Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells
Endothelial cells (ECs) are an important component of the tumor microenvironment, playing key roles in tumor development and progression that span from angiogenesis to immune regulation and drug resistance. Heterotypic tumor spheroids are one of the most widely used in vitro tumor microenvironment m...
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| Language: | English |
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Frontiers Media S.A.
2021-06-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2021.649949/full |
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| author | Teresa Franchi-Mendes Teresa Franchi-Mendes Nuno Lopes Nuno Lopes Catarina Brito Catarina Brito Catarina Brito |
| author_facet | Teresa Franchi-Mendes Teresa Franchi-Mendes Nuno Lopes Nuno Lopes Catarina Brito Catarina Brito Catarina Brito |
| author_sort | Teresa Franchi-Mendes |
| collection | DOAJ |
| description | Endothelial cells (ECs) are an important component of the tumor microenvironment, playing key roles in tumor development and progression that span from angiogenesis to immune regulation and drug resistance. Heterotypic tumor spheroids are one of the most widely used in vitro tumor microenvironment models, presenting improved recapitulation of tumor microenvironments compared to 2D cultures, in a simple and low-cost setup. Heterotypic tumor spheroid models incorporating endothelial cells have been proposed but present multiple limitations, such as the short culture duration typically obtained, the use of animal-derived matrices, and poor reproducibility; the diversity of culture conditions employed hinders comparison between studies and standardization of relevant culture parameters. Herein, we developed long-term cultures of triple heterotypic spheroids composed of the HCC1954 tumor cell line, human fibroblasts, and ECs. We explored culture parameters potentially relevant for EC maintenance, such as tumor cell line, seeding cell number, cell ratio, and agitation vs. static culture. In HCC1954-based spheroids, we observed maintenance of viable EC for up to 1 month of culture in agitation, with retention of the identity markers CD31 and von Willebrand factor. At the optimized tumor cell:fibroblast:EC ratio of 1:3:10, HCC1954-based spheroids had a higher EC area/total spheroid area at 1 month of culture than the other cell ratios tested. EC maintenance was tumor cell line-dependent, and in HCC1954-based spheroids it was also dependent on the presence of fibroblasts and agitation. Moreover, vascular endothelial growth factor (VEGF) supplementation was not required for maintenance of EC, as the factor was endogenously produced. ECs co-localized with fibroblasts, which accumulated preferentially in the core of the spheroids and secreted EC-relevant extracellular matrix proteins, such as collagen I and IV. This simple model setup does not rely on artificial or animal-derived scaffolds and can serve as a useful tool to explore the culture parameters influencing heterotypic spheroids, contributing to model standardization, as well as to explore molecular cross talk of ECs within the tumor microenvironment, and potentially its effects on drug response. |
| first_indexed | 2024-12-16T13:14:19Z |
| format | Article |
| id | doaj.art-b6b29b156f1f4c86909cf3a8ddead809 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-12-16T13:14:19Z |
| publishDate | 2021-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-b6b29b156f1f4c86909cf3a8ddead8092022-12-21T22:30:32ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852021-06-01910.3389/fbioe.2021.649949649949Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial CellsTeresa Franchi-Mendes0Teresa Franchi-Mendes1Nuno Lopes2Nuno Lopes3Catarina Brito4Catarina Brito5Catarina Brito6iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, PortugaliBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, PortugaliBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, PortugalThe Discoveries Centre for Regenerative and Precision Medicine, Oeiras, PortugalEndothelial cells (ECs) are an important component of the tumor microenvironment, playing key roles in tumor development and progression that span from angiogenesis to immune regulation and drug resistance. Heterotypic tumor spheroids are one of the most widely used in vitro tumor microenvironment models, presenting improved recapitulation of tumor microenvironments compared to 2D cultures, in a simple and low-cost setup. Heterotypic tumor spheroid models incorporating endothelial cells have been proposed but present multiple limitations, such as the short culture duration typically obtained, the use of animal-derived matrices, and poor reproducibility; the diversity of culture conditions employed hinders comparison between studies and standardization of relevant culture parameters. Herein, we developed long-term cultures of triple heterotypic spheroids composed of the HCC1954 tumor cell line, human fibroblasts, and ECs. We explored culture parameters potentially relevant for EC maintenance, such as tumor cell line, seeding cell number, cell ratio, and agitation vs. static culture. In HCC1954-based spheroids, we observed maintenance of viable EC for up to 1 month of culture in agitation, with retention of the identity markers CD31 and von Willebrand factor. At the optimized tumor cell:fibroblast:EC ratio of 1:3:10, HCC1954-based spheroids had a higher EC area/total spheroid area at 1 month of culture than the other cell ratios tested. EC maintenance was tumor cell line-dependent, and in HCC1954-based spheroids it was also dependent on the presence of fibroblasts and agitation. Moreover, vascular endothelial growth factor (VEGF) supplementation was not required for maintenance of EC, as the factor was endogenously produced. ECs co-localized with fibroblasts, which accumulated preferentially in the core of the spheroids and secreted EC-relevant extracellular matrix proteins, such as collagen I and IV. This simple model setup does not rely on artificial or animal-derived scaffolds and can serve as a useful tool to explore the culture parameters influencing heterotypic spheroids, contributing to model standardization, as well as to explore molecular cross talk of ECs within the tumor microenvironment, and potentially its effects on drug response.https://www.frontiersin.org/articles/10.3389/fbioe.2021.649949/full3D modelsspheroidsendothelial cellsfibroblastsbreast cancer |
| spellingShingle | Teresa Franchi-Mendes Teresa Franchi-Mendes Nuno Lopes Nuno Lopes Catarina Brito Catarina Brito Catarina Brito Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells Frontiers in Bioengineering and Biotechnology 3D models spheroids endothelial cells fibroblasts breast cancer |
| title | Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells |
| title_full | Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells |
| title_fullStr | Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells |
| title_full_unstemmed | Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells |
| title_short | Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells |
| title_sort | heterotypic tumor spheroids in agitation based cultures a scaffold free cell model that sustains long term survival of endothelial cells |
| topic | 3D models spheroids endothelial cells fibroblasts breast cancer |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2021.649949/full |
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