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

BACKGROUND: Anti-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. METHODS: Here, we describe a fixed end-point and kinetic, high-throughput stem cell co-culture model of cord formation. RESULTS: In 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. CONCLUSION: Together, these models may be used to identify novel drugs targeting tumor vessels that are not sensitive to VEGF inhibition

An <i>In Vitro</i> Cord Formation Assay Identifies Unique Vascular Phenotypes Associated with Angiogenic Growth Factors

<div><p>Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous <i>in vitro</i> assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into <i>in vivo</i> tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.</p></div

The role of endogenous growth factors in basal cord formation.

<p>(<b>A</b>) Basal cords made with ADSCs and ECFCs without the addition of growth factors were treated with IgG, or anti-VEGF or anti-HGF antibodies and stained for cords (CD31; green), smooth muscle actin (SMA; red), and nuclei (Hoechst 33342; blue). (<b>B</b>) Basal cords treated with IgG, anti-VEGF, or anti-HGF were quantified on the ArrayScan and the total tube area (left) and SMA index (right) are shown. * p<0.0001 vs basal. n = 3 per group. Scale bars are 250 µm.</p

Induction of cord formation with a TGF-β inhibitor.

<p>(<b>A</b>) Co-cultures of ADSCs and ECFCs were treated with a TGF-β inhibitor (LY2157299) and stained for cords (CD31; green), smooth muscle actin (SMA; red), and nuclei (Hoechst 33342; blue). (<b>B</b>) ArrayScan quantifications of total tube area following treatment with LY2157299 in basal medium (left) versus VEGF driven cords (right). n = 6 per group. (<b>C</b>) Mice harboring A549 tumor xenografts were treated with a TGF-β inhibitor (LY2157299), an inactive control (LY596144), or sunitinib (Sutent; SU11248) for 5 days. Tumors were fixed, sectioned, stained for tumor vessels (CD31; green), and quantified using Image J. * = p<0.05 vs. control. n = 4–6 per treatment group. Scale bars in A are 250 µm. Scale bars in B are 50 µm.</p

Cross-talk between angiogenic growth factors.

<p>Basal, VEGF-, bFGF-, or EGF-driven cords were treated with a VEGFR-2 antibody (IMC-1121B; Ramucirumab; Ram), a bFGF antibody (anti-bFGF), or an EGFR inhibitor (Gefitinib) and the percent inhibition of total tube area from the ArrayScan was graphed. n = 3 per group.</p

Characterization of growth factors present in the ADSC/ECFC co-culture system.

<p>(<b>A</b>) Basal or VEGF driven co-cultures of ADSCs and ECFCs were stained for cords (CD31; green), smooth muscle actin (SMA; red), and nuclei (Hoechst 33342; blue). (<b>B</b>) Media was collected from ADSCs alone or ADSC/ECFC co-cultures in basal or VEGF driven conditions at 48 (blue bar) and 96 hrs (red bar). Examination of angiogenic growth factors present in the collected media was measured using Luminex. VEGF, HGF, TGF-β, Ang1, and Ang2 had detectable levels above basal media, while bFGF, EGF, and PDGF were not detected (data not shown). n = 3–4 per group with similar results found on two separate experiments. * = p<0.05 vs. all other treatment groups. † = p<0.05 vs. basal media. Scale bars are 250 µm.</p

Phenotypes of angiogenic growth factor combinations.

<p>(<b>A</b>) ADSC and ECFC co-cultures were treated with combinations of angiogenic growth factors (V = VEGF, H = HGF, F = bFGF, E = EGF) with or without anti-VEGF (Bevacizumab; Bev) and stained for cords (CD31; green), smooth muscle actin (SMA; red), and nuclei (Hoechst 33342; blue). (<b>B</b>) ArrayScan quantifications of total tube area, branching index, length to width ratio, and SMA index of samples treated with combinations of growth factors with and without bevacizumab. n = 3 per group. * = p<0.05 vs. V+H. † = p<0.05 vs. V+F. ‡ = p<0.05 vs. H+E. # = p<0.05 vs. F+E. ₊ = p<0.05 vs. H+E+F. Scale bars are 250 µm.</p