Organoid-based Pharmacotyping Assay

Organoids were dissociated into single cells. 500 viable cells were plated per well in in 20μL 10% Matrigel / human complete organoid media. Therapeutic compounds were added 24 hours post plating, after the reformation of organoids was visually verified. Chemotherapeutic were tested in triplicates: gemcitabine, paclitaxel, SN38 range from 8.1x10⁻¹² M to 2.0x10⁻⁶ M, and 5-FU and Oxaliplatin range from 1.0x10⁻⁸ M to 5.0x10⁻⁵ M. Targeted drugs were tested in singlicates (range from 1.0x10⁻⁸ M to 1.0x10⁻⁵ M). Compounds were dissolved in DMSO and all treatment wells were normalized to 0.5% DMSO content. After 5 days cell viability was assessed using CellTiter-Glo as per manufacturer’s instruction (Promega) on a SpectraMax I3 (Molecular Devices) plate reader. A three-parameter log-logistic function with upper limit equal to the mean of the DMSO values was fit to the pharmacotyping data (viability vs. dose) with CRAN package drc v3.0-1 (61 (link)). Quality control was performed on the curve fitness: rejection of the curve if 100% plateau is located beyond 2 standard deviation of the mean DMSO control and visual inspection, leading to possible rejection, of the top 5% curves ranked with the highest sum of the squared differences between triplicate measurements and fitted curve. The area under the curve (AUC) was calculated using CRAN package Bolstad2 v1.0-28 (https://cran.r-project.org/web/packages/Bolstad2/). Normalized AUC was obtained by dividing the AUC value by the maximum area for the concentration range measured for each drug. The range of the normalized AUC is between 0 and 1.

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Tiriac H., Belleau P., Engle D.D., Plenker D., Deschênes A., Somerville T.D., Froeling F.E., Burkhart R.A., Denroche R.E., Jang G.H., Miyabayashi K., Young C.M., Patel H., Ma M., LaComb J.F., Palmaira R.L., Javed A.A., Huynh J.C., Johnson M., Arora K., Robine N., Shah M., Sanghvi R., Goetz A.B., Lowder C.Y., Martello L., Driehuis E., LeComte N., Askan G., Iacobuzio-Donahue C.A., Clevers H., Wood L.D., Hruban R.H., Thompson E., Aguirre A.J., Wolpin B.M., Sasson A., Kim J., Wu M., Bucobo J.C., Allen P., Sejpal D.V., Nealon W., Sullivan J.D., Winter J.M., Gimotty P.A., Grem J.L., DiMaio D.J., Buscaglia J.M., Grandgenett P.M., Brody J.R., Hollingsworth M.A., O’Kane G.M., Notta F., Kim E., Crawford J.M., Devoe C., Ocean A., Wolfgang C.L., Yu K.H., Li E., Vakoc C.R., Hubert B., Fischer S.E., Wilson J.M., Moffitt R., Knox J., Krasnitz A., Gallinger S, & Tuveson D.A. (2018). Organoid profiling identifies common responders to chemotherapy in pancreatic cancer. Cancer discovery, 8(9), 1112-1129.

Publication 2018

Cell viability Cells Chemotherapeutic Devices Dmso Drug Gemcitabine Human Matrigel Organoids Oxaliplatin Paclitaxel Promega Targeted drugs

Corresponding Organization : Mount Sinai Hospital

Other organizations : Johns Hopkins University, University of Baltimore, École Polytechnique Fédérale de Lausanne, Stony Brook University, Kettering University, Memorial Sloan Kettering Cancer Center, University of California, Davis, New York Genome Center, Thomas Jefferson University, SUNY Downstate Health Sciences University, University Medical Center Utrecht, Royal Netherlands Academy of Arts and Sciences, Princess Máxima Center, Broad Institute, Dana-Farber Cancer Institute, Hofstra University, Donald & Barbara Zucker School of Medicine at Hofstra/Northwell, University of Pennsylvania, University of Nebraska Medical Center, Cornell University

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Variable analysis

independent variables

Chemotherapeutic compounds (gemcitabine, paclitaxel, SN38) tested in a range from 8.1x10^-12 M to 2.0x10^-6 M
Chemotherapeutic compounds (5-FU, Oxaliplatin) tested in a range from 1.0x10^-8 M to 5.0x10^-5 M
Targeted drugs tested in a range from 1.0x10^-8 M to 1.0x10^-5 M

dependent variables

Cell viability assessed using CellTiter-Glo assay after 5 days of treatment

control variables

Organoids were dissociated into single cells
500 viable cells were plated per well in 20μL 10% Matrigel / human complete organoid media
Therapeutic compounds were added 24 hours post plating, after the reformation of organoids was visually verified
Compounds were dissolved in DMSO and all treatment wells were normalized to 0.5% DMSO content

positive controls

Not explicitly mentioned

negative controls

DMSO control wells

Annotations

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