RAIN-Droplet: a novel 3D in vitro angiogenesis model

Abstract

Angiogenesis is fundamentally required for the initialization, development and metastatic spread of cancer. A rapidly expanding number of new experimental, chemical modulators of endothelial cell function have been described for the therapeutic inhibition of angiogenesis in cancer. Despite this expansion, there has been very limited parallel growth of in vitro angiogenesis models or experimental tools. Here we present the Responsive Angiogenic Implanted Network (RAIN)-Droplet model and novel angiogenesis assay using an endothelial cell culture model of microvascular endothelial cells encapsulated in a spontaneously self-assembling, toroidal hydrogel droplet uniquely yielding discrete, pre-formed, angiogenic networks that may be embedded in 3D matrices. On embedding, radial growth of capillary-like sprouts and cell invasion was observed. The sprouts formed not only as outgrowths from endothelial cells on the surface of the droplets, but also, uniquely, from the pre-formed network structures within the droplet. We demonstrate proof of principle for the utility of the model showing significant inhibition of sprout formation (P<0.001) in the presence of bevacizumab, an anti-angiogenic antibody. Using the RAIN-Droplet assay, we also demonstrate a novel dose-dependent pro-angiogenic function for the characteristically anti-angiogenic multi-kinase inhibitor sorafenib. Exposure of endothelial cells in 3D culture to low, non-lethal doses (<1 μM) of sorafenib after initiation of sprouting resulted in the formation of significantly (P<0.05) more endothelial sprouts compared with controls over a 48-h period. Higher doses of sorafenib (5 μM) resulted in a significant (P<0.05) reduction of sprouting over the same time period. The RAIN-Droplet model is a highly versatile and simply constructed 3D focal sprouting approach well suited for the study of vascular morphogenesis and for preclinical testing of drugs. Furthermore, the RAIN-Droplet model has facilitated the discovery of a novel pro-angiogenic capacity for sorafenib, which may impact the clinical application and dosing regimen of that drug.

Figure 1. Schematic illustration of the RAIN-Droplet model

Depicting the three primary stages involved in construction of this 3-D culture model. Note that model requires only micropipette and common culture-ware for construction.

Figure 2. Formation of toroids was dependent on PM concentration

(upper panels), scale bar, 400 μm. Toroids formed from concentrations of 0.1% w/v PM and below. Toroids contracted as sprouting HDMEC networks were formed within the droplet. The same toroidal droplet is shown after 48 h incubation in culture medium (lower panel).

Figure 3. Brightfield and transmission electron micrographs of RAIN-droplets

(a) Continuous coverage of 0.05% PM toroid circumference by HDMEC (arrows), scale bar 200 μm. (b) Cell junctions at circumference, matrix is clearly visible within toroid to left of cells. Arrows indicate electron dense regions at cell-cell interface, scale bar 500 nm. (c) Structures in contracted toroids included a variety of cell structures, some bearing lumen-like vacuoles empty of surrounding matrix (arrow), scale bar 2 μm.

Figure 4. RAIN-Droplet model in 3-D culture and assay for effect of angiogenesis inhibitors on endothelial sprouting

(a), Actin was labelled with Alexafluor-488 Phalloidin (green) and nuclear staining with DAPI (bright blue), representative of multiple labeling studies. HDMEC sprouts originating from sprouting structures within the toroid (arrowhead) and from cells along the toroid circumference (arrow). The RAIN-Droplet toroid is visible as a diffuse, dark blue arch at the top right of the panel bordered by arches of green actin phalloidin stained cells. The droplet is potentially fluorescent due to either slight autofluorescence or differential retention of DAPI after labeling compared to the surrounding collagen. (b), Embedded RAIN-droplet toroids were exposed to VEGF (50 ng/ml) for 24 h then bevacizumab, 25 μg/ml, for up to 48 h. Scale bar is 100 μm. Bevacizumab (center panel) significantly inhibited endothelial sprouts compared to control (right panel) and caused marked attenuation of cell invasion (insets) (n=7-8, p<0.001).

Figure 5. Concentration dependent bi-phasic stimulatory and inhibitory effects of the multi-kinase inhibitor sorafenib on endothelial capillary sprout formation

(a) Proliferation assay for HDMEC exposed to varying concentrations of sorafenib. Cells were plated in 96-well plates in the presence or absence of sorafenib at indicated concentrations. Tetrazolium dye WST-1 was added at 24, 48 and 72 hours to individual plates and absorbance was determined by microplate reader at 540 nm. Test readings were normalized against absorbance at initial plating density and also as a percentage of untreated time-point controls, (n=6 for all points). (b) HDMEC were encapsulated in RAIN-Droplet gels, embedded in collagen with VEGF (5 ng in 0.1 ml collagen). Sorafenib was added to the wells in fresh culture medium after initial formation of capillary sprouts had occurred (24-72h). Capillary sprout number was assessed by observation under high power microscope (200x). (c) HDMEC were treated as for panel (b) but sorafenib was added immediately after embedding and prior to capillary sprout formation (n=4 for all sprouting assays data points).

Figure 6. Sorafenib promotes formation of robust endothelial sprouts during sprouting anastomosis between two RAIN-droplets

HDMEC were prepared for the RAIN-Droplet model as previously described and embedded in collagen containing VEGF, 50 ng/ml. Droplets were incubated 72 h to establish sprouts then were treated plus or minus sorafenib (0.5 μM) for a further 72 h with images of the same field captured every 24h. Notably before treatment the fields look very similar however the sorafenib appears to drive the HDMEC to rapidly organize and aggregate in one or two robust sprouts. The control cells form thinner, less coherent sprouts which become disorganized without further stimulation.

Figure 7. Effect of low concentration sorafenib on endothelial cell cord formation in the matrigel angiogenesis assay

HDMEC were plated onto matrigel in the presence or absence of sorafenib, 0.5 and 5 μM. Cells were incubated for up to 72 h with the same field being photographed per well every 24h. Cord numbers were counted manually using the Image J (NIH) point counter function, n ≥ 3 wells per concentration. Cords were defined as a discrete and linearly directional cord of cells stretching between two junction nodes. Sorafenib was found to increase cord number relative to controls with 5 μM sorafenib inducing significantly greater numbers of cords (p≤0.01).