Development and characterization of a high-throughput in vitro cord formation model insensitive to VEGF inhibition

Abstract

BackgroundAnti-VEGF therapy reduces tumor blood vessels, however, some vessels always remain. These VEGF insensitive vessels may help support continued tumor growth and metastases. Many in vitro assays examining multiple steps of the angiogenic process have been described, but the majority of these assays are sensitive to VEGF inhibition. There has been little focus on the development of high-throughput, in vitro assays to model the vessels that are insensitive to VEGF inhibition.MethodsHere, we describe a fixed end-point and kinetic, high-throughput stem cell co-culture model of cord formation.ResultsIn this system, cords develop within 24 hours, at which point they begin to lose sensitivity to VEGF inhibitors, bevacizumab, and ramucirumab. Consistent with the hypothesis that other angiogenic factors maintain VEGF-independent vessels, pharmacologic intervention with a broad spectrum anti-angiogenic antagonist (suramin), a vascular disrupting agent (combretastatin), or a combination of VEGF and Notch pathway inhibitors reduced the established networks. In addition, we used our in vitro approach to develop an in vivo co-implant vasculogenesis model that connects with the endogenous vasculature to form functional blood vessels. Similar to the in vitro system, over time these vessels become insensitive to VEGF inhibition.ConclusionTogether, these models may be used to identify novel drugs targeting tumor vessels that are not sensitive to VEGF inhibition.

An In Vitro Cord Formation Assay Identifies Unique Vascular Phenotypes Associated with Angiogenic Growth Factors

“…This increase in Ang1 is likely from pericytes associated with the endothelial cells. Ang1 is secreted by pericytes and previous studies have shown that pericyte association with cords began on day 3 and increased with time [7]. Therefore, there are likely only enough pericytes present at the 96 hr time point to effectively increase the Ang1 concentration.…”

“…This assay can be run in 96 and 384 well format with qualitative analysis on either an ArrayScan or an Acumen ([7] and Figure S2 ). Quantitative validation was performed on a similar assay ran on the Acumen eX3 in a 384 well format (Z′ score  = 0.68 or 0.70 and MSR  = 1.69 or 1.99 for tube area and SMA area analysis respectively; Figure S2 ).…”

“…Cells were stained essentially as previously described [7]. Endothelial cells were detected with a sheep anti-CD31 (PECAM-1; Sigma; 1∶200) antibody, smooth muscle actin (SMA) was detected with either a Cy3 conjugated mouse anti-smooth muscle actin (Sigma; 1∶200) or a mouse anti-human SMA antibody (Sigma; 1∶200), and nuclei was detected with Hoechst 33342 (Invitrogen; 1∶1000).…”

Human iPSC-derived endothelial cell sprouting assay in synthetic hydrogel arrays

“…EC functional assays are useful to recapitulate one or multiple events of the angiogenesis cascade, but many existing assays lack the ability to recapitulate VEGF-dependent[5,15,62] and MMP-dependent[13,14] angiogenesis and currently lack chemically-defined substrates in a quantitative assay format relevant to angiogenesis. Therefore, there is a motivation to develop in vitro platforms in which EC behavior is dependent on VEGF signaling and MMP activity in a chemically-defined array format.…”

“…The lack of dependence on MMP activity is a limitation of two-dimensional configurations that are commonly used in small molecule screening applications[13,14]. Furthermore, the vascular endothelial growth factor (VEGF)-dependence is unclear in some tubulogenesis assay platforms[5,15], and this further limits comparisons of EC tubulogenesis to VEGF-dependent in vivo vascular formation[1]. …”