Cancer cell angiogenic capability is regulated by 3D culture and integrin engagement

Abstract

Three-dimensional culture alters cancer cell signaling; however, the underlying mechanisms and importance of these changes on tumor vascularization remain unclear. A hydrogel system was used to examine the role of the transition from 2D to 3D culture, with and without integrin engagement, on cancer cell angiogenic capability. Three-dimensional culture recreated tumor microenvironmental cues and led to enhanced interleukin 8 (IL-8) secretion that depended on integrin engagement with adhesion peptides coupled to the polymer. In contrast, vascular endothelial growth factor (VEGF) secretion was unaffected by 3D culture with or without substrate adhesion. IL-8 diffused greater distances and was present in higher concentrations in the systemic circulation, relative to VEGF. Implantation of a polymeric IL-8 delivery system into GFP bone marrow-transplanted mice revealed that localized IL-8 up-regulation was critical to both the local and systemic control of tumor vascularization in vivo. In summary, 3D integrin engagement within tumor microenvironments regulates cancer cell angiogenic signaling, and controlled local and systemic blockade of both IL-8 and VEGF signaling may improve antiangiogenic therapies.

Fig. 1.

Cell binding and integrin engagement. (A) Two-dimensional adhesion studies verified that OSCC-3 adhered to RGD-functionalized alginate in a manner comparable with TCPS and TCPS coated with fibronectin (fibronectin), whereas seeding cells on nonmodified alginate matrices (alginate) did not result in cell adhesion. (B) Inhibition of α5β1 integrins with function blocking antibodies (a5b1 ab) decreased adhesion and proliferation of OSCC-3 on 2D RGD-alginate substrates compared with control conditions, whereas no statistically significant differences were detected after inhibition of αvβ3 integrins (avb3 ab). (Scale bar: 50 μm.) **, P < 0.01. (C) FRET analysis to quantify the number of receptor–ligand bonds formed between cells in 3D culture and RGD peptides coupled to the polymer. Increasing the number of RGD peptides significantly increased integrin engagement by RGD-functionalized alginates as detected by increased energy transfer (decreased donor fluorescein emission intensity at λ = 520 nm; increased acceptor emission at λ = 580 nm). *, P < 0.05. Changes in energy transfer were translated into absolute bond numbers per cell as a function of the number of RGD ligands available to each cell (n RGD per cell), as described in ref. , and no bonds were noted in the absence of RGD ligands.

Fig. 2.

Histological characteristics and cell proliferation in 2D and 3D alginate cultures. (A) Images of cells cultured within gels prepared from unmodified (alginate) or RGD-modified alginate (RGD-alginate) displayed qualitatively similar morphologies and cellularity as detected by microscopic evaluation of tumor spheroids and H&E-stained histological cross-sections, respectively. Hypoxia, as determined by immunohistological staining for the hypoxia marker Hypoxyprobe (brown stain; most visible in magnified Insets) developed in both culture systems. (Inset scale bars: 10 μm.) (B) Integrin engagement allowed for tumor cell proliferation when cells were cultured on (2D) or in (3D) RGD-modified alginate gels, whereas no proliferation occurred in the absence of cell–matrix interactions (alginate) in either 2D or 3D culture. Cellularity at later stages of 3D culture was controlled by integrin engagement. *, P < 0.05; **, P < 0.01.

Fig. 3.

Angiogenic characteristics of tumor cells cultured in 2D and 3D alginate systems. (A) Three-dimensional culture (3D, RGD-alginate) resulted in dramatically enhanced IL-8 levels relative to 2D culture (2D, RGD-alginate), and integrin engagement was critical to this end as determined by ELISA of conditioned medium from 3D cultures within unmodified (3D, alginate) and RGD-modified alginate (3D, RGD-alginate). No significant differences were detected between VEGF secretion in 2D (2D, RGD-alginate) and 3D cell culture (3D, RGD-alginate), and 3D integrin engagement increased VEGF secretion in 3D cultures only slightly. (B) Three-dimensional control experiments with nonadhesive RGE-alginate resulted in diminished secretion of IL-8 (*, P < 0.05; **, P < 0.01), whereas VEGF secretion was similar for cultures within nonadhesive (alginate, RGE-alginate) and adhesive (RGD-alginate) matrices. (C) Analysis of the contribution of the 3D environment and 3D integrin engagement. Cytodex beads were coated in a 2D manner with OSCC-3 (cell-coated beads) and subsequently encapsulated within 3D alginate (alginate, w/beads). IL-8 secretion for this condition was enhanced relative to 2D cultures (2D, RGD-alginate) but was lower compared with both 3D conditions (3D, alginate and 3D, RGD-alginate). No remarkable differences were detected for VEGF and bFGF secretion under these conditions. (D) VEGF, bFGF, and IL-8 secretion presented as a fraction of the total secretion of angiogenic factors (in picograms per 10,000 cells). The effects of 3D microenvironmental conditions and 3D integrin engagement were determined by comparing 2D cultures on RGD alginate, 3D culture within alginate and RGD-alginate, and tumors formed in vivo. Data bars are small where not visible.

Fig. 4.

Analysis of local and systemic proangiogenic signaling. (A) Mathematical modeling of VEGF and IL-8 concentrations in the tissue surrounding the tumor as a result of diffusion, elimination, and secretion predicts that IL-8 will be present at greater concentrations, compared with VEGF, and is more likely to reach the systemic circulation. (B) IL-8 is more broadly distributed throughout the surrounding pseudotissue (Matrigel) than VEGF in vitro. Specifically, IL-8 diffuses away from an initial point source (0 h) more readily, relative to VEGF, as determined by diffusion analysis of fluorescently labeled VEGF and IL-8 within Matrigel after 0.5 h, 1 h, and 2 h. Spatial distribution curves generated by densitometric image analysis show average values (n = 4). (C) Calculation of the normalized systemic concentrations of angiogenic factors (fraction of the sum of the concentration in the local tumor microenvironment and the systemic circulation as calculated based on the total volume of each tumor and a mouse blood volume of ≈72 mL/kg) confirmed that IL-8 is available in the systemic circulation to a much greater extent than the other two factors. *, P < 0.05.

Fig. 5.

Systemic and local effects of IL-8 on tumor vascularization and progression in vivo. (A) Antiangiogenic therapy using delivery of antibodies blocking IL-8 and VEGF either individually (IL-8 ab, VEGF ab) or simultaneously (both ab, both ab i.p.) inhibited tumor growth compared with the no-antibody control condition. Localized delivery of neutralizing IL-8 antibody (IL-8 ab) inhibited tumor progression more significantly compared with delivery of VEGF antibody (VEGF ab) (P < 0.05) and localized delivery of a regimen that consisted of both neutralizing IL-8 and neutralizing VEGF antibody (both ab) inhibited tumor formation more extensively relative to the same therapy systemically applied via i.p. injection (both ab i.p.) (P < 0.05). (B) Localized and sustained delivery of IL-8 from polymeric scaffolds as a mimic of IL-8 secretion from tumors (IL-8) resulted in increased blood vessel formation at the muscle–scaffold interface relative to implantation of blank control scaffolds (control) as determined by image analysis of histological cross-sections stained for the endothelial cell marker CD31 (P < 0.05). Inside the scaffold, blood vessel density was similar in both the control and IL-8 condition (data not shown). Dashed lines indicate the muscle–scaffold boundary. (C) Localized IL-8 release increased recruitment of bone marrow-derived cells (green) to the vasculature (red). IL-8 delivery from polymer scaffolds (IL-8) in mice transplanted with EGFP bone marrow led to more cells that costained for CD31 and EGFP, compared with mice transplanted with blank control scaffolds (control) (P < 0.05) as analyzed by coimmunofluorescence of cryosections (see Fig. S9 for confirmation of colocalization of staining). Arrows and dashed lines indicate double-labeled cells and the muscle–scaffold boundary, respectively.