was 15.8% in the high-risk group vs. 5.4% in the low-risk group according to Semmler et al. Results were similar when limiting prediction to events occurring >3 years after HCV-cure.

Conclusion

In patients with cACLD, the risks of hepatic decompensation, and more importantly, HCC remain constant after HCV-cure, even in the long-term (>3 years). A one-time post-treatment risk stratification based on non-invasive criteria confers important prognostic information that is maintained during long-time follow-up, as the hazards over time remain proportional.

THE RISKS OF FIRST HEPATIC DECOMPENSATION AND HCC REMAIN CONSTANT DURING LONG-TERM FOLLOW-UP AND CAN BE STRATIFIED BY A ONE-TIME ASSESSMENT AFTER HCV-CURE

Georg Semmler 1, Sonia Alonso Lopez 2, Monica Pons 3, Sabela Lens 4, Elton Dajti 5, Marie Griemsmann 6, Alberto Zanetto 7, Lukas Burghart 8, Stephanie Hametner-Schreil 9, Lukas Hartl 1, Marisa Manzano 10, Sergio Rodriguez Tajes 4, Paola Zanaga 7, Michael Schwarz 11, Maria Luisa Gutierrez 12, Mathias Jachs 1, Anna Pocurull 13, Benjamin Polo 14, Dominik Ecker 9, Beatriz Mateos 15, Sonia Izquierdo 16, Yolanda Real 17, Michael Gschwantler 18, Francesco Paolo Russo 7, Francesco Azzaroli 5, Benjamin Maasoumy 6, Thomas Reiberger 1, Xavier Forns 19, Joan Genesca 20, Rafael Banares 2, Mattias Mandorfer 1

1 Medical University of Vienna, Vienna, Austria|||Medical University of Vienna, Vienna, Austria

2 Hospital General Universitario Gregorio Marañón, Madrid, Spain|||Instituto De Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain|||Universidad Complutense de Madrid, Madrid, Spain

3 Vall d’Hebron University Hospital, Vall d’Hebron Institut of Research (VHIR), Vall d’Hebron Barcelona Hospital Campus, Universitat Autònoma de Barcelona, Barcelona, Spain

4 Hospital Clínic, IDIBAPS, Universitat de Barcelona, Barcelona, Spain|||Centro de Investigación Biomédica En Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain

5 University of Bologna, Bologna, Italy

6 Hannover Medical School, Hannover, Germany

7 Padua University Hospital, Padua, Italy

8 Medical University of Vienna, Vienna, Austria|||Klinikum Ottakring, Vienna, Austria

9 Ordensklinikum Linz Barmherzige Schwestern, Linz, Austria

10 Hospital Universitario 12 De Octubre, Madrid, Spain

11 Medical University of Vienna, Vienna, Austria|||Medical University of Vienna, Vienna, Austria|||Klinikum Ottakring, Vienna, Austria

12 Hospital Universitario Fundación Alcorcón, Madrid, Spain

13 Hospital Clínic, IDIBAPS, Universitat de Barcelona, Barcelona, Spain|||Centro de Investigacíon Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain

14 Hospital Universitario Fundación Jimenez Díaz, Madrid, Spain

15 Hospital Universitario Ramón y Cajal, Madrid, Spain

16 Hospital Universitario Clínico San Carlos, Madrid, Spain

17 Hospital Universitario La Princesa, Madrid, Spain

18 Klinikum Ottakring, Vienna, Austria

19 Centro de Investigación Biomédica En Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain|||Hospital Clínic, IDIBAPS, Universitat de Barcelona, Barcelona, Spain

20 Vall d’Hebron University Hospital, Vall d’Hebron Institut of Research (VHIR), Vall d’Hebron Barcelona Hospital Campus, Universitat Autònoma de Barcelona, Barcelona, Spain|||Centro de Investigacíon Biomédica en Red de Enfermedades Hepáticas y Digestivas (C

Conference

UEG Week Copenhagen 2023

Topics

Hepatobiliary

Submission format

Abstract

Session

Novel aspects in liver cirrhosis (Posters)

Citation

United European Gastroenterology Journal 2023; 11 (Supplement 8)

Published

2023

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Introduction

Despite the usefulness of white light endoscopy (WLE) and nonmagnified narrow band imaging (NBI) for screening superficial esophageal squamous cell carcinoma (SESCC) and precancerous lesions, these lesions may be missed due to their subtle features and interpretation variations among endoscopists.

Aims & Methods

Our team has developed an artificial intelligence (AI) system to detect SESCC and precancerous lesions using WLE and nonmagnified NBI. This study aims to evaluate the impact of this AI system on clinical diagnosis.
A prospective tandem study was conducted to evaluate the auxiliary diagnostic performance of the AI system in a real clinical setting. Consecutive patients undergoing sedated upper gastrointestinal endoscopy at 12 hospitals in China were recruited. Patients were randomly assigned to either the AI-assisted first group or the routine first group at a 1:1 ratio. The same endoscopist at each center performed tandem upper gastrointestinal endoscopy for each eligible patient on the same day. In the AI-assisted group, the endoscopist performed the first examination with the assistance of the AI system and the second examination without it. In the routine first group, the order of examinations was reversed. The primary outcome was the miss rate of SESCC and precancerous lesions. The secondary outcome including the detection rate of SESCC and precancerous lesions.

Results

Between October 2021 and June 2022, a total of 5,856 eligible patients were included and randomly assigned to the AI-assisted first group and 5,850 were assigned to the routine first group. The per-lesion miss rate was significantly lower in the AI-assisted first group than in the routine first group (1.7% vs 6.7%; Risk ratio (RR)=0.25; p=0.08). The detection rate in the AI-assisted first group was significantly higher than that in the routine first group (1.8% vs 1.3%; RR=1.38; p=0.03).

Conclusion

The proposed AI system can reduce the miss rate and improve the detection rate of SESCC and precancerous lesions during upper gastrointestinal endoscopy under WLE and non-magnified NBI.

ARTIFICIAL INTELLIGENCE-ASSISTED SYSTEM IMPROVES THE ENDOSCOPIC DIAGNOSIS OF SUPERFICIAL ESOPHAGEAL SQUAMOUS CELL CARCINOMA AND PRECANCEROUS LESIONS: A PROSPECTIVE, MULTICENTER, TANDEM, RANDOMIZED CONTROLLED STUDY

Xianglei Yuan 1, Wei Liu 1, Yixiu Lin 1, Xianhui Zeng 2, Bing Hu 1

1 West China Hospital, Sichuan University, Chengdu, China

2 West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, China

Conference

UEG Week Copenhagen 2023

Topics

Oesophagus

Submission format

Abstract

Session

Innovations in upper GI endoscopy (Posters)

Citation

United European Gastroenterology Journal 2023; 11 (Supplement 8)

Published

2023

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