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

Author(s):  
Teresa Franchi-Mendes ◽  
Nuno Lopes ◽  
Catarina Brito

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.

Tumor Biology ◽  
2012 ◽  
Vol 33 (3) ◽  
pp. 739-748 ◽  
Author(s):  
Abdullah Onul ◽  
Kim M. Elseth ◽  
Humberto De Vitto ◽  
William A. Paradise ◽  
Benjamin J. Vesper ◽  
...  

2021 ◽  
Vol 9 (11) ◽  
pp. e003134
Author(s):  
Shusuke Kawashima ◽  
Takashi Inozume ◽  
Masahito Kawazu ◽  
Toshihide Ueno ◽  
Joji Nagasaki ◽  
...  

BackgroundPatients with cancer benefit from treatment with immune checkpoint inhibitors (ICIs), and those with an inflamed tumor microenvironment (TME) and/or high tumor mutation burden (TMB), particularly, tend to respond to ICIs; however, some patients fail, whereas others acquire resistance after initial response despite the inflamed TME and/or high TMB. We assessed the detailed biological mechanisms of resistance to ICIs such as programmed death 1 and/or cytotoxic T-lymphocyte-associated protein 4 blockade therapies using clinical samples.MethodsWe established four pairs of autologous tumor cell lines and tumor-infiltrating lymphocytes (TILs) from patients with melanoma treated with ICIs. These tumor cell lines and TILs were subjected to comprehensive analyses and in vitro functional assays. We assessed tumor volume and TILs in vivo mouse models to validate identified mechanism. Furthermore, we analyzed additional clinical samples from another large melanoma cohort.ResultsTwo patients were super-responders, and the others acquired resistance: the first patient had a non-inflamed TME and acquired resistance due to the loss of the beta-2 microglobulin gene, and the other acquired resistance despite having inflamed TME and extremely high TMB which are reportedly predictive biomarkers. Tumor cell line and paired TIL analyses showed high CD155, TIGIT ligand, and TIGIT expression in the tumor cell line and tumor-infiltrating T cells, respectively. TIGIT blockade or CD155-deletion activated T cells in a functional assay using an autologous cell line and paired TILs from this patient. CD155 expression increased in surviving tumor cells after coculturing with TILs from a responder, which suppressed TIGIT+ T-cell activation. Consistently, TIGIT blockade or CD155-deletion could aid in overcoming resistance to ICIs in vivo mouse models. In clinical samples, CD155 was related to resistance to ICIs in patients with melanoma with an inflamed TME, including both primary and acquired resistance.ConclusionsThe TIGIT/CD155 axis mediates resistance to ICIs in patients with melanoma with an inflamed TME, promoting the development of TIGIT blockade therapies in such patients with cancer.


2017 ◽  
Vol 68 (6) ◽  
pp. 1341-1344
Author(s):  
Grigore Berea ◽  
Gheorghe Gh. Balan ◽  
Vasile Sandru ◽  
Paul Dan Sirbu

Complex interactions between stem cells, vascular cells and fibroblasts represent the substrate of building microenvironment-embedded 3D structures that can be grafted or added to bone substitute scaffolds in tissue engineering or clinical bone repair. Human Adipose-derived Stem Cells (hASCs), human umbilical vein endothelial cells (HUVECs) and normal dermal human fibroblasts (NDHF) can be mixed together in three dimensional scaffold free constructs and their behaviour will emphasize their potential use as seeding points in bone tissue engineering. Various combinations of the aforementioned cell lines were compared to single cell line culture in terms of size, viability and cell proliferation. At 5 weeks, viability dropped for single cell line spheroids while addition of NDHF to hASC maintained the viability at the same level at 5 weeks Fibroblasts addition to the 3D construct of stem cells and endothelial cells improves viability and reduces proliferation as a marker of cell differentiation toward osteogenic line.


1983 ◽  
Vol 48 (2) ◽  
pp. 377-383 ◽  
Author(s):  
E Knust ◽  
W Dietrich ◽  
B Fleckenstein ◽  
W Bodemer

2019 ◽  
Vol 26 (09) ◽  
pp. 1950058
Author(s):  
SADEQ H. LAFTA ◽  
ALI ABDULRAHMAN TAHA ◽  
MUHAMMAD M. FARHAN ◽  
SHAIMA Y. ABDULFATTAH

Nanoparticles of alpha ferric oxide ([Formula: see text]-Fe2O3) were prepared by the hydrothermal method. Structural properties of [Formula: see text]-Fe2O3 were determined by XRD, SEM and AFM measurements. The particles had a good matching with standard pattern. Average particle size was about 90[Formula: see text]nm and the distribution extended from about 20[Formula: see text]nm to 120[Formula: see text]nm. Biocompatibility study of ferric oxide nanoparticles against bacteria, parasites, tumor cell line and normal cells was determined. No antibacterial activity was observed for the concentration, of ferric oxide nanoparticles in distilled water, up to 1.5[Formula: see text]mg/ml vs. E. coli and S. aureus. Moreover, MTT assay was used to determine the cytotoxicity against parasites and cells. Intermediate cytotoxicity (53.30%) of 1.5[Formula: see text]mg/ml of prepared nanoparticles was noted against L. tropica, while weak cytotoxicity of 5.20% was observed against L. donovani at the same concentration of ferric oxide nanoparticles. On the other hand, the prepared nanoparticles revealed low cytotoxicity (47.28%) against SR tumor cell line, while no cytotoxicity was shown against lymphocytes, as a model of normal cells.


Endocrine ◽  
2008 ◽  
Vol 33 (1) ◽  
pp. 62-76 ◽  
Author(s):  
Katharina H. Ruebel ◽  
Alexey A. Leontovich ◽  
Yoshinori Tanizaki ◽  
Long Jin ◽  
Gail A. Stilling ◽  
...  

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