The cancer angiogenesis co-culture assay: In vitro quantification of the angiogenic potential of tumoroids

Truelsen, Sarah Line Bring; Mousavi, Nabi; Wei, Haoche; Harvey, Lucy; Stausholm, Rikke; Spillum, Erik; Hagel, Grith; Qvortrup, Klaus; Thastrup, Ole; Harling, Henrik; Mellor, Harry; Thastrup, Jacob

doi:10.1371/journal.pone.0253258

Cited by 12 publications

(7 citation statements)

References 56 publications

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“…Besides matrix composition, an appropriate supportive cell type is important for endothelial cells to form stable pre-vascular networks. Fibroblasts have been widely used in angiogenesis models since they produce a supportive matrix [48] and have been shown to promote migration and proliferation of co-cultured HUVEC [49][50][51]. Fibroblasts have also been demonstrated to support hepatocytes, e.g.…”

Section: Development Of the Cfg Bioink Supporting Pre-vascular Networ...mentioning

confidence: 99%

Core–shell bioprinting of vascularized in vitro liver sinusoid models

et al. 2022

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In vitro liver models allow the investigation of the cell behavior in disease conditions or in response to changes in the microenvironment. A major challenge in liver tissue engineering is to mimic the tissue-level complexity: Besides the selection of suitable biomaterial(s) replacing the extracellular matrix (ECM) and cell sources, the three-dimensional (3D) microarchitecture defined by the fabrication method is a critical factor to achieve functional constructs. In this study, coaxial extrusion-based 3D bioprinting has been applied to develop a liver sinusoid-like model that consists of a core compartment containing pre-vascular structures and a shell compartment containing hepatocytes. The shell ink was composed of alginate and methylcellulose (algMC), dissolved in human fresh frozen plasma. The algMC blend conferred high printing fidelity and stability to the core-shell constructs and the plasma as biologically active component enhanced viability and supported cluster formation and biomarker expression of HepG2 embedded in the shell. For the core, a natural ECM-like ink based on angiogenesis-supporting collagen-fibrin (CF) matrices was developed; the addition of gelatin (G) enabled 3D printing in combination with the plasma-algMC shell ink. Human endothelial cells (HUVEC), laden in the CFG core ink together with human fibroblasts as supportive cells, formed a pre-vascular network in the core in the absence and presence of HepG2 in the shell. The cellular interactions occurring in the triple culture model enhanced the albumin secretion. In conclusion, core-shell bioprinting was shown to be a valuable tool to study cell-cell-interactions and to develop complex tissue-like models.

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Section: Development Of the Cfg Bioink Supporting Pre-vascular Networ...mentioning

confidence: 99%

Core–shell bioprinting of vascularized in vitro liver sinusoid models

et al. 2022

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“…Previous evidence has demonstrated that up-regulation of genes encoding angiogenic factors correlates with the increased secretion of angiogenic factors from the cells [ 49 – 51 ]. Our present study also addressed this by using ELISA to measure the secreted levels of VEGF and TGF-β1 (the representatives for the up-regulated and unaffected angiogenic factors, respectively).…”

Section: Discussionmentioning

confidence: 99%

“…In consistent with those previously reported findings, our present data confirmed that changes in expression of the angiogenesis-related genes really affected the secretion of their protein products. Among the secreted angiogenic factors, VEGF and FGF2 have been reported to trigger ECs activity [ 50 , 51 ]. Due to the up-regulation of VEGF and FGF2 in our present study, we thus performed in vitro experiments to assess the angiogenesis features induced by CM from MDCK cells with ARID1A and/or ACTB knockdowns.…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of ARID1A-interacting proteins in renal tubular cells and their molecular regulation of angiogenesis

Yoodee,

Peerapen,

Plumworasawat

et al. 2023

J Transl Med

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Background Defects and deficiency of AT-rich interactive domain-containing protein 1A (ARID1A) encoded by a tumor suppressor gene ARID1A have recently been suggested to get involved in angiogenesis, a crucial process in carcinogenesis. However, molecular mechanisms of ARID1A deficiency to induce angiogenesis in kidney cancer remain underinvestigated. Methods We performed large-scale identification of ARID1A protein interactors in renal tubular epithelial cells (RTECs) using immunoprecipitation (IP) followed by nanoLC-ESI-LTQ-Orbitrap tandem mass spectrometry (MS/MS). Their roles in angiogenesis were investigated using various assays. Results A total of 74 ARID1A-interacting proteins were identified. Protein–protein interactions analysis revealed that these identified proteins interacted directly or indirectly with ARID1A. Among them, the direct interaction between ARID1A and β-actin was validated by IP and reciprocal IP followed by Western blotting. Small interfering RNA (siRNA) was used for single and double knockdowns of ARID1A and ACTB. Semi-quantitative RT-PCR demonstrated that deficiency of ARID1A, but not ACTB, significantly affected expression of angiogenesis-related genes in RTECs (VEGF and FGF2 were increased, whereas PDGF and EGF were decreased). However, the knockdowns did not affect TGFB1 and FGF1 levels. The quantitative mRNA expression data of VEGF and TGFB1 were consistent with the secreted levels of their protein products as measured by ELISA. Only secreted products derived from ARID1A-deficient RTECs significantly increased endothelial cells (ECs) migration and tube formation. Some of the other carcinogenic features could also be confirmed in the ARID1A-deficient RTECs, including increased cell migration and chemoresistance. Double knockdowns of both ARID1A and ACTB did not enhance the effects of single ARID1A knockdown in all assays. Conclusions We report herein a large dataset of the ARID1A-interacting proteins in RTECs using an IP-MS/MS approach and confirm the direct interaction between ARID1A and β-actin. However, the role of ARID1A deficiency in angiogenesis is independent of β-actin.

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“…Curiously, when HUVECs were themselves also incorporated into the spheroids, as a tri-culture of cancer cells, fibroblasts and HUVECs, the angiogenic potential of the spheroids were reduced compared to the co-cultures of only cancer cells and fibroblasts (Nashimoto et al, 2020). In addition, co-culture assays have been developed for quantification of angiogenesis that can be adapted to screen for novel anti-angiogenic compounds or even used in a personalized medicine context by screening known anti-angiogenic therapeutics against patient-derived tumor organoids Truelsen et al, 2021). Altogether, these co-culture models are a useful tool for studying angiogenesis as well as identifying effective therapeutics and underscoring the importance of multi-cellular interactions in this process.…”

Section: Vasculature and Angiogenesismentioning

confidence: 99%

Key aspects for conception and construction of co-culture models of tumor-stroma interactions

Mason

Öhlund

2023

Front. Bioeng. Biotechnol.

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The tumor microenvironment is crucial in the initiation and progression of cancers. The interplay between cancer cells and the surrounding stroma shapes the tumor biology and dictates the response to cancer therapies. Consequently, a better understanding of the interactions between cancer cells and different components of the tumor microenvironment will drive progress in developing novel, effective, treatment strategies. Co-cultures can be used to study various aspects of these interactions in detail. This includes studies of paracrine relationships between cancer cells and stromal cells such as fibroblasts, endothelial cells, and immune cells, as well as the influence of physical and mechanical interactions with the extracellular matrix of the tumor microenvironment. The development of novel co-culture models to study the tumor microenvironment has progressed rapidly over recent years. Many of these models have already been shown to be powerful tools for further understanding of the pathophysiological role of the stroma and provide mechanistic insights into tumor-stromal interactions. Here we give a structured overview of different co-culture models that have been established to study tumor-stromal interactions and what we have learnt from these models. We also introduce a set of guidelines for generating and reporting co-culture experiments to facilitate experimental robustness and reproducibility.

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The cancer angiogenesis co-culture assay: In vitro quantification of the angiogenic potential of tumoroids

Cited by 12 publications

References 56 publications

Core–shell bioprinting of vascularized in vitro liver sinusoid models

Core–shell bioprinting of vascularized in vitro liver sinusoid models

Identification and characterization of ARID1A-interacting proteins in renal tubular cells and their molecular regulation of angiogenesis

Key aspects for conception and construction of co-culture models of tumor-stroma interactions

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