Cancer cell angiogenic capability is regulated by 3D culture and integrin engagement Claudia Fischbacha,b, Hyun Joon Konga,c, Susan X. Hsionga, Marta B. Evangelistaa,d,e, Will Yuena, and David J. Mooneya,f,1 aSchool of Engineering and Applied Sciences, Harvard University, 40 Oxford Street, Cambridge, MA 02138; bDepartment of Biomedical Engineering, Cornell University, 157 Weill Hall, Ithaca, NY 14853; cDepartment of Chemical and Biomolecular Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801; dInstituto de Engenharia Biomedica, Divisao de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal; eUniversidade do Porto, Faculdade de Engenharia, Departamento de Engenharia Metalu´rgica e de Materiais, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal; and fWyss Institute of Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138 Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved November 26, 2008 (received for review September 9, 2008) Three-dimensional culture alters cancer cell signaling; however, the regulate VEGF and IL-8 secretion and that these differences underlying mechanisms and importance of these changes on tumor impact tumor vascularization in vivo. These findings serve to vascularization remain unclear. A hydrogel system was used to examine identify 3D integrin engagement as a mechanism that alters cancer the role of the transition from 2D to 3D culture, with and without cell angiogenic signaling and that may be explored toward more integrin engagement, on cancer cell angiogenic capability. Three- efficacious antiangiogenic therapies. dimensional culture recreated tumor microenvironmental cues and led to enhanced interleukin 8 (IL-8) secretion that depended on integrin Results and Discussion engagement with adhesion peptides coupled to the polymer. In contrast, The roles of 3D culture and integrin engagement on cancer cell vascular endothelial growth factor (VEGF) secretion was unaffected by angiogenic potential were examined with oral squamous cell car- 3D culture with or without substrate adhesion. IL-8 diffused greater cinoma cells (OSCC-3) and a synthetic ECM of alginate hydrogels distances and was present in higher concentrations in the systemic containing covalently coupled RGD adhesion peptides at a density circulation, relative to VEGF. Implantation of a polymeric IL-8 delivery that correlates to the number of RGD adhesion sites in tumors in system into GFP bone marrow-transplanted mice revealed that localized vivo (9). OSCC-3 cells were chosen because they are representative IL-8 up-regulation was critical to both the local and systemic control of of a particularly aggressive epithelial malignancy that is mediated in tumor vascularization in vivo. In summary, 3D integrin engagement large part by its microenvironmentally controlled angiogenic po- within tumor microenvironments regulates cancer cell angiogenic tential (10). Alginates gel in the presence of divalent cations (e.g., signaling, and controlled local and systemic blockade of both IL-8 and Ca2ϩ), yielding matrices with controlled mechanical properties VEGF signaling may improve antiangiogenic therapies. (Ϸ60 kDa in our studies) and pore sizes that degrade very slowly and by a mechanism that is independent of cell-secreted proteolytic ECM ͉ microenvironment ͉ tumor vascularization ͉ drug delivery ͉ IL-8 enzymes (11). Consequently, alginate hydrogels provide controlla- ble and constant chemical and physical conditions to adherent or hanges of the microenvironment are fundamental to tumori- encapsulated cells, as contrasted with many other natural biopoly- Cgenesis, and 3D cultures may be used to study the mechanisms mers (e.g., Matrigel, collagen). and effects by which these alterations modulate cancer cell signaling Alginates are typically nonadhesive to cells, but chemical mod- (1). Pathologically relevant cancer models have been developed by ification with RGD, the primary integrin recognition site contained culturing tumor cells within natural and synthetic extracellular in many ECM proteins (e.g., fibronectin) (12), readily allowed matrix (ECM) mimics, and 3D microenvironmental conditions OSCC-3 to adhere onto topographically flat films of these gels (Fig. within these culture systems clearly alter tumor cell signaling (2, 3). 1A). To develop a pathologically relevant mimic of the ECM found However, the multiple variables typically inherent in the transition in tumors in vivo, alginates were modified with RGD peptides to from 2D to 3D make it difficult to define the underlying mecha- yield matrices that recreated the density of RGD adhesion sites of nisms and importance of these changes. For example, there may tumor-associated ECM in vivo (RGD-alginates, 8.0 ϫ 1016 per mL simultaneously exist differences in the soluble environment because matrix; tumor-associated ECM, 8.6 ϫ 1016 per mL matrix). OSCC-3 of substances diffusing through or from the adhesion substrate, attached onto RGD-alginate surfaces in a quantitatively similar but central hypoxia, and polarity of cell adhesion. Comparison between less spread manner compared with tissue culture plastic (TCPS) tumor cells seeded onto ECM-coated 2D culture dishes and and fibronectin-coated TCPS (Fig. 1A). Inhibition of ␣5␤1 and encapsulated within 3D ECM gels can be further complicated by ␣v␤3 integrins (i.e., the two predominant receptors involved in differing mechanical properties of the adhesion substrate in each binding ECM molecules with RGD domains) by specific antibodies situation that can alter tumor cell behavior (4). indicated that cells predominantly engage ␣5␤1 integrins to interact Tumor vascularization represents a hallmark of cancer, and we with RGD-modified alginate substrates because decreased OSCC-3 have developed an artificial ECM culture system to examine the adhesion and proliferation were observed in response to anti-␣5␤1 role of the transition from 2D to 3D culture on tumor vasculariza- treatment, but not blockade of ␣v␤3 (Fig. 1B). Additionally, tion, in the absence or presence of integrin engagement under OSCC-3 cultured on these matrices secreted the proangiogenic constant chemical and physical properties. Tumor vascularization is factors VEGF, IL-8, and bFGF in a manner comparable with other mediated by an overbalance of proangiogenic molecules that may SCIENCES be caused by dysregulated cell–ECM interactions (5). Vascular endothelial growth factor (VEGF), basic fibroblast growth factor Author contributions: C.F. and D.J.M. designed research; C.F., H.J.K., S.X.H., M.B.E., and APPLIED BIOLOGICAL (bFGF), and IL-8, in particular, have been widely investigated for W.Y. performed research; H.J.K., S.X.H., and W.Y. contributed new reagents/analytic tools; their role in tumor vascularization, and their signaling is regulated C.F., H.J.K., and D.J.M. analyzed data; and C.F. and D.J.M. wrote the paper. by both cell–ECM interactions and 3D culture conditions (3, 6, 7). The authors declare no conflict of interest. Because integrin engagement with ECM molecules is differentially This article is a PNAS Direct Submission. regulated in 2D relative to 3D culture (3, 8), it is likely that these 1To whom correspondence should be addressed. E-mail: [email protected]. changes underlie the tumor angiogenic switch. We provide exper- This article contains supporting information online at www.pnas.org/cgi/content/full/ imental evidence that the integrated effects of 3D tumor micro- 0808932106/DCSupplemental. environmental cues and 3D integrin engagement differentially © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808932106 PNAS ͉ January 13, 2009 ͉ vol. 106 ͉ no. 2 ͉ 399–404 Downloaded by guest on September 26, 2021 A TCPS fibronectin RGD-alginate alginate A morphology cellularity hypoxia o n io n s e h alginate d 50 um a 100 um 25 um 50 um ) B Adhesion 30 Proliferation 25 control a5b1 ab avb3 ab 10,000 20 el (x RGD-alginate 100 um 25 um 50 um r g 15 ** e p 10 5 umber B 2-D 3-D 3.0 0 1) Cell n control a5b1 ab avb3 ab 70 alginate ** day 2.5 ** 60 o 1E+06 16 2.0 50 ve t C ) RGD-alginate no peptide 4 * ati 1E+06 l * nits) * 40 1.5 ** re x10 12 u ( l 30 r ( 8E+05 4.8 and 1.0 8 cel itrary 9.7x10 day to 1) ber (relative / 8 20 b 6E+05 RGD/cell 0.5 alginate ds 10 97.5 x108 * RGD-alginate 4E+05 4 ll numbe 0 e 0.0 sity (ar RGD/cell C n # bon 2E+05 num Cell 0 day 1 day 5 day 10 day 1 day 5 day 10 day 15 Inte 0E+00 0 4,815 9,650 96,500 508 528 548 568 588 608 628 648 Fig. 2. Histological characteristics and cell proliferation in 2D and 3D alginate 5 Wavelength (nm) n RGD per cell (x10 ) cultures. (A) Images of cells cultured within gels prepared from unmodified (alginate) or RGD-modified alginate (RGD-alginate) displayed qualitatively sim- Fig. 1. Cell binding and integrin engagement. (A) Two-dimensional adhesion ilar morphologies and cellularity as detected by microscopic evaluation of tumor studies verified that OSCC-3 adhered to RGD-functionalized alginate in a manner spheroids and H&E-stained histological cross-sections, respectively. Hypoxia, as comparable with TCPS and TCPS coated with fibronectin (fibronectin), whereas determined by immunohistological staining for the hypoxia marker Hy- seeding cells on nonmodified alginate matrices (alginate) did not result in cell poxyprobe (brown stain; most visible in magnified Insets) developed in both adhesion. (B) Inhibition of ␣5␤1 integrins with function blocking antibodies (a5b1 culture systems. (Inset scale bars: 10 ␮m.) (B) Integrin engagement allowed for ab) decreased adhesion and proliferation of OSCC-3 on 2D RGD-alginate sub- tumor cell proliferation when cells were cultured on (2D) or in (3D) RGD-modified strates compared with control conditions, whereas no statistically significant alginate gels, whereas no proliferation occurred in the absence of cell–matrix differences were detected after inhibition of ␣v␤3 integrins (avb3 ab).