Cargando…

Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models

The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of t...

Descripción completa

Detalles Bibliográficos
Autores principales: Lotsberg, Maria L., Røsland, Gro V., Rayford, Austin J., Dyrstad, Sissel E., Ekanger, Camilla T., Lu, Ning, Frantz, Kirstine, Stuhr, Linda E. B., Ditzel, Henrik J., Thiery, Jean Paul, Akslen, Lars A., Lorens, James B., Engelsen, Agnete S. T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9076321/
https://www.ncbi.nlm.nih.gov/pubmed/35530312
http://dx.doi.org/10.3389/fonc.2022.818437
_version_ 1784701896790900736
author Lotsberg, Maria L.
Røsland, Gro V.
Rayford, Austin J.
Dyrstad, Sissel E.
Ekanger, Camilla T.
Lu, Ning
Frantz, Kirstine
Stuhr, Linda E. B.
Ditzel, Henrik J.
Thiery, Jean Paul
Akslen, Lars A.
Lorens, James B.
Engelsen, Agnete S. T.
author_facet Lotsberg, Maria L.
Røsland, Gro V.
Rayford, Austin J.
Dyrstad, Sissel E.
Ekanger, Camilla T.
Lu, Ning
Frantz, Kirstine
Stuhr, Linda E. B.
Ditzel, Henrik J.
Thiery, Jean Paul
Akslen, Lars A.
Lorens, James B.
Engelsen, Agnete S. T.
author_sort Lotsberg, Maria L.
collection PubMed
description The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of two or more cell types. Compared to traditional monolayer cell culture models, the organization of cells into a 3D tissue-like structure favors relevant physiological conditions with chemical and physical gradients as well as cell-cell and cell-extracellular matrix (ECM) interactions that recapitulate many of the hallmarks of cancer in situ. Epidermal growth factor receptor (EGFR) mutations are prevalent in non-small cell lung cancer (NSCLC), yet various mechanisms of acquired resistance, including epithelial-to-mesenchymal transition (EMT), limit the clinical benefit of EGFR tyrosine kinase inhibitors (EGFRi). Improved preclinical models that incorporate the complexity induced by epithelial-to-mesenchymal plasticity (EMP) are urgently needed to advance new therapeutics for clinical NSCLC management. This study was designed to provide a thorough characterization of multicellular spheroids of isogenic cancer cells of various phenotypes and demonstrate proof-of-principle for the applicability of the presented spheroid model to evaluate the impact of cancer cell phenotype in drug screening experiments through high-dimensional and spatially resolved imaging mass cytometry (IMC) analyses. First, we developed and characterized 3D homotypic and heterotypic spheroid models comprising EGFRi-sensitive or EGFRi-resistant NSCLC cells. We observed that the degree of EMT correlated with the spheroid generation efficiency in monocultures. In-depth characterization of the multicellular heterotypic spheroids using immunohistochemistry and high-dimensional single-cell analyses by IMC revealed intrinsic differences between epithelial and mesenchymal-like cancer cells with respect to self-sorting, spatiotemporal organization, and stromal cell interactions when co-cultured with fibroblasts. While the carcinoma cells harboring an epithelial phenotype self-organized into a barrier sheet surrounding the fibroblasts, mesenchymal-like carcinoma cells localized to the central hypoxic and collagen-rich areas of the compact heterotypic spheroids. Further, deep-learning-based single-cell segmentation of IMC images and application of dimensionality reduction algorithms allowed a detailed visualization and multiparametric analysis of marker expression across the different cell subsets. We observed a high level of heterogeneity in the expression of EMT markers in both the carcinoma cell populations and the fibroblasts. Our study supports further application of these models in pre-clinical drug testing combined with complementary high-dimensional single-cell analyses, which in turn can advance our understanding of the impact of cancer-stroma interactions and epithelial phenotypic plasticity on innate and acquired therapy resistance in NSCLC.
format Online
Article
Text
id pubmed-9076321
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-90763212022-05-07 Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models Lotsberg, Maria L. Røsland, Gro V. Rayford, Austin J. Dyrstad, Sissel E. Ekanger, Camilla T. Lu, Ning Frantz, Kirstine Stuhr, Linda E. B. Ditzel, Henrik J. Thiery, Jean Paul Akslen, Lars A. Lorens, James B. Engelsen, Agnete S. T. Front Oncol Oncology The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of two or more cell types. Compared to traditional monolayer cell culture models, the organization of cells into a 3D tissue-like structure favors relevant physiological conditions with chemical and physical gradients as well as cell-cell and cell-extracellular matrix (ECM) interactions that recapitulate many of the hallmarks of cancer in situ. Epidermal growth factor receptor (EGFR) mutations are prevalent in non-small cell lung cancer (NSCLC), yet various mechanisms of acquired resistance, including epithelial-to-mesenchymal transition (EMT), limit the clinical benefit of EGFR tyrosine kinase inhibitors (EGFRi). Improved preclinical models that incorporate the complexity induced by epithelial-to-mesenchymal plasticity (EMP) are urgently needed to advance new therapeutics for clinical NSCLC management. This study was designed to provide a thorough characterization of multicellular spheroids of isogenic cancer cells of various phenotypes and demonstrate proof-of-principle for the applicability of the presented spheroid model to evaluate the impact of cancer cell phenotype in drug screening experiments through high-dimensional and spatially resolved imaging mass cytometry (IMC) analyses. First, we developed and characterized 3D homotypic and heterotypic spheroid models comprising EGFRi-sensitive or EGFRi-resistant NSCLC cells. We observed that the degree of EMT correlated with the spheroid generation efficiency in monocultures. In-depth characterization of the multicellular heterotypic spheroids using immunohistochemistry and high-dimensional single-cell analyses by IMC revealed intrinsic differences between epithelial and mesenchymal-like cancer cells with respect to self-sorting, spatiotemporal organization, and stromal cell interactions when co-cultured with fibroblasts. While the carcinoma cells harboring an epithelial phenotype self-organized into a barrier sheet surrounding the fibroblasts, mesenchymal-like carcinoma cells localized to the central hypoxic and collagen-rich areas of the compact heterotypic spheroids. Further, deep-learning-based single-cell segmentation of IMC images and application of dimensionality reduction algorithms allowed a detailed visualization and multiparametric analysis of marker expression across the different cell subsets. We observed a high level of heterogeneity in the expression of EMT markers in both the carcinoma cell populations and the fibroblasts. Our study supports further application of these models in pre-clinical drug testing combined with complementary high-dimensional single-cell analyses, which in turn can advance our understanding of the impact of cancer-stroma interactions and epithelial phenotypic plasticity on innate and acquired therapy resistance in NSCLC. Frontiers Media S.A. 2022-04-22 /pmc/articles/PMC9076321/ /pubmed/35530312 http://dx.doi.org/10.3389/fonc.2022.818437 Text en Copyright © 2022 Lotsberg, Røsland, Rayford, Dyrstad, Ekanger, Lu, Frantz, Stuhr, Ditzel, Thiery, Akslen, Lorens and Engelsen https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Oncology
Lotsberg, Maria L.
Røsland, Gro V.
Rayford, Austin J.
Dyrstad, Sissel E.
Ekanger, Camilla T.
Lu, Ning
Frantz, Kirstine
Stuhr, Linda E. B.
Ditzel, Henrik J.
Thiery, Jean Paul
Akslen, Lars A.
Lorens, James B.
Engelsen, Agnete S. T.
Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models
title Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models
title_full Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models
title_fullStr Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models
title_full_unstemmed Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models
title_short Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models
title_sort intrinsic differences in spatiotemporal organization and stromal cell interactions between isogenic lung cancer cells of epithelial and mesenchymal phenotypes revealed by high-dimensional single-cell analysis of heterotypic 3d spheroid models
topic Oncology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9076321/
https://www.ncbi.nlm.nih.gov/pubmed/35530312
http://dx.doi.org/10.3389/fonc.2022.818437
work_keys_str_mv AT lotsbergmarial intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT røslandgrov intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT rayfordaustinj intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT dyrstadsissele intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT ekangercamillat intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT luning intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT frantzkirstine intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT stuhrlindaeb intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT ditzelhenrikj intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT thieryjeanpaul intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT akslenlarsa intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT lorensjamesb intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels
AT engelsenagnetest intrinsicdifferencesinspatiotemporalorganizationandstromalcellinteractionsbetweenisogeniclungcancercellsofepithelialandmesenchymalphenotypesrevealedbyhighdimensionalsinglecellanalysisofheterotypic3dspheroidmodels