Erratum: Corrigendum: High-throughput RNAi screen for essential genes and drug synergistic combinations in colorectal cancer

Abstract Metastatic colorectal cancer is a leading cause of cancer death. However, current therapy options are limited to chemotherapy, with the addition of anti-EGFR antibodies for patients with RAS wild-type tumours. Novel drug targets, or drug combinations that induce a synergistic response, would be of great benefit to patients. The identification of genes that are essential for cell survival can be undertaken using functional genomics screens. Furthermore, performing such screens in the presence of a targeted agent would allow the identification of combinations that result in a synthetic lethal interaction. Here, we present a dataset containing the results of a large scale RNAi screen (815 genes) to detect essential genes as well as synergistic combinations with targeted therapeutic agents using a panel of 27 colorectal cancer cell lines. These data identify genes that are essential for colorectal cancer cell survival as well as synthetic lethal treatment combinations using novel computational approaches. Moreover, this dataset could be utilised in combination with genomic profiling to identify predictive biomarkers of response.

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Combined flow cytometry and high throughput image analysis for the study of essential genes in Caenorhabditis elegans

10.1101/218735 ◽

2017 ◽

Author(s):

Blanca Hernando-Rodríguez ◽

Annmary Paul Erinjeri ◽

María Jesús Rodríguez-Palero ◽

Val Millar ◽

Sara González-Hernández ◽

...

Keyword(s):

Image Analysis ◽

Caenorhabditis Elegans ◽

High Throughput ◽

Rnai Screen ◽

Essential Genes ◽

Genetic Interactions ◽

Automated Image Analysis ◽

C Elegans ◽

Larval Stages ◽

Lethal Mutations

ABSTRACTBackgroundThe advancement in automated image based microscopy platforms coupled with high throughput liquid workflows has facilitated the design of large scale screens utilizing multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high throughput approaches.ResultsIn C. elegans, non-conditional lethal mutations can be maintained in heterozygosis using chromosome balancers, commonly labelled with GFP in the pharynx. Moreover gene-expression is typically monitored by the use of fluorescent reporters marked with the same fluorophore. Therefore, the separation of the different populations of animals at early larval stages represents a challenge. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at early larval stages. Because sorting is not completely error-free, we develop an automated high-throughput image-analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image-analysis in a functional genomic RNAi screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while, homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both, known and new PHB genetic interactors affecting the UPRmt and growth.ConclusionsA systematic way to analyse genetic interactions of essential genes in multicellular organisms is lacking. The method presented here allows the study of balanced lethal mutations in a high-throughput manner and can be easily adapted depending on the user’s requirements. Therefore, it will serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

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Revealing the changes of IgG subclass‐specific N ‐glycosylation in colorectal cancer progression by high‐throughput assay

PROTEOMICS - CLINICAL APPLICATIONS ◽

10.1002/prca.202000022 ◽

2021 ◽

pp. 2000022

Author(s):

Si Liu ◽

Yuting Yu ◽

Yuanyuan Liu ◽

Jiajing Lin ◽

Yang Fu ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Progression ◽

High Throughput ◽

Igg Subclass ◽

Colorectal Cancer Progression ◽

High Throughput Assay

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A high-throughput genome-wide RNAi screen identifies modifiers of survival motor neuron protein

Cell Reports ◽

10.1016/j.celrep.2021.109125 ◽

2021 ◽

Vol 35 (6) ◽

pp. 109125

Author(s):

Nikki M. McCormack ◽

Mahlet B. Abera ◽

Eveline S. Arnold ◽

Rebecca M. Gibbs ◽

Scott E. Martin ◽

...

Keyword(s):

Motor Neuron ◽

High Throughput ◽

Rnai Screen ◽

Survival Motor Neuron ◽

Survival Motor Neuron Protein ◽

Genome Wide ◽

Motor Neuron Protein

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O23: CHARACTERISING PATIENT-DERIVED COLORECTAL CANCER TISSUE-ORIGINATED ORGANOIDAL SPHEROIDS FOR HIGH-THROUGHPUT MICROFLUIDIC APPLICATIONS

British Journal of Surgery ◽

10.1093/bjs/znab117.023 ◽

2021 ◽

Vol 108 (Supplement_1) ◽

Author(s):

MI Khot ◽

M Levenstein ◽

R Coppo ◽

J Kondo ◽

M Inoue ◽

...

Keyword(s):

Colorectal Cancer ◽

Fluid Flow ◽

High Throughput ◽

Microfluidic Devices ◽

In Vitro Models ◽

Fluorescent Imaging ◽

Cancer Tissue ◽

Cell Models

Abstract Introduction Three-dimensional (3D) cell models have gained reputation as better representations of in vivo cancers as compared to monolayered cultures. Recently, patient tumour tissue-derived organoids have advanced the scope of complex in vitro models, by allowing patient-specific tumour cultures to be generated for developing new medicines and patient-tailored treatments. Integrating 3D cell and organoid culturing into microfluidics, can streamline traditional protocols and allow complex and precise high-throughput experiments to be performed with ease. Method Patient-derived colorectal cancer tissue-originated organoidal spheroids (CTOS) cultures were acquired from Kyoto University, Japan. CTOS were cultured in Matrigel and stem-cell media. CTOS were treated with 5-fluorouracil and cytotoxicity evaluated via fluorescent imaging and ATP assay. CTOS were embedded, sectioned and subjected to H&E staining and immunofluorescence for ABCG2 and Ki67 proteins. HT29 colorectal cancer spheroids were produced on microfluidic devices using cell suspensions and subjected to 5-fluorouracil treatment via fluid flow. Cytotoxicity was evaluated through fluorescent imaging and LDH assay. Result 5-fluorouracil dose-dependent reduction in cell viability was observed in CTOS cultures (p<0.01). Colorectal CTOS cultures retained the histology, tissue architecture and protein expression of the colonic epithelial structure. Uniform 3D HT29 spheroids were generated in the microfluidic devices. 5-fluorouracil treatment of spheroids and cytotoxic analysis was achieved conveniently through fluid flow. Conclusion Patient-derived CTOS are better complex models of in vivo cancers than 3D cell models and can improve the clinical translation of novel treatments. Microfluidics can streamline high-throughput screening and reduce the practical difficulties of conventional organoid and 3D cell culturing. Take-home message Organoids are the most advanced in vitro models of clinical cancers. Microfluidics can streamline and improve traditional laboratory experiments.

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