Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis

Abstract

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration 1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2) 3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens 4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting. Endothelial cells (ECs) in zonated capillaries sustain tissue-specific homeostasis and supply angiocrine factors to guide organ regeneration 1,2. By contrast, maladaptation of ECs contributes to fibrosis and tumour progression 6,7. The mechanism(s) by which ECs acquire adaptive tissue-specific heterogeneity or maladapt within the scarred tissues or tumour microenvironment are unknown. Identifying the molecular determinants of vascular heterogeneity requires the generation of malleable and perfusable vascular networks that are

In vitro 3D Systems to Model Tumor Angiogenesis and Interactions With Stromal Cells

“…Endothelial progenitor cells (EPC) harvested as endothelial colony-forming cells (ECFC) from cord blood can also be used, but those from adult peripheral blood exhibit limited proliferation potential (Ferratge et al, 2017). Recently, Palikuqi et al (2020) reported "reset" vascular EC that transiently express ETS variant transcription factor 2 (ETV2) and that self-assemble into vascular networks and arborized cancerous human colon organoids. Quite remarkably, only cells from human origin are used in relevant 3D models; while isolation and culture of mouse EC have been developed for years now, their ability to form capillaries has proven to be more challenging (Nowak-Sliwinska et al, 2018).…”

“…Quite remarkably, using mixtures of Matrigel TM with natural polymers like fibrin turned out to noticeably improve capillary formation and resulted in efficient inosculation with the host vasculature and further blood perfusion after implantation in mice (Laib et al, 2009). Recently, Palikuqi et al (2020) developed matrices made of mixture of laminin, entactin, and collagen IV that support vasculogenesis.…”

“…Frontiers in Cell and Developmental Biology | www.frontiersin.org often respond very differently to the same microenvironment cues. Thus, efficient and robust induction of EC from human pluripotent stem cells and multiple human iPSC lines or reset EC might also constitute a renewable and infinite cell source (Jahan and McCloskey, 2020;Palikuqi et al, 2020). These findings open new perspectives which should facilitate the study of epigenetic changes in human cancer and angiogenesis studies using preclinical complex in vitro 3D systems.…”

Organotypic Modeling of the Tumor Landscape

“…However, in such approaches, endothelial cells may not always be free to interact with tumor cells because of the artificial membranes used in the organ-on-a-chip devices. To address this issue, endothelial cells were modified to produce 'reset' vascular endothelial cells (R-VECs) that grew into 3D branching vessels capable of transporting human blood in microfluidic chambers and when transplanted into mice (Palikuqi et al, 2020). These R-VECs adapted their growth upon their coculture with either normal colon organoids or patient-derived colorectal organoids.…”

Liquid-based 3D Cultured Tumoroids Modeling Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer

“…In organoids and tumoroids, complicated structures of organs and tumors can be reconstructed in vitro. For example, tumor microenvironment such as vasculatures [16,17], immune cells [12], fibroblastic stromal cells, and normal epithelial cells can be reconstructed in vitro within organoids and tumoroids [18][19][20]; (6) Gene expression signatures, molecular profiles, and secretory phenotypes are particular in 3D tumoroids according to their physical properties, e.g., moonlighting metalloproteinases (MMPs) [6,13,[21][22][23], ATP-binding cassette (ABC) transporters including ABC-G1 and ABC-G2 [15], and intercellular adhesion molecules such as EpCAM/CD326 and E-cadherin [7,12], stem cell markers such as CD44 variants and CD133 [7], other factors potentially involving stemness such as ESRP1/2, MUC1, and Notch/delta signal [7]. The molecular profiles of tumoroids involve properties of CSCs [7].…”