Published OnlineFirst December 8, 2017; DOI: 10.1158/1541-7786.MCR-17-0272 Signal Transduction Molecular Cancer Research Nanoscale Tuning of VCAM-1 Determines VLA-4–Dependent Melanoma Cell Plasticity on RGD Motifs Katharina Amschler1, Eugen Kossmann1, Luise Erpenbeck1, Sebastian Kruss2, Tillmann Schill1, Margarete Schon€ 1, Sigrid M.C. Mockel€ 1, Joachim P. Spatz3, and Michael P. Schon€ 1 Abstract The biophysical fine-tuning of cancer cell plasticity is crucial for 1 in a dichotomic and density-dependent fashion. This was tumor progression but remains largely enigmatic. Although vas- accompanied by concordant regulation of F-actin cytoskeleton cular cell adhesion molecule-1 (VCAM-1/CD106) has been impli- remodeling, Rac1-expression, and paxillin-related adhesion for- cated in melanoma progression, here its presentation on endo- mation. The novel function of VCAM-1 was corroborated in vivo thelial cells was associated with diminished melanoma cell using two murine models of pulmonary metastasis. The regula- spreading. Using a specific nanoscale modulation of VCAM-1 tion of melanoma cell plasticity by VCAM-1 highlights the com- (tunable from 70 to 670 ligands/mm2) next to integrin ligands plex regulation of tumor–matrix interactions. (RGD motifs) in a bifunctional system, reciprocal regulation of integrin a4 (ITGA4/VLA-4/CD49d)-dependent adhesion and Implications: Nanotechnology has revealed a novel dichotomic spreading of melanoma cells was found. As the VCAM-1/VLA-4 function of the VCAM-1/VLA-4 interaction on melanoma cell receptor pair facilitated adhesion, while at the same time antag- plasticity, as nanoscale tuning of this interaction reciprocally onizing RGD-mediated spreading, melanoma cell morphogene- determines adhesion and spreading in a ligand density-depen- sis on these bifunctional matrices was directly regulated by VCAM- dent manner. Mol Cancer Res; 16(3); 528–42. Ó2017 AACR. Introduction progression and metastasis through binding to its predominant receptor, VLA-4 (very late antigen-4, a b integrin, CD29/CD49d) Notwithstanding recent advances in the treatment of melano- 4 1 on melanoma cells (11–14). Evidence for this notion came from ma, this aggressive skin tumor is fatal in most cases once distant both murine (12, 15) and human studies, with the latter linking metastases occur (1–5). The formation of metastases depends on VCAM-mediated interactions with expression of its ligand, VLA-4, extravasation of tumor cells from blood or lymph vessels, a and "aggressiveness" of the tumor cells (16). However, emerging complex process that is orchestrated by a plethora of accessory evidence suggests a more complex and versatile role of VCAM-1/ cells, soluble and sessile mediators as well as nanoscale adhesive VLA-4 interactions, at least in certain tumors (17), a notion that is interactions (6). supported by a recent study showing that strong upregulation of VCAM-1 (vascular cell adhesion molecule-1, CD106) was one VCAM-1 on pulmonary microvessels did not lead to increased of the first adhesion molecules implicated in metastasis-promot- experimental melanoma metastasis in vivo (18). ing endothelial cell interactions with malignant tumors, including A number of influencing factors have been implicated in the melanoma (7–10). Indeed, it has been a paradigm for 25 years focal regulation of VCAM-1 expression in vivo. These include now that endothelial expression of VCAM-1 facilitates melanoma shear stress on the vessel wall (19–22), tumor cell, and immune cell–produced cytokines and chemokines (23) as well as direct or indirect contact of tumor cells with the endothelial lining 1Department of Dermatology, Venereology and Allergology, University Medical (24–26). Under certain experimental conditions, melanoma cells Center, Gottingen,€ Germany. 2Department of Physical Chemistry, Georg August € 3 may stimulate expression of VCAM-1 directly at pulmonary University, Gottingen, Germany. Department of Cellular Biophysics, Max Planck fl Institute for Medical Research, Heidelberg and Laboratory of Biophysical metastatic sites (24). In addition to upregulation during in am- Chemistry, University of Heidelberg, Heidelberg, Germany. mation or metastasis, VCAM-1 can undergo ectodomain shedding leading to a net reduction of its density (27, 28). Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). As density, clustering, or neighborhood relation of cell surface molecules can hardly be determined precisely on the nanoscale K. Amschler and E. Kossmann contributed equally to this article. under in vivo or conventional in vitro conditions, such as random Corresponding Author: Michael P. Schon,€ Department of Dermatology, coating of plastic tissue culture ware, the exact biophysical com- € € University Medical Center, Gottingen, Gottingen 37075, Germany. Phone: 49- position and consecutive functional relevance of VCAM-1- or 551-3966401; E-mail: [email protected] VLA-4–containing microdomains still remain unknown. Current- doi: 10.1158/1541-7786.MCR-17-0272 ly available model systems do not allow the study of isolated Ó2017 American Association for Cancer Research. VCAM-1 functions, let alone VCAM-1 functions in conjunction 528 Mol Cancer Res; 16(3) March 2018 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst December 8, 2017; DOI: 10.1158/1541-7786.MCR-17-0272 Nanoscale VCAM-1 and Melanoma Cell Plasticity with other adhesion receptors in bifunctional systems, with exact matrices with adherent cells were washed with PBS for 5 minutes. and measurable adjustment of biophysical properties. As a con- To ascertain the validity of the results with short adhesion periods, sequence of this less-than-optimal situation, the role of VCAM-1 the experiments were repeated with observation periods of up to 3 for the dynamics and morphogenesis of malignant cells cannot be hours. Phase-contrast images from random positions were taken understood. at different time points using an Axiovert 200 system (Zeiss). On the basis of nanopatterning and biofunctionalization con- cepts, we now provide experimental evidence for an unexpected Melanoma cell adhesion on endothelial cells dichotomic function of VCAM-1 toward human melanoma cells. Human umbilical vein endothelial cells (HUVEC; Lonza) were Experimental platforms modeling density variation of relevant cultured in endothelial cell growth medium (EGM; Lonza) sup- fi receptors and extracellular signals in a nanoscopically de ned plemented with gentamycin at 37 C and 5% CO2 in a humidified spatial arrangement allow the study of functional consequences of atmosphere. HUVECs were used at passages four to six. Upon important (patho)physiologic regulations, such as receptor dis- confluence, endothelial cells were exposed for 4 hours to recom- tribution. Therefore, such nanotechnological approaches can binant human TNFa (25 ng/mL; ImmunoTools). Binding sites of refine our models of tumor cell biology. To mimic the variable VCAM-1 were blocked for 1 hour using a function-blocking presentation of VCAM-1, we have generated mono- and bifunc- VCAM-1–directed antibody, or the cultures were exposed to an tional matrices based on gold nanopatterns functionalized with equal amount of an unrelated isotype-matched control antibody. VCAM-1 alone or interspersed with RGD-binding motifs, respec- PKH26 (Sigma)-labeled human melanoma cells (105) were tively. These nanoscopically defined matrices are models of endo- allowed to attach for 40 minutes. Wells were washed three times thelial cell surfaces in different states of activation. VCAM-1 site in a standardized manner with PBS. Adhesion and spreading were densities were tunable over one order of magnitude (from 70 documented using an inverted fluorescence microscope (Zeiss, ligand sites/mm2 to 670 ligand sites/mm2). We demonstrate that Axiovert 200) and quantitated using ImageJ (http://rsb.info.nih. nanoscopic VCAM-1 mediates firm attachment of melanoma cells gov/ij/). but, unexpectedly, simultaneously inhibits RGD-induced mela- noma cell spreading as well as the associated cytoskeletal reor- Nanopatterning ganization and focal contact formation in a density-dependent Nanostructured matrices were produced by block copolymer fashion. These results may shift the paradigm of a unidirectional micellar nanolithography (BCML; refs. 30–33). In short, glass function of VCAM-1 by demonstrating that its interaction with cover slides (24 Â 24, no. 1; Carl Roth) were cleaned in caroic acid VLA-4 can elicit a seemingly paradoxical inhibition of melanoma [1 hour, 1:3 H2O2 (30%): H2SO4 (p.A., Merck)]. These glass slides cell spreading. The inhibitory activity was specific for the VCAM-1/ were then sonicated four times for 5 minutes each and dried with VLA-4 interaction. Thus, our results indicate a new, density- N2. Micellar solutions were prepared in a Glove Box (Braun) by dependent function of VCAM-1 toward melanoma cells with dissolving the polystyrene-polyvinylpyridine-block-copolymers implications for the fine-tuning of integrin-mediated spreading. (Polysciences) in toluene (Merck). The micelles' cores were then loaded with HAuCl4 Á 3H2O (Sigma). The cleaned glass cover slips Materials and Methods were dip coated into the micelle solution in a standardized fashion. Organic parts of the micelles were removed by plasma Cell culture and preparation for functional experiments treatment (10% H2, 90% Argon, 0.4 mbar, 150 W, TePla 100; The