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Cited by 1103D Printing in Biomedical ResearchCancer Cells and MetastasisCellular Mechanics and Interactions

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Application of Concave Microwells to Pancreatic Tumor Spheroids Enabling Anticancer Drug Evaluation in a Clinically Relevant Drug Resistance Model

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Yeon, Sang-Eun, No, Da Yoon, Lee, Sang-Hoon, Nam, Suk Woo, Oh, Il-Hoan, Lee, Jaehwi, Kuh, Hyo-Jeong·2013·PLoS ONE
DOI 10.1371/journal.pone.0073345PMC PMC3769301
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Abstract

Intrinsic drug resistance of pancreatic ductal adenocarcinoma (PDAC) warrants studies using models that are more clinically relevant for identifying novel resistance mechanisms as well as for drug development. Tumor spheroids (TS) mimic in vivo tumor conditions associated with multicellular resistance and represent a promising model for efficient drug screening, however, pancreatic cancer cells often fail to form spheroids using conventional methods such as liquid overlay. This study describes the induction of TS of human pancreatic cancer cells (Panc-1, Aspc-1, Capan-2) in concave polydimethylsiloxane (PDMS) microwell plates and evaluation of their usefulness as an anticancer efficacy test model. All three cell lines showed TS formation with varying degree of necrosis inside TS. Among these, Panc-1 spheroid with spherical morphology, a rather rough surface, and unique adhesion structures were successfully produced with no notable necrosis in concave microwell plates. Panc-1 TS contained growth factors or enzymes such as TGF-β1, CTGF, and MT1-MMP, and extracellular matrix proteins such as collagen type I, fibronectin, and laminin. Panc-1 cells grown as TS showed changes in stem cell populations and in expression levels of miRNAs that may play roles in chemoresistance. Visualization of drug penetration and detection of viability indicators, such as Ki-67 and MitoSOX, were optimized for TS for quantitative analysis. Water-soluble tetrazolium (MTS) and acid phosphatase (APH) assays were also successfully optimized. Overall, we demonstrated that concave PDMS microwell plates are a novel platform for preparation of TS of weakly aggregating cells and that Panc-1 spheroids may represent a novel three-dimensional model for anti-pancreatic cancer drug screening.

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Figure 1 Schematic overview of pancreatic tumor spheroids (TS) generated in concave microwells. Pancreatic cancer cells cultured in agarose-coated 96 well plates produced only loose aggregates (A). The TS formation process in a BSA-coated concave microwell plate (B). The TS structure, compared with an in vivo tumor, showed a close resemblance to the in vivo condition. Cells in the TS retained the characteristics of in vivo tumors under 3D culture conditions.
Figure 1 Schematic overview of pancreatic tumor spheroids (TS) generated in concave microwells. Pancreatic cancer cells cultured in agarose-coated 96 well plates produced only loose aggregates (A). The TS formation process in a BSA-coated concave microwell plate (B). The TS structure, compared with an in vivo tumor, showed a close resemblance to the in vivo condition. Cells in the TS retained the characteristics of in vivo tumors under 3D culture conditions.CC BY · PubMed Central
Figure 2 Concave PDMS microwell plate for culture and growth of pancreatic tumor spheroids (TS). Shape and dimension of concave microwell plate 600 (A). Changes in size of three different pancreatic spheroids cultured in microwell 600 (B). Size distribution of Panc-1 spheroids cultured in three different sized-microwell plate during 13 days of culture (C). Data are expressed as mean ± SE of a minimum of 10 spheroids cultured in one microwell plate.
Figure 2 Concave PDMS microwell plate for culture and growth of pancreatic tumor spheroids (TS). Shape and dimension of concave microwell plate 600 (A). Changes in size of three different pancreatic spheroids cultured in microwell 600 (B). Size distribution of Panc-1 spheroids cultured in three different sized-microwell plate during 13 days of culture (C). Data are expressed as mean ± SE of a minimum of 10 spheroids cultured in one microwell plate.CC BY · PubMed Central
Figure 3 Morphology and histological examination of tumor spheroids (TS) cultured in concave microwell 600 or in 96 well plates. Representative images of H&E stained paraffin sections or toluidine blue stained semi-thin sections, SEM and TEM images of HT-29 (A), Panc-1 (B), Aspc-1 (C), and Capan-2 (D) spheroids cultured in concave microwell 600 plates for 5 and 13 days. Aggregates of Panc-1, Aspc-1, and Capan-2 cells formed in agarose-coated 96 well plates shown as an H&E stained paraffin section or bright field images (E). Arrow: desmosome; dotted lines: gap junctions; arrow head: tight junction; cross: lipid droplet; I: invagination structure; N: necrotic regions. The scale bars indicate 100 μm, 50 μm, 2 μm and 500 μm, in H&E or toluidine blue stained, SEM, TEM and bright-field images, respectively.
Figure 3 Morphology and histological examination of tumor spheroids (TS) cultured in concave microwell 600 or in 96 well plates. Representative images of H&E stained paraffin sections or toluidine blue stained semi-thin sections, SEM and TEM images of HT-29 (A), Panc-1 (B), Aspc-1 (C), and Capan-2 (D) spheroids cultured in concave microwell 600 plates for 5 and 13 days. Aggregates of Panc-1, Aspc-1, and Capan-2 cells formed in agarose-coated 96 well plates shown as an H&E stained paraffin section or bright field images (E). Arrow: desmosome; dotted lines: gap junctions; arrow head: tight junction; cross: lipid droplet; I: invagination structure; N: necrotic regions. The scale bars indicate 100 μm, 50 μm, 2 μm and 500 μm, in H&E or toluidine blue stained, SEM, TEM and bright-field images, respectively.CC BY · PubMed Central
Figure 4 Expression of proteins associated with tumor growth and drug resistance. Spheroids were cultured for 5-β1, CTGF, and MT1-MMP (A) and for ECM proteins such as collagen type I, fibronectin, and laminin (B). (Scale bar = 20 μm).
Figure 4 Expression of proteins associated with tumor growth and drug resistance. Spheroids were cultured for 5-β1, CTGF, and MT1-MMP (A) and for ECM proteins such as collagen type I, fibronectin, and laminin (B). (Scale bar = 20 μm).CC BY · PubMed Central
Figure 5 Analysis of pancreatic cancer stem cell markers. Expression patterns of stem cell markers such as CD44, CD24, and ESA were compared between 2D (A) and 3D cultures (B). Percentages of the cell population expressing CD44, CD24, and ESA in Panc-1 cells cultured under 2D and 3D conditions are summarized in the table.
Figure 5 Analysis of pancreatic cancer stem cell markers. Expression patterns of stem cell markers such as CD44, CD24, and ESA were compared between 2D (A) and 3D cultures (B). Percentages of the cell population expressing CD44, CD24, and ESA in Panc-1 cells cultured under 2D and 3D conditions are summarized in the table.CC BY · PubMed Central
Figure 6 The miRNA expression profiles were compared between 2D and 3D cultures. A heatmap of differentially expressed miRNAs generated by unsupervised hierarchical cluster analysis (A). Differential expression levels of miRNAs between 2D and 3D cultures were confirmed using qRT-PCR for several genes selected from microarray data. Among the differentially expressed miRNAs, up-regulated miRNAs including miR-34b-5p, miR-578, miR-1304, and miR-324-5p and down-regulated miRNAs including miR-7-5p, and miR-34b-3p were analyzed using primers for mature mRNAs (B). Data are expressed as the mean ± SE of triplicates. * p<0.05, n = 3.
Figure 6 The miRNA expression profiles were compared between 2D and 3D cultures. A heatmap of differentially expressed miRNAs generated by unsupervised hierarchical cluster analysis (A). Differential expression levels of miRNAs between 2D and 3D cultures were confirmed using qRT-PCR for several genes selected from microarray data. Among the differentially expressed miRNAs, up-regulated miRNAs including miR-34b-5p, miR-578, miR-1304, and miR-324-5p and down-regulated miRNAs including miR-7-5p, and miR-34b-3p were analyzed using primers for mature mRNAs (B). Data are expressed as the mean ± SE of triplicates. * p<0.05, n = 3.CC BY · PubMed Central
Figure 7 Penetration of DOX into Panc-1 spheroids. Penetration was evaluated by imaging DOX autofluorescence in sections of spheroids. Spheroids were cultured in a concave microwell 600 plate for 5 μM for 12 h. Fluorescence intensity across sections was measured and expressed as the mean ± SE of 5 replicates. (Scale bar = 100 μm) * and **, p<0.01 and p<0.001, respectively.
Figure 7 Penetration of DOX into Panc-1 spheroids. Penetration was evaluated by imaging DOX autofluorescence in sections of spheroids. Spheroids were cultured in a concave microwell 600 plate for 5 μM for 12 h. Fluorescence intensity across sections was measured and expressed as the mean ± SE of 5 replicates. (Scale bar = 100 μm) * and **, p<0.01 and p<0.001, respectively.CC BY · PubMed Central
Figure 8 Cytotoxicity/viability assays for Panc-1 spheroids. Changes in Ki-67 expression were measured following exposure to DOX at the indicated concentrations for 12 h (A). Changes in MitoSOX were measured following exposure to GEM at the indicated concentration for 12 h (B). MTS (C) and APH assay (D) after exposure to GEM for 72 h. Data are expressed as the mean ± SE of 6 replicates. (Scale bar = 100 μm) * p<0.001.
Figure 8 Cytotoxicity/viability assays for Panc-1 spheroids. Changes in Ki-67 expression were measured following exposure to DOX at the indicated concentrations for 12 h (A). Changes in MitoSOX were measured following exposure to GEM at the indicated concentration for 12 h (B). MTS (C) and APH assay (D) after exposure to GEM for 72 h. Data are expressed as the mean ± SE of 6 replicates. (Scale bar = 100 μm) * p<0.001.CC BY · PubMed Central

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Metadata

Authors
Yeon, S.-E., No, D. Y., Lee, S.-H., Nam, S. W., Oh, I.-H., Lee, J., & Kuh, H.-J.
Affiliations
  • Catholic University of Korea (KR)
  • Korea University (KR)
  • Chung-Ang University (KR)
Corresponding author
Kuh, Hyo-Jeong
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-09-10
Volume
8
Issue
9
Pages
e73345
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0073345
PMC
PMC3769301
OpenAlex
W2025183054
Cited by
110
References
42
Citations by year
2026: 1 · 2025: 6 · 2024: 6 · 2023: 4 · 2022: 4 · 2021: 12 · 2020: 14 · 2019: 12 · 2018: 11 · 2017: 13 · 2016: 11 · 2015: 9 · 2014: 6
FWCI
4.79
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Research Foundation
  • National Research Foundation of Korea (2012R1A5A2047939, 2012R1A2A2A01003361)
Topics
  • Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
Keywords
pancreatic ductal adenocarcinoma, drug resistance, anticancer drug screening, PANC-1 cells, 3D tumor models, extracellular matrix, chemoresistance, drug penetration, necrosis, stem cell populations, microRNA expression, MTS assay
MeSH terms
Antinematodal Agents, Drug Screening Assays, Antitumor, Equipment Design, Humans, Pancreatic Neoplasms, Spheroids, Cellular, Cell Line, Tumor

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