Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells

Abstract

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.

Heterotypic Multicellular Spheroids as Experimental and Preclinical Models of Sprouting Angiogenesis

“…As pointed out by Franchi-Mendes et al [ 60 ], one of the advantages of spheroid assays is the possibility of focused manipulations with cells to assist elucidation of specific hypotheses. One of more cell types can be genetically altered or specifically labeled enhancing the probative value of the obtained results.…”

“…Next, the cadherin-cadherin interactions between neighboring cells tighten the cell-cell connections further and promote additional spheroid compaction. The tissue spheroids sharing many common biological features with a number of normal or diseased tissues, such as vascularized tumors, blood-brain barrier, and cardiac tissue, were produced [ 58 , 59 , 60 ]. The ability to co-culture two or more cell types makes 3D tissue spheroids a promising tool to model heterotypic cell-cell interactions and brings this 3D system to the cutting edge of tissue engineering and drug screening [ 55 , 56 , 61 , 62 ].…”

Three-Dimensional in Vitro Models: A Promising Tool To Scale-Up Breast Cancer Research

“…Their results showed that tumor cells are essential for endothelial cell maintenance, and at the optimized ratio of 1:3:10 tumor cell/ fibroblast/endothelial cell, the ratio of endothelial cell area to total area of the spheroid was higher in HCC1954-based spheroids during one month of culture. 29 They found that central colocalization of endothelial cells and fibroblast cells in the spheroids and secretion of collagen I and IV as endothelial cell-relevant extracellular matrix proteins were evident. Based on these results, it seems that spheroid models can be considered as a precious tool for identifying the molecular crosstalk of endothelial cells concerning the tumor microenvironment and its efficacy on the action and response of drugs.…”

“…The number of vessels was higher in spheroids infiltrated with macrophages than in spheroids without macrophages. In another study, Franchi-Mendes et al 29 explored the essential parameters needed for endothelial cell maintenance in cell culture conditions. For this purpose, they cocultured breast cancer cell line HCC1954 with human fibroblasts and endothelial cells and developed a culture of triple heterotypic spheroids in the long term.…”

One-Step Generation and Purification of Cell-Encapsulated Hydrogel Microsphere With an Easily Assembled Microfluidic Device

“…Scaffold-free approaches include using hanging drop plates (Kelm et al, 2003;Guo et al, 2014), low cell attachment plates (Ivanov et al, 2015), and emerging techniques such as magnetic levitation (Türker et al, 2018), ultrasound waves, and acoustic waves. Scaffold-free approaches are easy to perform and can be applied to various cell types (Franchi-Mendes et al, 2021). Microfabrication techniques such as photolithography (Du et al, 2008;Brandenberg et al, 2020) and micromolding (Tekin et al, 2011;Tao et al, 2019) have also been used to create spheroids, allowing the volume and shape of spheroids to be precisely controlled.…”