High-Throughput siRNA Screening of Breast Cancer

The gene library for the siRNA screen was chosen from a meta-analysis of breast cancer published sequencing studies (1 (link),12 (link)–16 (link)), choosing the top 200 recurrently mutated genes that encompassed the top 100 genes in ER+, HER2+ and TNBC respectively (Table S1). Reverse transfection siRNA spheroid screens were performed as previously described in triplicate (5 ,11 (link)). MCF10a, MCF10NeoT, MCF10AT1, MCF10DCIS.com, MCF10Ca1a, MCF10Ca1d, MCF10Ca1h cell lines were reverse-transfected with 37.5nM of Dharmacon siGENOME siRNA using Lullaby reagent (Oz biosciences). BT20, CAL-51, HCC70, HCC1937, HCC1806, Hs578t and MDA-MB-157 cell lines were reverse-transfected with 37.5nM of Dharmacon siGENOME siRNA using Viromer (lipocalyx). Cell viability was measured after 5 days by CellTiter-Glo. The progression series screen was analysed using z-score analysis. The TNBC cell line targeted screen was analysed by plate median normalised values. Spheroids were imaged using a Nikon TE 2000 inverted wide field microscope fitted with a motorised stage, filter wheels and a Pro-Scan controller (Prior Scientific), Shutter (Sutter instruments), Orca R2 camera (Hamamatsu), 84000v2 DAPI/FITC/TRITC/Cy5 Quad (Chroma technology). The microscope is operated by HCI imaging software 4.3.1.33. Experiments are carried out at 37⁰C (Solent Scientific) and 10% CO₂.

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Peck B., Bland P., Mavrommati I., Muirhead G., Cottom H., Wai P.T., Maguire S.L., Barker H.E., Morrison E., Kriplani D., Yu L., Gibson A., Falgari G., Brennan K., Farnie G., Buus R., Marlow R., Novo D., Knight E., Guppy N., Kolarevic D., Susnjar S., Milijic N.M., Naidoo K., Gazinska P., Roxanis I., Pancholi S., Martin L.A., Holgersen E.M., Cheang M.C., Noor F., Postel-Vinay S., Quinn G., McDade S., Krasny L., Huang P., Daley F., Wallberg F., Choudhary J.S., Haider S., Tutt A.N, & Natrajan R. (2021). 3D functional genomics screens identify CREBBP as a targetable driver in aggressive triple-negative breast cancer. Cancer research, 81(4), 847-859.

Publication 2021

Lullaby reagent Orca R2 camera

Cancer of breast Cell line Cell viability Dapi Fitc Gene library Genes Her2 Microscope Orca Progression Scan Sirna Transfection Tritc

Corresponding Organization :

Other organizations : Breast Cancer Now, Institute of Cancer Research, Queen's University Belfast, Walter and Eliza Hall Institute of Medical Research, University of Manchester, University of Oxford, King's College London, Royal Marsden NHS Foundation Trust, Oncology Institute of Vojvodina, Université Paris-Saclay, Inserm, Institut Gustave Roussy, Fondation de l'Avenir

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Variable analysis

independent variables

SiRNA (Dharmacon siGENOME siRNA)

dependent variables

Cell viability (measured by CellTiter-Glo)

control variables

Cell lines (MCF10a, MCF10NeoT, MCF10AT1, MCF10DCIS.com, MCF10Ca1a, MCF10Ca1d, MCF10Ca1h, BT20, CAL-51, HCC70, HCC1937, HCC1806, Hs578t, MDA-MB-157)
Transfection reagent (Lullaby reagent (Oz biosciences) for MCF10a, MCF10NeoT, MCF10AT1, MCF10DCIS.com, MCF10Ca1a, MCF10Ca1d, MCF10Ca1h; Viromer (lipocalyx) for BT20, CAL-51, HCC70, HCC1937, HCC1806, Hs578t, MDA-MB-157)
Incubation conditions (37°C, 10% CO2)

controls

No positive or negative controls were explicitly mentioned.

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