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Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip

SIMPLE SUMMARY: Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) recapitulates solid tumors in vivo. This microtumor model is particularly useful for multiplex phenotypic analysis, but requires tissue optical clearing (TOC) for 3D visualization. We develo...

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Autores principales: Oh, Min-Suk, Khawar, Iftikhar Ali, Lee, Dong Woo, Park, Jong Kook, Kuh, Hyo-Jeong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762293/
https://www.ncbi.nlm.nih.gov/pubmed/33297288
http://dx.doi.org/10.3390/cancers12123662
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author Oh, Min-Suk
Khawar, Iftikhar Ali
Lee, Dong Woo
Park, Jong Kook
Kuh, Hyo-Jeong
author_facet Oh, Min-Suk
Khawar, Iftikhar Ali
Lee, Dong Woo
Park, Jong Kook
Kuh, Hyo-Jeong
author_sort Oh, Min-Suk
collection PubMed
description SIMPLE SUMMARY: Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) recapitulates solid tumors in vivo. This microtumor model is particularly useful for multiplex phenotypic analysis, but requires tissue optical clearing (TOC) for 3D visualization. We developed a transfer-free 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with the TOC method. Our method succeeded in improving immunostaining and optical transmission in each TS as well as the entire microtumor specimen. The utility of this method was demonstrated by showing phenotypic changes, such as increased levels of membrane protrusion, single-cell dissemination, and ECM remodeling, and changes in the expression of epithelial–mesenchymal transition–related proteins and drug-induced apoptosis in TSs of human pancreatic cancer cells co-cultured with cancer-associated fibroblasts and M2-type tumor-associated macrophages. ABSTRACT: Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) represents a microtumor model that recapitulates human solid tumors in vivo, and is useful for 3D multiplex phenotypic analysis. However, the low efficiency of 3D culture and limited 3D visualization of microtumor specimens impose technical hurdles for the evaluation of TS-based phenotypic analysis. Here, we report a 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with a tissue optical clearing (TOC) method for high-content phenotypic analysis of microtumors. To prove the utility of this method, phenotypic changes in TSs of human pancreatic cancer cells were determined by co-culture with cancer-associated fibroblasts and M2-type tumor-associated macrophages. Significant improvement was achieved in immunostaining and optical transmission in each TS as well as the entire microtumor specimen, enabling optimization in image-based analysis of the morphology, structural organization, and protein expression in cancer cells and the ECM. Changes in the invasive phenotype, including cellular morphology and expression of epithelial–mesenchymal transition-related proteins and drug-induced apoptosis under stromal cell co-culture were also successfully analyzed. Overall, our study demonstrates that a minipillar array chip combined with TOC offers a novel system for 3D culture-to-3D visualization of microtumors to facilitate high-content phenotypic analysis.
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spelling pubmed-77622932020-12-26 Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip Oh, Min-Suk Khawar, Iftikhar Ali Lee, Dong Woo Park, Jong Kook Kuh, Hyo-Jeong Cancers (Basel) Article SIMPLE SUMMARY: Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) recapitulates solid tumors in vivo. This microtumor model is particularly useful for multiplex phenotypic analysis, but requires tissue optical clearing (TOC) for 3D visualization. We developed a transfer-free 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with the TOC method. Our method succeeded in improving immunostaining and optical transmission in each TS as well as the entire microtumor specimen. The utility of this method was demonstrated by showing phenotypic changes, such as increased levels of membrane protrusion, single-cell dissemination, and ECM remodeling, and changes in the expression of epithelial–mesenchymal transition–related proteins and drug-induced apoptosis in TSs of human pancreatic cancer cells co-cultured with cancer-associated fibroblasts and M2-type tumor-associated macrophages. ABSTRACT: Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) represents a microtumor model that recapitulates human solid tumors in vivo, and is useful for 3D multiplex phenotypic analysis. However, the low efficiency of 3D culture and limited 3D visualization of microtumor specimens impose technical hurdles for the evaluation of TS-based phenotypic analysis. Here, we report a 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with a tissue optical clearing (TOC) method for high-content phenotypic analysis of microtumors. To prove the utility of this method, phenotypic changes in TSs of human pancreatic cancer cells were determined by co-culture with cancer-associated fibroblasts and M2-type tumor-associated macrophages. Significant improvement was achieved in immunostaining and optical transmission in each TS as well as the entire microtumor specimen, enabling optimization in image-based analysis of the morphology, structural organization, and protein expression in cancer cells and the ECM. Changes in the invasive phenotype, including cellular morphology and expression of epithelial–mesenchymal transition-related proteins and drug-induced apoptosis under stromal cell co-culture were also successfully analyzed. Overall, our study demonstrates that a minipillar array chip combined with TOC offers a novel system for 3D culture-to-3D visualization of microtumors to facilitate high-content phenotypic analysis. MDPI 2020-12-07 /pmc/articles/PMC7762293/ /pubmed/33297288 http://dx.doi.org/10.3390/cancers12123662 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Oh, Min-Suk
Khawar, Iftikhar Ali
Lee, Dong Woo
Park, Jong Kook
Kuh, Hyo-Jeong
Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip
title Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip
title_full Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip
title_fullStr Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip
title_full_unstemmed Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip
title_short Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip
title_sort three-dimensional imaging for multiplex phenotypic analysis of pancreatic microtumors grown on a minipillar array chip
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762293/
https://www.ncbi.nlm.nih.gov/pubmed/33297288
http://dx.doi.org/10.3390/cancers12123662
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