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A simple high-throughput approach identifies actionable drug sensitivities in patient-derived tumor organoids.

ABSTRACT: Tumor organoids maintain cell-cell interactions, heterogeneity, microenvironment, and drug response of the sample they originate from. Thus, there is increasing interest in developing tumor organoid models for drug development and personalized medicine applications. Although organoids are in principle amenable to high-throughput screenings, progress has been hampered by technical constraints and extensive manipulations required by current methods. Here we introduce a miniaturized method that uses a simplified geometry by seeding cells around the rim of the wells (mini-rings). This allows high-throughput screenings in a format compatible with automation as shown using four patient-derived tumor organoids established from two ovarian and one peritoneal high-grade serous carcinomas and one carcinosarcoma of the ovary. Using our automated screening platform, we identified personalized responses by measuring viability, number, and size of organoids after exposure to 240 kinase inhibitors. Results are available within a week from surgery, a timeline compatible with therapeutic decision-making.

SUBMITTER: Phan N

PROVIDER: S-EPMC6389967 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A simple high-throughput approach identifies actionable drug sensitivities in patient-derived tumor organoids.

Phan Nhan N Hong Jenny J JJ Tofig Bobby B Mapua Matthew M Elashoff David D Moatamed Neda A NA Huang Jin J Memarzadeh Sanaz S Damoiseaux Robert R Soragni Alice A

Communications biology 20190225

Tumor organoids maintain cell-cell interactions, heterogeneity, microenvironment, and drug response of the sample they originate from. Thus, there is increasing interest in developing tumor organoid models for drug development and personalized medicine applications. Although organoids are in principle amenable to high-throughput screenings, progress has been hampered by technical constraints and extensive manipulations required by current methods. Here we introduce a miniaturized method that use ...[more]

PMID: 30820473

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Project description:The long culture duration of patient-derived organoids (PDOs) have severely limited their clinical applications. The aim of this study was to determine the effect of lactate supplementation on the growth, genetic profiles and drug sensitivities of PDOs from hepatopancreatobiliary tumors. LM3, Huh7, Panc02, and RBE cell lines were cultured as organoids in the presence or absence of lactate, and total protein was extracted to measure the expression of α-enolase (ENO1), hypoxia-inducible factor-1α (HIF1α), AKT, and PI3 kinase (PI3K). Thirteen hepatopancreatobiliary tumor specimens were collected during surgical resection and cultured as PDOs with or without L-lactate. Hematoxylin and eosin (H&E) staining and immunohistochemical staining were performed on the original tissues and PDOs to compare their pathological structures, and their genetic profiles were analyzed by whole-exome sequencing (WES). The sensitivity of the PDOs to gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infigratinib, and lenvatinib were evaluated in terms of cell viability. Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with PDOs to test the sensitivity of PDOs to tislelizumab. The addition of 20 mM lactate significantly promoted the growth of LM3 and Huh 7 organoids by 217% and 36%, respectively, compared to the control group, and the inhibition of lactate transporter decreased their growth. The HIF1α/ENO1/AKT/PI3K pathway was also activated by lactate. The inhibition of enolase also partly decreased the growth of organoids treated with lactate. Furthermore, 20 mM lactate increased the viability of 9 PDOs from 135% to 317% without affecting their pathological features. The genetic similarity, in terms of single nucleotide variations, insertions, and deletions, between original tissues and lactate-treated PDOs ranged from 83.2% to 94.1%, and that between the untreated and lactate-treated PDOs was at least 93.2%. Furthermore, the addition of lactate did not significantly change the dose-response curves of the PDOs to chemotherapeutic drugs, targeted drugs, and immune checkpoint inhibitor, especially for the drugs to which the cells were sensitive. Thus, lactate can be added to the culture medium of PDOs to promote their growth without altering their genetic profiles and drug sensitivities.

Project description:BackgroundPatient-derived organoids (PDO) have been proposed as a novel in vitro method of drug screening for different types of cancer. However, to date, extrahepatic biliary tract carcinoma (eBTC) PDOs have not yet been fully established.MethodsWe collected six samples of gallbladder carcinoma (GBC) and one sample of extrahepatic cholangiocarcinoma (eCCA) from seven patients to attempt to establish eBTC PDOs for drug screening. We successfully established five GBC and one eCCA PDOs. Histological staining was used to compare structural features between the original tissues and cancer PDOs. Whole exome sequencing (WES) was performed to analyze the genetic profiles of original tissues and cancer PDOs. Drug screening, including gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, infigratinib, and ivosidenib, was measured and verified by clinical effects in certain cases.ResultsDifferent PDOs exhibited diverse growth rates during in vitro culture. Hematoxylin and eosin staining demonstrated that the structures of most cancer PDOs retained the original structures of adenocarcinoma. Immunohistological and periodic acid-schiff staining revealed that marker expression in cancer PDOs was similar to that of the original specimens. Genetic profiles from the four original specimens, as well as paired cancer PDOs, were analyzed using whole exome sequencing. Three of the four PDOs exhibited a high degree of similarity when compared to the original specimens, except for GBC2 PDO, which only had a concordance of 74% in the proportion of single nucleotide polymorphisms in the coding sequence. In general, gemcitabine was found to be the most efficient drug for eBTC treatment, as it showed moderate or significant inhibitory impact on cancer growth. Results from drug screening were confirmed to a certain extent by three clinical cases.ConclusionsOur study successfully established a series of eBTC PDOs, which contributed to the field of eBTC PDOs. Additional enhancements should be explored to improve the growth rate of PDOs and to preserve their immune microenvironment.

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A simple high-throughput approach identifies actionable drug sensitivities in patient-derived tumor organoids.

Publications

A simple high-throughput approach identifies actionable drug sensitivities in patient-derived tumor organoids.

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