The biophysical fine-tuning of cancer cell plasticity is crucial for tumor progression but remains largely enigmatic. Although vascular cell adhesion molecule-1 (VCAM-1/CD106) has been implicated in melanoma progression, here its presentation on endothelial cells was associated with diminished melanoma cell spreading. Using a specific nanoscale modulation of VCAM-1 (tunable from 70 to 670 ligands/μm²) next to integrin ligands (RGD motifs) in a bifunctional system, reciprocal regulation of integrin α4 (ITGA4/VLA-4/CD49d)-dependent adhesion and spreading of melanoma cells was found. As the VCAM-1/VLA-4 receptor pair facilitated adhesion, while at the same time antagonizing RGD-mediated spreading, melanoma cell morphogenesis on these bifunctional matrices was directly regulated by VCAM-1 in a dichotomic and density-dependent fashion. This was accompanied by concordant regulation of F-actin cytoskeleton remodeling, Rac1-expression, and paxillin-related adhesion formation. The novel function of VCAM-1 was corroborated in vivo using two murine models of pulmonary metastasis. The regulation of melanoma cell plasticity by VCAM-1 highlights the complex regulation of tumor–matrix interactions.

Implications: Nanotechnology has revealed a novel dichotomic function of the VCAM-1/VLA-4 interaction on melanoma cell plasticity, as nanoscale tuning of this interaction reciprocally determines adhesion and spreading in a ligand density-dependent manner. Mol Cancer Res; 16(3); 528–42. ©2017 AACR.

Notwithstanding recent advances in the treatment of melanoma, this aggressive skin tumor is fatal in most cases once distant metastases occur (1–5). The formation of metastases depends on extravasation of tumor cells from blood or lymph vessels, a complex process that is orchestrated by a plethora of accessory cells, soluble and sessile mediators as well as nanoscale adhesive interactions (6).

VCAM-1 (vascular cell adhesion molecule-1, CD106) was one of the first adhesion molecules implicated in metastasis-promoting endothelial cell interactions with malignant tumors, including melanoma (7–10). Indeed, it has been a paradigm for 25 years now that endothelial expression of VCAM-1 facilitates melanoma progression and metastasis through binding to its predominant receptor, VLA-4 (very late antigen-4, α4β1 integrin, CD29/CD49d) on melanoma cells (11–14). Evidence for this notion came from both murine (12, 15) and human studies, with the latter linking VCAM-mediated interactions with expression of its ligand, VLA-4, and “aggressiveness” of the tumor cells (16). However, emerging evidence suggests a more complex and versatile role of VCAM-1/VLA-4 interactions, at least in certain tumors (17), a notion that is supported by a recent study showing that strong upregulation of VCAM-1 on pulmonary microvessels did not lead to increased experimental melanoma metastasis in vivo (18).

A number of influencing factors have been implicated in the focal regulation of VCAM-1 expression in vivo. These include 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 (24–26). Under certain experimental conditions, melanoma cells may stimulate expression of VCAM-1 directly at pulmonary metastatic sites (24). In addition to upregulation during inflammation or metastasis, VCAM-1 can undergo ectodomain shedding leading to a net reduction of its density (27, 28).

As density, clustering, or neighborhood relation of cell surface molecules can hardly be determined precisely on the nanoscale under in vivo or conventional in vitro conditions, such as random coating of plastic tissue culture ware, the exact biophysical composition and consecutive functional relevance of VCAM-1- or VLA-4–containing microdomains still remain unknown. Currently available model systems do not allow the study of isolated VCAM-1 functions, let alone VCAM-1 functions in conjunction with other adhesion receptors in bifunctional systems, with exact and measurable adjustment of biophysical properties. As a consequence of this less-than-optimal situation, the role of VCAM-1 for the dynamics and morphogenesis of malignant cells cannot be understood.

On the basis of nanopatterning and biofunctionalization concepts, we now provide experimental evidence for an unexpected dichotomic function of VCAM-1 toward human melanoma cells. Experimental platforms modeling density variation of relevant receptors and extracellular signals in a nanoscopically defined spatial arrangement allow the study of functional consequences of important (patho)physiologic regulations, such as receptor distribution. Therefore, such nanotechnological approaches can refine our models of tumor cell biology. To mimic the variable presentation of VCAM-1, we have generated mono- and bifunctional matrices based on gold nanopatterns functionalized with VCAM-1 alone or interspersed with RGD-binding motifs, respectively. These nanoscopically defined matrices are models of endothelial cell surfaces in different states of activation. VCAM-1 site densities were tunable over one order of magnitude (from 70 ligand sites/μm² to 670 ligand sites/μm²). We demonstrate that nanoscopic VCAM-1 mediates firm attachment of melanoma cells but, unexpectedly, simultaneously inhibits RGD-induced melanoma cell spreading as well as the associated cytoskeletal reorganization and focal contact formation in a density-dependent fashion. These results may shift the paradigm of a unidirectional function of VCAM-1 by demonstrating that its interaction with VLA-4 can elicit a seemingly paradoxical inhibition of melanoma cell spreading. The inhibitory activity was specific for the VCAM-1/VLA-4 interaction. Thus, our results indicate a new, density-dependent function of VCAM-1 toward melanoma cells with implications for the fine-tuning of integrin-mediated spreading.

Cell culture and preparation for functional experiments

The human melanoma cell lines A375 and MeWo as well as the murine melanoma line B16F10 were grown in DMEM supplemented with 10% FCS, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (all from Invitrogen), at 37°C in a humidified 5% CO2 atmosphere. Culture medium and solutions were prewarmed before use. Cells were subcultured twice a week using trypsin (0.05% w/v)/EDTA (5 mmol/L) for detachment. All cells were easily recognizable as melanoma cells by their melanin production. Sources (ATCC, catalog no. CRL-1619), identity, and routine mycoplasma testing of the cells have been described previously (29). Cells were expanded and multiple aliquots were stored frozen. Cells used for experiments were not passaged more than five times. PCR testing for mycoplasma contamination was performed on a routine basis according to the manufacturer's instructions (Biontex), and cells used for experiments were negative for mycoplasma.

Functional experiments with human melanoma cells were performed in 6-well culture plates (Greiner Bio-One). The cells were harvested at a confluence of approximately 70% using 1 mL Accutase Solution (PromoCell) for 5 minutes at 37°C. Cells were then resuspended in complete DMEM and counted. After washing with PBS and resuspension in DMEM containing 10% FCS, the cells were placed in the incubator for 20 minutes before being added to the functionalized matrices at a final concentration of 105 cells/mL. After incubation for 30 to 45 minutes at 37°C, the matrices with adherent cells were washed with PBS for 5 minutes. To ascertain the validity of the results with short adhesion periods, the experiments were repeated with observation periods of up to 3 hours. Phase-contrast images from random positions were taken at different time points using an Axiovert 200 system (Zeiss).

Melanoma cell adhesion on endothelial cells

Human umbilical vein endothelial cells (HUVEC; Lonza) were cultured in endothelial cell growth medium (EGM; Lonza) supplemented with gentamycin at 37°C and 5% CO2 in a humidified atmosphere. HUVECs were used at passages four to six. Upon confluence, endothelial cells were exposed for 4 hours to recombinant human TNFα (25 ng/mL; ImmunoTools). Binding sites of VCAM-1 were blocked for 1 hour using a function-blocking VCAM-1–directed antibody, or the cultures were exposed to an equal amount of an unrelated isotype-matched control antibody. PKH26 (Sigma)-labeled human melanoma cells (105) were allowed to attach for 40 minutes. Wells were washed three times in a standardized manner with PBS. Adhesion and spreading were documented using an inverted fluorescence microscope (Zeiss, Axiovert 200) and quantitated using ImageJ (http://rsb.info.nih.gov/ij/).

Nanopatterning

Nanostructured matrices were produced by block copolymer micellar nanolithography (BCML; refs. 30–33). In short, glass cover slides (24 × 24, no. 1; Carl Roth) were cleaned in caroic acid [1 hour, 1:3 H2O2 (30%): H2SO4 (p.A., Merck)]. These glass slides were then sonicated four times for 5 minutes each and dried with N2. Micellar solutions were prepared in a Glove Box (Braun) by dissolving the polystyrene-polyvinylpyridine-block-copolymers (Polysciences) in toluene (Merck). The micelles' cores were then loaded with HAuCl4 · 3H2O (Sigma). The cleaned glass cover slips were dip coated into the micelle solution in a standardized fashion. Organic parts of the micelles were removed by plasma treatment (10% H2, 90% Argon, 0.4 mbar, 150 W, TePla 100; PVA). Gold nanoparticles, herein referred to as Au-NPs, were thus firmly bound to the glass surface.

Nanoscopically defined matrices were generated with a wide range of Au-NP spacings (mean distances between adjacent nanoparticle sites). The actual densities of each batch of nano-matrices were determined precisely by scanning electron microscopy (Ultra 55; Zeiss) and covered the range from 70/μm² to 670/μm² (corresponding to ligand site distances ranging from 40 to 120 nm). As micellar block copolymer nanolithography results in a quasi-hexagonal assembly of the nanoparticles, hence the ligand site density can also be approximated by applying the formula x = 2/(d2√3).

Biofunctionalization

Biofunctionalization was performed in 60-mm Petri dishes (Greiner). Nanostructured glass surfaces were UV ozone cleaned (UV-Ozone Cleaning System UVOH 150 LAB; FHR Anlagenbau) for 10 minutes (0.8 slm). Afterwards, matrices were incubated with 100 μL of 0.5 mg/mL PLL(20kDa)-g-[3.5]-PEG(2kDa) and PLL(20kDa)-g-[3.5]-PEG(2kDa)/PEG-RGD(3.4kDa) (SuSoS), respectively, and 10 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Calbiochem) at pH 7.4 for 45 minutes on parafilm. Then, the matrices were washed in aqua dest. for 30 minutes and were carefully dried. Thereafter, they were incubated with 200 μL of 1 mg/mL HS-(CH2)11-EG3–NTA (NTA, nitrilotriacetic acid; Prochimia) in ethanol solution (1:5) for 90 minutes. Ni2+ was bound to the NTA-group by incubating the matrices for 20 minutes with 120 μL of a 10 mmol/L NiCl2 (Merck) solution in HBS (10 mmol/L HEPES, 75 mmol/L NaCl; Carl Roth) at pH 7.5. Finally, the matrices were incubated with 10 μg/mL VCAM-1 (VCAM-1 recombinant protein His tagged, HEK293-derived, MyBioSource) or PECAM-1 (PECAM-1 recombinant protein, His tagged, HEK293-derived, MyBioSource), respectively, in PBS for 2.5 hours. It had been established in additional adhesion experiments that His-tagged and non–His-tagged VCAM-1 exerted identical activities toward adhesion and spreading of human melanoma cells (cell lines A375 and MeWo). After biomolecule functionalization, the matrices were washed with PBS for 15 minutes before being used in cell experiments. Matrices were generally equilibrated in the corresponding solvent before and washed with the corresponding solvent for 10 minutes after each functionalization step. It had been established by adhesion experiments with A375 melanoma cells that treatment with NE did not reduce the passivating capacity of the PEG coating.

Surface characterization

Quartz crystal microbalance with dissipation (QCM-D) is an electromechanical method measuring frequency changes of (coated) oscillating quartz crystals. Such frequency changes can provide information about adsorption processes taking place on the coated surface. For QCM-D measurements, an E4 System (Q-Sense) was used. Gold-coated quartz crystals (4.95 MHz, Q-Sense) were cleaned for 45 minutes/150 W in O2-plasma (TePla 100). The same molecules and solvents were used for surface characterization and biofunctionalization.

To prove successful biomolecule binding to the Au-NPs, functionalized matrices were incubated with 10 μg/mL of mouse anti-human VCAM-1 IgG1 antibody (clone 1.G11B1; LifeSpan BioSciences) for 2 hours. After further incubation with 8 μg/mL of a second fluorescein-labeled anti-mouse antibody (Cell Signaling Technology) for 1 hour, the margin of functionalization was detected with fluorescence microscopy (Axiovert 200; Zeiss)

siRNA transfection of human melanoma cells

The siRNA directed against the human integrin subunit α4 (Hs_ITGA4_6; Qiagen) as well as the nontargeting control siRNA (AllStars Neg. Control siRNA; Qiagen) were diluted according to the manufacturer's instructions and stored at −20°C. Melanoma cells were transfected with siRNA at a concentration of 125 nmol/L using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Cells were added to the siRNA–lipofectamine suspension and DMEM without antibiotics. The transfection was repeated after 24 hours of incubation. Transfected cells were harvested after a further incubation of 72 hours and directly used in experiments with parallel flow cytometry analysis of α4-integrin expression.

Flow cytometry

Melanoma cells (in their exponential growth phase) were harvested using Accutase (PromoCell), washed with PBS, resuspended in PBS containing 5% human male AB-serum (Sigma), and incubated with 1 μg/mL of either anti-α4 (clone P1H4, Abcam), anti-αVβ3 (clone LM609, Merck), anti-α5 (clone SAM-1, Beckman Coulter), anti-β1 (clone 4B4LDC9LDH8, Beckman Coulter), or an IgG1 isotype control (Zymed). Surface-bound primary antibodies were detected using 0.5 μL of Alexa Fluor 488–conjugated goat-derived anti-mouse IgG (H+L) as a secondary reagent (Cell Signaling Technology). Analysis was performed using the FACSCanto II and the FACS Diva software (BD Biosciences).

Cleavage of VCAM-1 by neutrophil elastase

Human neutrophil elastase (NE; Elastin Products) was diluted in PBS at a stock concentration of 100 μg/mL. Biofunctionalized VCAM-1 matrices were incubated with 10 μg/mL of human NE dissolved in 100 μL PBS for 30 minutes at 37°C. Control matrices were incubated with 100 μL PBS. Subsequently, both matrices were washed twice in PBS prior to their use in experiments.

Immunofluorescence assays and microscopy

Matrices were washed in PBS, and attached cells were fixed with 4% formaldehyde/PBS for 30 minutes at room temperature. Subsequently, the samples were blocked with 5% FCS in PBS for 60 minutes, followed by anti-paxillin (clone Y133, Abcam) for 3 hours at room temperature. After 3 washing steps in PBS, cells were incubated with Alexa Fluor 488–conjugated anti-mouse IgG F(ab')2 (Cell Signaling Technology) for 1 hour. F-actin was then stained with phalloidin (PromoFluor-55; PromoCell) for 30 minutes. Fluorescence Mounting Medium (Dako) containing 0.5 μg/mL DAPI (Sigma) resulted in nuclear staining. Fluorescence microscopy was performed on an Axio Imager M.1 system equipped with an AxioCam MRm (Zeiss).

PCR

Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Two micrograms of total RNA were transcribed using First Strand cDNA Synthesis Kit (Fermentas). For DNA amplification, Taq-DNA Polymerase (PeqLab) was used. Primers and conditions used are detailed in Supplemental Table 1.

Data analysis

After defined time intervals (specified in the Results section), standardized overview photomicrographs were analyzed morphometrically using ImageJ. For measurement of cell areas, a grid (220 × 290 μm) was set on the image center of a 16× field. All cells within this grid were accepted for measurement granting a standardized and randomized analysis.

For the analysis of focal adhesions, images of cells stained with phalloidin and a paxillin-directed antibody were split into different color channels via ImageJ. Within the green (paxillin stain) channel, the threshold of brightness and contrast was adjusted individually for every cell to distinguish between circumferential focal adhesions and background signals. Particles between 0.1 and 5 μm2 (this range includes the cut-off value of 2 μm², which distinguishes between mature and immature focal adhesions; ref. 34) were considered as focal adhesions and counted using ImageJ. The method was confirmed by manual counting and manual size measurements of focal adhesions showing similar results.

GFP transfection of melanoma cells and short-term pulmonary metastasis in mice

GFP was stably transfected into B16F10 melanoma cells according to an optimized protocol using 1.5 × 106 cells, 2 μg, and 100 μL of Nucleofector solution V (Amaxa). Transfected cells expressing high levels of GFP were subcloned thrice, selected in DMEM (Invitrogen) supplemented with 1 mg/mL of G418 selection compound (Sigma) and sorted by FACS. In general, 106 GFP-labeled melanoma cells were injected via the lateral tail vein. Mice were sacrificed 1, 5, or 30 minutes after tumor cell injection (n = 3 mice in each group), and the lungs were immediately snap-frozen in liquid nitrogen. Two complementary lines of experiments were performed to achieve different expression states of VCAM-1 in pulmonary capillaries: The animals were either untreated or received an intratracheal instillation of 0.5 μg of the TLR2/6 agonist, MALP-2 (macrophage-activating lipopeptide-2), 24 hours prior to tumor cell injection as described previously (18). Cryostat-cut sections of the lungs were assessed by double fluorescence analysis (with GFP identifying melanoma cells and PE-labeled antibodies detecting VCAM-1). Animal experiments were approved by the appropriate governmental authority (LAVES, permission number 33.9-42502-04-14).

Statistical analysis

Data represent means ± SEM of at least three independent experiments. One-way ANOVA test was performed to detect differences between the four estimated VCAM-1 densities of 670, 280, 120, and 70 proteins per μm2. After confirming normal distribution of the data points, further individual comparisons between two matrices were performed by the Student two-tailed, unpaired t test. Means determined to be significantly different (P < 0.05) were subjected to post hoc tests using Bonferroni correction analysis.

Inhibition of VCAM-1 facilitates melanoma cell spreading on activated endothelial cells

As the consequences of endothelial cell expression of VCAM-1 for cancer cell functions have not been explored in detail, we were initially interested in direct functional activities of VCAM-1 in the interaction with human melanoma cells. Toward this end, human endothelial cells were exposed to TNFα (25 ng/mL) to stimulate a robust expression of VCAM-1 (Fig. 1A; ref. 35).

Figure 1.

VCAM-1 inhibition facilitates melanoma cell spreading on endothelial cells. A, Human endothelial cells were cultured without (top) or in the presence of TNFα (25 ng/mL, bottom) to stimulate expression of adhesion molecules. Expression of VCAM-1 was visualized by immunofluorescence. B, Human endothelial cells stimulated with TNFα and expressing VCAM-1 were treated with an isotype-matched control antibody (left photomicrographs) or with a function-blocking antibody directed against VCAM-1 (right photomicrographs). Human melanoma cells (A375) fluorescently labeled with PKH26 were allowed to adhere for 40 minutes to these endothelial cell cultures. Images depict low (top) and high magnification (bottom), respectively. In the top two photomicrographs, filled arrows indicate examples of round, nonspread melanoma cells, and open arrows indicate examples of spread melanoma cells. C, The total numbers of melanoma cells adhered to TNF-stimulated endothelial cell cultures were quantitated (left graph). In addition, the numbers of nonspread (round) and spread melanoma cells, respectively, were quantitated separately both on endothelial cells treated with isotype-matched and VCAM-directed antibodies (right graph). The values shown represent averages from 12 random microscopic fields for each condition. The experiment was repeated twice. **, P < 0.01; ***, P < 0.001.

Figure 1.

VCAM-1 inhibition facilitates melanoma cell spreading on endothelial cells. A, Human endothelial cells were cultured without (top) or in the presence of TNFα (25 ng/mL, bottom) to stimulate expression of adhesion molecules. Expression of VCAM-1 was visualized by immunofluorescence. B, Human endothelial cells stimulated with TNFα and expressing VCAM-1 were treated with an isotype-matched control antibody (left photomicrographs) or with a function-blocking antibody directed against VCAM-1 (right photomicrographs). Human melanoma cells (A375) fluorescently labeled with PKH26 were allowed to adhere for 40 minutes to these endothelial cell cultures. Images depict low (top) and high magnification (bottom), respectively. In the top two photomicrographs, filled arrows indicate examples of round, nonspread melanoma cells, and open arrows indicate examples of spread melanoma cells. C, The total numbers of melanoma cells adhered to TNF-stimulated endothelial cell cultures were quantitated (left graph). In addition, the numbers of nonspread (round) and spread melanoma cells, respectively, were quantitated separately both on endothelial cells treated with isotype-matched and VCAM-directed antibodies (right graph). The values shown represent averages from 12 random microscopic fields for each condition. The experiment was repeated twice. **, P < 0.01; ***, P < 0.001.

Close modal

Melanoma cell adhesion was not affected by antibody-mediated inhibition of VCAM-1 [isotype, 29.3 (±6.9) cells/0.1 mm2, vs. anti–VCAM-1, 33.3 (±7.1) cells/0.1 mm2, difference not significant; Figure 1B and C, left graph], presumably due to compensation by other endothelial adhesion molecules induced by TNFα. However, we observed a striking and hitherto unknown morphologic effect inasmuch as a conspicuously higher number of melanoma cells spread on the endothelial layer when VCAM-1 had been blocked (Fig. 1B and C, right graph): Although endothelial cell cultures treated with an isotype-matched control antibody facilitated spreading of 17.2 (±5.1) A375 melanoma cells/ 0.1 mm2, exposure to a function-blocking antibody directed against VCAM-1 resulted in spreading of 27.0 (±5.7) melanoma cells/0.1 mm2 (P = 0.00034). In contrast, melanoma cells that firmly adhered but did not spread were 12.0 (±5.1)/0.1 mm2 on isotype-treated but only 6.3 (±2.7)/0.1 mm2 on anti–VCAM-1–treated endothelial cells (P = 0.00351). In addition, spread melanoma cells were significantly larger by 13% when VCAM-1 was blocked (based on individual morphometric measurements of 392 cells on control cultures and 356 cells on cultures with VCAM-1 inhibition; P = 0.001). These significant VCAM-1 function-associated differences of melanoma cell behavior were not discernable from previous data and opened up the possibility of a dichotomic function of VCAM-1 for static melanoma cell adhesion on the one hand and spreading on the other.

Given that the molecular elucidation of this intriguing dichotomy was not possible by conventional methods, we have devised and utilized a tunable nanotechnology-based experimental system to investigate the unexpected traits of VCAM-1 on the nanoscale.

Defined VCAM-1 nanoscale patterns specifically support melanoma cell adhesion

Matrices presenting different site densities of VCAM-1 were created by combining nanopatterning and subsequent biofunctionalization. We used BCML as a platform to control lateral distribution and density of Au-NPs (30, 31, 36). Each Au-NP ideally serves as an anchor point for a single VCAM-1 molecule, which is covalently bound in a site-directed manner using subsequent steps of biofunctionalization: In the first step, glass slides were covered with Au-NPs using BCML (Fig. 2A shows an example of a nanopatterned glass substrate with 6-nm Au-NP-dots). For this study, we created different distances from 40 to 120 nm, corresponding to ligand site densities ranging from 670/μm² to 70/μm², that is, covering one order of magnitude. Next, we covalently bound HS-(CH2)11-EG3-NTA to the Au-NPs using nitrilotriacetic acid (NTA) head groups and NiCl2 for site-directed immobilization of human recombinant His-tagged VCAM-1 (Fig. 2B). To control for successful biofunctionalization, we performed Quartz Crystal Microbalance with Dissipation monitoring on homogeneous gold, demonstrating that VCAM-1 was bound correctly to gold through its terminal His6 sequence, thus mimicking the physiologic orientation of a transmembrane type I protein (Fig. 2C). Passivation (PLL-g-PEG) in-between the Au-NPs prevented nonspecific binding of cells or proteins. The margin of functionalization on the slides provided an internal control for both the functional presentation of VCAM-1 and the passivation, as immunofluorescence microscopy demonstrated binding of VCAM-1–directed antibodies exclusively to the nanopatterned areas. Because of the passivation layer between the Au-NPs, neither VCAM-1 nor VCAM-1–directed antibodies adsorbed to areas without Au-NPs (Fig. 2D, left). When two human melanoma cell lines (A375 and MeWo) were incubated with a VCAM-1–functionalized surface, cells did not adhere beyond the margin of functionalization, again showing the low nonspecific background of our model system (Fig. 2D, right).

Figure 2.

Nanoscopic presentation of VCAM-1 facilitates attachment and diminishes spreading of human melanoma cells. A, Representative scanning electron microscopy image of gold nanopatterns (Au-NPs) on glass (d=98 ±11nm). B, Schematic of biofunctionalization: Via its C-terminal 6His tag, VCAM-1 is immobilized in a site-directed manner to NTA-groups bound on gold nanoparticles (Au-NPs). C, The subsequent steps of biofunctionalization on gold were monitored by Quartz Crystal Microbalance with Dissipation (QCMD) measurements. The frequency/dissipation changes show successful conjugation of the VCAM-1-molecule. D, Immunofluorescence image of the border region between nanostructured and nonnanostructured areas using a fluorescent antibody–directed against VCAM-1, which creates a sharply demarcated line (margin of functionalization; left photomicrograph). The clear contrast indicates immobilization of VCAM-1 on the Au-NPs and shows a low nonspecific background. The right hand phase-contrast image again depicts the margin of functionalization and demonstrates that A375 melanoma cells adhere almost exclusively to the side functionalized with VCAM-1 nanopatterns but not to the pegylation side. E, Defined gold nanoparticle matrices were biofunctionalized with VCAM-1, yielding ligand presentations at the indicated densities. The spaces between the ligand sites were passivated by (PLL-g-PEG) without RGD. Human melanoma cells adhered to these matrices for 45 minutes, and the matrices were then washed in a standardized fashion. F, Melanoma cells that had firmly adhered to the matrices depicted in E were quantitated microscopically. The values shown represent mean numbers from 10 standardized microscopic fields (SD). *, P < 0.05; ***, P < 0.001. The experiment was repeated with similar results. G, Matrices with nanoscopically defined VCAM-1 (left), RGD motifs in between the passivating pegylation (middle) or both (right, bifunctional matrix) were generated as detailed in Materials and Methods. Representative phase-contrast photomicrographs of A375-melanoma cells on nanoscopic-VCAM-1 (65 nm) after a washing step with PBS for 10 minutes showing attachment but no spreading on nanoscopic VCAM-1 (top left), full spreading when RGD motifs are interspersed within the pegylation (middle) and reduced spreading when nanosopic VCAM-1 and RGD are combined in a bifunctional matrix (right), respectively. H, Flow cytometry histograms depicting the melanoma cell expression of integrins relevant for binding to RGD (α5β1, αVβ3) or VCAM-1 (α4β1).

Figure 2.

Nanoscopic presentation of VCAM-1 facilitates attachment and diminishes spreading of human melanoma cells. A, Representative scanning electron microscopy image of gold nanopatterns (Au-NPs) on glass (d=98 ±11nm). B, Schematic of biofunctionalization: Via its C-terminal 6His tag, VCAM-1 is immobilized in a site-directed manner to NTA-groups bound on gold nanoparticles (Au-NPs). C, The subsequent steps of biofunctionalization on gold were monitored by Quartz Crystal Microbalance with Dissipation (QCMD) measurements. The frequency/dissipation changes show successful conjugation of the VCAM-1-molecule. D, Immunofluorescence image of the border region between nanostructured and nonnanostructured areas using a fluorescent antibody–directed against VCAM-1, which creates a sharply demarcated line (margin of functionalization; left photomicrograph). The clear contrast indicates immobilization of VCAM-1 on the Au-NPs and shows a low nonspecific background. The right hand phase-contrast image again depicts the margin of functionalization and demonstrates that A375 melanoma cells adhere almost exclusively to the side functionalized with VCAM-1 nanopatterns but not to the pegylation side. E, Defined gold nanoparticle matrices were biofunctionalized with VCAM-1, yielding ligand presentations at the indicated densities. The spaces between the ligand sites were passivated by (PLL-g-PEG) without RGD. Human melanoma cells adhered to these matrices for 45 minutes, and the matrices were then washed in a standardized fashion. F, Melanoma cells that had firmly adhered to the matrices depicted in E were quantitated microscopically. The values shown represent mean numbers from 10 standardized microscopic fields (SD). *, P < 0.05; ***, P < 0.001. The experiment was repeated with similar results. G, Matrices with nanoscopically defined VCAM-1 (left), RGD motifs in between the passivating pegylation (middle) or both (right, bifunctional matrix) were generated as detailed in Materials and Methods. Representative phase-contrast photomicrographs of A375-melanoma cells on nanoscopic-VCAM-1 (65 nm) after a washing step with PBS for 10 minutes showing attachment but no spreading on nanoscopic VCAM-1 (top left), full spreading when RGD motifs are interspersed within the pegylation (middle) and reduced spreading when nanosopic VCAM-1 and RGD are combined in a bifunctional matrix (right), respectively. H, Flow cytometry histograms depicting the melanoma cell expression of integrins relevant for binding to RGD (α5β1, αVβ3) or VCAM-1 (α4β1).

Close modal

Nanoscale VCAM-1 presentation facilitates attachment but not spreading of melanoma cells

Two human melanoma cell lines (A375 and MeWo) expressing the dimeric α4β1 integrin (VLA-4, CD29/CD49d) at high levels (i.e., MFI > 2,000) bound firmly to nanoscopic VCAM-1 matrices in a strictly density-dependent fashion (Fig. 2E and F) but, surprisingly, did not spread on this ligand (Fig. 2G, left). In striking contrast, a matrix with 10% to 15% RGD (arginine-glycine-aspartic acid) motifs interspersed within the pegylation areas readily facilitated profound melanoma cell spreading (Fig. 2G, middle). RGD is known to induce spreading via integrin activation (37), with the linear form used here binding preferentially to the α5β1 and αVβ3 integrins, respectively (38). However, when both integrin-directed signals (RGD and VCAM-1) were combined within the same matrix, melanoma cells adopted an intermediate phenotype (Fig. 2A, right). Human melanoma cells showed strong expression of all integrins implicated in the interaction with VCAM-1 and RGD, respectively (Fig. 2h).

To better understand these apparently distinct and opposite integrin-mediated functions in human melanoma cells, the following experiments were designed to analyze different site densities of VCAM-1 in bifunctional association with the RGD peptide as a second signal presented within the pegylation areas.

Tuning of VCAM-1 densities in bifunctional matrices controls melanoma cell behavior

When human melanoma cells were exposed to bifunctional matrices displaying RGD in combination with nanostructured VCAM-1 at varying and tunable densities, it became apparent that RGD-mediated spreading could be precisely downmodulated by the VLA-4–directed activity of VCAM-1. This function was density dependent in a manner reciprocal to the VLA-4/VCAM-1–mediated attachment:

As expected, control matrices (RGD alone) enabled full spreading of human melanoma cells (37). However, tuning VCAM-1 to higher densities linearly inhibited cell spreading (Fig. 3A), while adhesion was not affected (Fig. 3B). When the cell surface areas were measured, VCAM-1 presentation at 70 ligand sites per μm² (standardized ligand site distance 120 nm) did not modify cell spreading, whereas 120 ligand sites per μm² (distance 90 nm) reduced the cell sizes moderately to 80.69% of the controls (SD ±13%, not statistically significant). Higher densities, however, significantly reduced cell spreading: At 280 ligand sites per μm² (distance 65 nm), we saw a reduction to 66.54% (SD ±16.88%; P = 0.02), and 670 ligand sites per μm² (distance 40 nm) decreased the cell sizes to 48.30% (SD ±0.09%; P = 0.0001; Fig. 3C). Confirming that these results hold true in general, similar results were obtained with a second unrelated melanoma cell line (MeWo). Again, VCAM-1 at a density of 70 ligand sites per μm² (distance 120 nm) did not affect cell spreading compared with presentation of RGD alone, whereas higher densities of VCAM-1 gradually inhibited cell spreading with 670 ligand sites per μm² (distance 40 nm) significantly reducing the cell size to 60.42% (SD ±18.34; P = 0.037; Fig. 3A, bottom row; Supplementary Fig. S1). No further spreading was observed with longer observation times of up to 3 hours.

Figure 3.

VCAM-1 density determines melanoma cell plasticity. A, Representative phase-contrast photomicrographs of A375 (top row) and MeWo (bottom row) melanoma cells interacting with bifunctional matrices (RGD + VCAM-1) with increasing site densities of VCAM-1 (ligand site distances of 120, 90, 65, and 40 nm, respectively, corresponding to ligand site densities ranging from 70/μm² to 670/μm² VCAM-1 ligands). B, A375 melanoma cells showing similar quantitative attachment independent of variations of VCAM-1 density (by one order of magnitude) next to RGD. C, Cell spreading as determined by measuring of the cell surface areas is reduced on matrices with high densities of VCAM-1, indicating an inverse linear correlation of cell surface and VCAM-1 density. Analysis of A375 melanoma cells was performed after 1 hour, and cell spreading on the control matrices (RGD alone) was set to 100%. Relative to this control, tunable nanoscopic presentation significantly inhibits cell spreading in a density-dependent manner. Values shown represent the mean (± SEM) of 6 independent experiments. [P = 0.0001 for RGD control vs. 670/μm2 (ligand distance: 40 nm), P = 0.0243 for RGD control vs. 280/μm2 (ligand distance: 65 nm), P = 0.0005 for 70/μm2 (ligand distance: 120 nm) vs. 670/μm2 (ligand distance: 65 nm), P = 0.0440 for 70/μm2 (ligand distance: 120 nm) vs. 280/μm2 (ligand distance: 65 nm)]; global effect between 40, 65, 90, and 120 nm, respectively, was assessed using one-way ANOVA, pairs were then analyzed with the two-sided Student t test and subsequent Bonferroni adjustment. *, P < 0.05; ***, P < 0.001. D, Anti-paxillin and phalloidin were used to stain focal adhesions (green) and F-actin fibers (red), respectively. VCAM-1 densities of 70 sites per μm² (ligand distances of 120 nm) and 120 sites per μm² (distances of 90 nm), respectively, support stress fiber formation in contrast to ligand site densities ≥280/μm² (distances ≤65 nm; top). Size and number of paxillin adhesions are regulated by VCAM-1 density (bottom). E, Statistical evaluation showing mean focal adhesion sizes of melanoma cells bound to VCAM-1 at the indicated site densities within a pegylated surface interspersed with RGD. Paxillin adhesions ranging from 0.1 to 5 μm2 were regarded as focal adhesions and analyzed morphometrically. The average size of paxillin adhesions directly correlates with increased VCAM-1 density. Values shown represent the mean (±SEM) of four independent experiments (P = 0.0201 for 70/μm2 vs. 670/μm2, P = 0.0111 for 120/μm2 vs. 670/μm2 and P = 0.0371 for 120/μm2 vs. 280/μm2; *, P < 0.05; analyzed with the two-sided Student t test). F, The number of focal adhesions (0.1–5 μm2) per melanoma cell bound to VCAM-1 at the indicated site densities next to RGD. The number of paxillin adhesions shows an inverse linear correlation with the densities of VCAM-1. Values shown represent the mean (±SEM) of four independent experiments (P = 0.0005 for 70/μm2 vs. 670/μm2, P = 0.0013 for 120/μm2 vs. 670/μm2 and P = 0.0441 for 280/μm2 vs. 670/μm2). For all statistical analysis, unpaired Bonferroni adjusted t tests were used. *, P < 0.05; **, P < 0.01; ***, P < 0.001. G, A375-melanoma cells were incubated on bifunctional matrices (RGD + VCAM-1 at the indicated site densities) for 3 hours prior to analyzing expression of RhoA, Rac-1, Cdc25, and Cyclin D1 using RT-PCR. Relative expression was determined using GAPDH for normalization. Values shown represent the mean of three independent experiments (P = 0.0130 for Rac-1 expression on 670 ligand sites/μm² vs. on 70 ligand sites/μm²). H, Human melanoma cells (A375) were incubated on bifunctional matrices (RGD + VCAM-1 at the indicated site densities) for 3 hours prior to analyzing gene expression by RT-PCR. The panels depict the results of a representative experiment (out of three experiments showing similar results). Only Rac-1 is expressed at higher levels on low-density VCAM-1 compared with high-density VCAM-1.

Figure 3.

VCAM-1 density determines melanoma cell plasticity. A, Representative phase-contrast photomicrographs of A375 (top row) and MeWo (bottom row) melanoma cells interacting with bifunctional matrices (RGD + VCAM-1) with increasing site densities of VCAM-1 (ligand site distances of 120, 90, 65, and 40 nm, respectively, corresponding to ligand site densities ranging from 70/μm² to 670/μm² VCAM-1 ligands). B, A375 melanoma cells showing similar quantitative attachment independent of variations of VCAM-1 density (by one order of magnitude) next to RGD. C, Cell spreading as determined by measuring of the cell surface areas is reduced on matrices with high densities of VCAM-1, indicating an inverse linear correlation of cell surface and VCAM-1 density. Analysis of A375 melanoma cells was performed after 1 hour, and cell spreading on the control matrices (RGD alone) was set to 100%. Relative to this control, tunable nanoscopic presentation significantly inhibits cell spreading in a density-dependent manner. Values shown represent the mean (± SEM) of 6 independent experiments. [P = 0.0001 for RGD control vs. 670/μm2 (ligand distance: 40 nm), P = 0.0243 for RGD control vs. 280/μm2 (ligand distance: 65 nm), P = 0.0005 for 70/μm2 (ligand distance: 120 nm) vs. 670/μm2 (ligand distance: 65 nm), P = 0.0440 for 70/μm2 (ligand distance: 120 nm) vs. 280/μm2 (ligand distance: 65 nm)]; global effect between 40, 65, 90, and 120 nm, respectively, was assessed using one-way ANOVA, pairs were then analyzed with the two-sided Student t test and subsequent Bonferroni adjustment. *, P < 0.05; ***, P < 0.001. D, Anti-paxillin and phalloidin were used to stain focal adhesions (green) and F-actin fibers (red), respectively. VCAM-1 densities of 70 sites per μm² (ligand distances of 120 nm) and 120 sites per μm² (distances of 90 nm), respectively, support stress fiber formation in contrast to ligand site densities ≥280/μm² (distances ≤65 nm; top). Size and number of paxillin adhesions are regulated by VCAM-1 density (bottom). E, Statistical evaluation showing mean focal adhesion sizes of melanoma cells bound to VCAM-1 at the indicated site densities within a pegylated surface interspersed with RGD. Paxillin adhesions ranging from 0.1 to 5 μm2 were regarded as focal adhesions and analyzed morphometrically. The average size of paxillin adhesions directly correlates with increased VCAM-1 density. Values shown represent the mean (±SEM) of four independent experiments (P = 0.0201 for 70/μm2 vs. 670/μm2, P = 0.0111 for 120/μm2 vs. 670/μm2 and P = 0.0371 for 120/μm2 vs. 280/μm2; *, P < 0.05; analyzed with the two-sided Student t test). F, The number of focal adhesions (0.1–5 μm2) per melanoma cell bound to VCAM-1 at the indicated site densities next to RGD. The number of paxillin adhesions shows an inverse linear correlation with the densities of VCAM-1. Values shown represent the mean (±SEM) of four independent experiments (P = 0.0005 for 70/μm2 vs. 670/μm2, P = 0.0013 for 120/μm2 vs. 670/μm2 and P = 0.0441 for 280/μm2 vs. 670/μm2). For all statistical analysis, unpaired Bonferroni adjusted t tests were used. *, P < 0.05; **, P < 0.01; ***, P < 0.001. G, A375-melanoma cells were incubated on bifunctional matrices (RGD + VCAM-1 at the indicated site densities) for 3 hours prior to analyzing expression of RhoA, Rac-1, Cdc25, and Cyclin D1 using RT-PCR. Relative expression was determined using GAPDH for normalization. Values shown represent the mean of three independent experiments (P = 0.0130 for Rac-1 expression on 670 ligand sites/μm² vs. on 70 ligand sites/μm²). H, Human melanoma cells (A375) were incubated on bifunctional matrices (RGD + VCAM-1 at the indicated site densities) for 3 hours prior to analyzing gene expression by RT-PCR. The panels depict the results of a representative experiment (out of three experiments showing similar results). Only Rac-1 is expressed at higher levels on low-density VCAM-1 compared with high-density VCAM-1.

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These results suggested that (i) VCAM-1 exerted an unexpected inhibitory function on RGD-mediated melanoma cell spreading when displayed at intermediate (280 ligand sites/μm²) to high (670 ligand sites/μm²) densities; (ii) that numeric variations of VCAM-1 densities governed this function in a unidirectional linear fashion; and (iii) that this novel function was exerted independently of or in addition to the adhesive function. The following experiments were designed to dissect and confirm this intriguing finding in more detail, because morphologic changes of tumor cells may affect a number of important functions such as migration, invasion (including extravasation as a particular form of invasion), and metastasis.

VCAM-1 density variation controls dichotomous features in paxillin-related adhesion formation

To understand the VCAM-1–dependent inhibition of cell spreading mechanistically, F-actin (a parameter for cytoskeletal reorganization) and paxillin (a focal adhesion marker specifically binding to the α4 integrin; ref. 39) were visualized in human melanoma cells (A375 and MeWo). Indeed, the formation of paxillin-related adhesions (defined to measure 0.1–5 μm²) was strongly dependent on VCAM-1 density: In line with the inhibition of cell spreading, F-actin accumulation was markedly reduced on high VCAM-1 densities, whereas densities of less than 280/μm² facilitated stress fiber formation (Fig. 3D, top). In addition, intermediate to high VCAM-1 densities (280–670 ligand sites/μm²) showed a clearly reduced number of paxillin-related focal adhesions, which were significantly larger compared with low densities of VCAM-1 (70–120 ligand sites/μm²). In contrast, low densities of VCAM-1 induced significantly more but smaller paxillin-related adhesions (Fig. 3D, bottom). In detail, a low VCAM-1 density of 70 ligand sites per μm² induced 32.82 (SD ±5.68) focal adhesions per cell with an average size of 0.57 μm² (SD ±0.1 μm²), and a VCAM-1 density of 120 ligand sites/μm² induced 25.55 (SD ± 4.73) focal adhesions per cell with a size of 0.65 μm² (SD ±0.02 μm²). A ligand site density of 280/μm² induced 22.66 (SD ±8.36) focal adhesions with a size of 0.73 μm² (SD ±0.07 μm²), which was significantly larger compared with the size of focal adhesions on matrices with 120 ligand sites per μm² (P = 0.0370). Finally, a very high VCAM-1 density of 670 sites/μm² resulted in only 8.29 (SD ±1.39) focal adhesions per cell with an average size of 0.91 μm² (SD ±0.12 μm²), thus being significantly fewer and larger compared with focal adhesions on matrices with 120 or 70 ligand sites per μm², respectively (P = 0.0111 and P = 0.0201, respectively; Fig. 3D and E). The reduced number of paxillin-related adhesions per cell correlated directly with increasing VCAM-1 density (P = 0.0441 comparing 670 ligand sites/μm² with 280 ligand sites/μm², P = 0.0013 comparing 670 ligand sites/μm² with 120 ligand sites/μm², and P = 0.0005 comparing 670 ligand sites/μm² with 70 ligand sites/μm², respectively; Fig. 3D and F). Thus, intermediate to high VCAM-1 densities (280–670 ligand sites/μm²) did not support stress fiber formation, which was accompanied by very few but significantly larger paxillin-related adhesions. In contrast, low VCAM-1 densities (70–120 ligand sites/μm²) permitted F-actin accumulation associated with a high number of small paxillin-related adhesions.

VCAM-1 density is associated with Rac-1 expression in human melanoma cells

Further mechanistic insight into the VCAM-1–dependent regulation of melanoma cell spreading was obtained by analyzing the transcription of tumor progression–related genes in melanoma cells interacting with bifunctional matrices with high or low VCAM-1 densities (670 vs. 70 ligand sites/μm², respectively): The two GTPases, RhoA and Rac-1, are involved in the cytoskeletal organization (40), whereas cyclin D1 and Cdc25 are players in the mitotic machinery (41). Although the expression of RhoA, cyclin D1, and Cdc25c was similar under both conditions, the expression of Rac-1 was higher by 41.9% at 70 compared with 670 VCAM-1 sites/μm2 (P = 0.013; Fig. 3G and H). Given that Rac-1 is an important regulator of lamellipodia formation (40), its isolated downregulation on high VCAM-1 densities correlated conspicuously with the inhibitory effect of VCAM-1 on spreading.

The regulation of melanoma cell morphogenesis is specific for the interaction of nanoscopically presented VCAM-1 with VLA-4 on melanoma cells

Three series of complementary control experiments were conducted to confirm that the regulation of melanoma cell spreading behavior was specific for VCAM-1 interacting with VLA-4 on melanoma cells:

First, VCAM-1 was cleaved off the bifunctional nanostructured matrices by NE, which degrades VCAM-1 (27, 28). It was established first that NE influenced neither the passivating pegylation nor the function of RGD interspersed within the pegylated surface (data not shown). Indeed, when matrices functionalized with VCAM-1 (280 ligand sites/μm2) were incubated with NE for 20 minutes, human melanoma cells were not significantly impaired in their RGD-mediated spreading behavior, which was in clear contrast to matrices with uncleaved VCAM-1 [the size of cells on uncleaved VCAM-1 matrices reached 60.31% (±14.97%) of cell sizes on RGD-only matrices (P = 0.0001), while the size of cells on NE-cleaved matrices was 89.35% (±9.52%; not significant; Fig. 4A and B)]. Thus, enzymatic cleavage of VCAM-1 abrogated its regulatory effect on melanoma cell spreading.

Figure 4.

Melanoma cell plasticity is regulated specifically by VCAM-1/VLA-4. A, Specificity controls for the functional effect of VCAM-1 with regard to the inhibition of RGD-induced cell spreading were performed by modification of VCAM-1 presentation, which is depicted schematically at the top (presentation of intact VCAM-1, left; enzymatic cleavage of VCAM-1 by neutrophil elastase, second from left; replacement of VCAM-1 with PECAM-1, third from left; RGD-only matrices, right). All experiments were performed on VCAM-1 ligand site distances of 65 nm. The bottom panels depict representative photomicrographs from each of the indicated matrices showing that only intact VCAM-1 can inhibit RGD-induced melanoma cell spreading. B, Melanoma cells adhered to the matriced specified in A for 60 minutes. The cell surface area was determined under each condition. Values shown represent the mean (±SEM) of three independent experiments (≥100 cells were analyzed per matrix/condition; P = 0.0076 for VCAM-1 vs. truncated VCAM-1, P = 0.0040 for VCAM-1 vs. PECAM-1 and P = 0.0001 for VCAM-1 vs. RGD control. **, P < 0.01; ***, P < 0.001, analyzed with the two-sided Student t test). C, Human A375 melanoma cells were subjected to downregulation of the α4 integrin subunit by siRNA. The histograms depict a representative flow cytometry experiment showing integrin VLA-4 expression after treatment with control siRNA (dark gray curve), α4-directed siRNA (light gray curve), and an isotype control (open curve). D, An average reduction of 50% in α4 expression was achieved by transfection of A375 melanoma cells with α4-directed siRNA compared with control siRNA, respectively. Values shown represent the mean (±SEM) of four independent experiments (**, P < 0.01, analyzed with the two-sided Student t test). E, Melanoma cells transfected with α4-directed siRNA are able to spread on a RGD-containing matrix with nanoscopic VCAM-1 (left photomicrograph). In contrast, melanoma cells treated with control siRNA are inhibited in cell spreading on nanoscopic VCAM-1 (right photomicrograph). The phalloidin/DAPI fluorescence image shows impaired F-actin filament organization. F, Statistical evaluation of cell spreading: melanoma cells treated with control-siRNA are clearly inhibited in cell spreading compared with melanoma cells treated with α4-siRNA on nanoscopic VCAM-1 within an RGD-containing matrix (black bars, relative to the respective RGD controls). Both subpopulations (control vs. α4 knockdown cells) are able to spread on the RGD control (light gray bars). Values shown represent the mean (±SEM) of three independent experiments (P = 0.0278 for control siRNA transfected cells vs. α4 siRNA knockdown cells, P = 0.0092 for control siRNA transfected melanoma cells on RGD + VCAM-1 vs. RGD-only presentation, Student two-tailed, unpaired t test).

Figure 4.

Melanoma cell plasticity is regulated specifically by VCAM-1/VLA-4. A, Specificity controls for the functional effect of VCAM-1 with regard to the inhibition of RGD-induced cell spreading were performed by modification of VCAM-1 presentation, which is depicted schematically at the top (presentation of intact VCAM-1, left; enzymatic cleavage of VCAM-1 by neutrophil elastase, second from left; replacement of VCAM-1 with PECAM-1, third from left; RGD-only matrices, right). All experiments were performed on VCAM-1 ligand site distances of 65 nm. The bottom panels depict representative photomicrographs from each of the indicated matrices showing that only intact VCAM-1 can inhibit RGD-induced melanoma cell spreading. B, Melanoma cells adhered to the matriced specified in A for 60 minutes. The cell surface area was determined under each condition. Values shown represent the mean (±SEM) of three independent experiments (≥100 cells were analyzed per matrix/condition; P = 0.0076 for VCAM-1 vs. truncated VCAM-1, P = 0.0040 for VCAM-1 vs. PECAM-1 and P = 0.0001 for VCAM-1 vs. RGD control. **, P < 0.01; ***, P < 0.001, analyzed with the two-sided Student t test). C, Human A375 melanoma cells were subjected to downregulation of the α4 integrin subunit by siRNA. The histograms depict a representative flow cytometry experiment showing integrin VLA-4 expression after treatment with control siRNA (dark gray curve), α4-directed siRNA (light gray curve), and an isotype control (open curve). D, An average reduction of 50% in α4 expression was achieved by transfection of A375 melanoma cells with α4-directed siRNA compared with control siRNA, respectively. Values shown represent the mean (±SEM) of four independent experiments (**, P < 0.01, analyzed with the two-sided Student t test). E, Melanoma cells transfected with α4-directed siRNA are able to spread on a RGD-containing matrix with nanoscopic VCAM-1 (left photomicrograph). In contrast, melanoma cells treated with control siRNA are inhibited in cell spreading on nanoscopic VCAM-1 (right photomicrograph). The phalloidin/DAPI fluorescence image shows impaired F-actin filament organization. F, Statistical evaluation of cell spreading: melanoma cells treated with control-siRNA are clearly inhibited in cell spreading compared with melanoma cells treated with α4-siRNA on nanoscopic VCAM-1 within an RGD-containing matrix (black bars, relative to the respective RGD controls). Both subpopulations (control vs. α4 knockdown cells) are able to spread on the RGD control (light gray bars). Values shown represent the mean (±SEM) of three independent experiments (P = 0.0278 for control siRNA transfected cells vs. α4 siRNA knockdown cells, P = 0.0092 for control siRNA transfected melanoma cells on RGD + VCAM-1 vs. RGD-only presentation, Student two-tailed, unpaired t test).

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Second, we replaced VCAM-1 with platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31), a related immunoglobulin superfamily ligand, which is similar in size. Successful functionalization was controlled using a PECAM-1–directed antibody to visualize the margin of functionalization (not shown). When PECAM-1- and VCAM-1–functionalized nanosurfaces, respectively, were incubated with melanoma cells, only VCAM-1 but not PECAM-1 inhibited RGD-induced cell spreading. Melanoma cells on VCAM-1 matrices (280 ligand sites/μm2) reached only the abovementioned 60.31% (±14.97%) of the size of cells on RGD-only matrices (P = 0.0001), while cells on PECAM-1 matrices (280 ligand sites/μm2) reached 100.74% (±29.36%, P = 0.0040) compared with VCAM-1–containing bifunctional matrices (Fig. 4A and B). These results indicate that a related adhesion molecule could not exert the regulatory function found for VCAM-1.

Third, we downregulated the major interaction partner of VCAM-1, the α4 integrin (VLA-4, CD49d), on melanoma cells by siRNA-based knockdown. Although untreated melanoma cells as well as melanoma cells transfected with control siRNA constructs showed high expression of α4 (MFI = 2,195.24, ±311.21), cells transfected with anti–α4-siRNA showed a significantly reduced expression of α4 (MFI = 1,087.75, ±270.25; reduction by 50.45%; P = 0.0017; Fig. 4C and D). Other integrins potentially relevant in our system, that is, α5, β1, and αVβ3, respectively, showed no alteration following α4 knockdown (data not shown). When the respective cells were incubated with nanostructured VCAM-1–presenting matrices (280 ligand sites/μm²), the control siRNA-transfected cells reached a cell size of 56.09% (±11.30%) compared with cells on RGD-only matrices. In contrast, downregulation of the α4 integrin subunit resulted in a cell size of 87.18% (±18.35%) relative to the RGD-only controls (P = 0.0278 for control siRNA transfected cells vs. α4-directed siRNA knockdown cells; Fig. 4E and F). Corresponding immunofluorescence analysis showed a rescue of F-actin accumulation in melanoma cells with reduced α4 expression on VCAM-1 matrices, in contrast to the control-transfected cells (Fig. 4e). Thus, the VCAM-1–mediated inhibition of melanoma cell spreading on RGD matrices depended on expression of the α4 integrin.

Contact with VCAM-1 expressed by pulmonary blood vessels is associated with globular morphology of melanoma cells in vivo

The next series of experiments was designed to address the in vivo relevance of the results of our nanotechnology-based in vitro experiments. Previous studies have demonstrated that blocking of VCAM-1 can diminish long-term experimental pulmonary melanoma metastasis (12, 15). To refine the aspect of VCAM-1 in very early steps of metastasis, we have established and utilized two complementary modified mouse models to address the association of circulating melanoma cells with VCAM-1–expressing endothelia in vivo. Because previous models addressing the role of VCAM-1 for melanoma metastasis have entailed inflammation (thus making the assessment of VCAM's role difficult), we have used one experimental setting without and another with a pulmonary inflammatory response. In the first setting, C57BL6/J mice were not pretreated, while in a complimentary setting, VCAM-1 expression on pulmonary capillary vessels was induced by intratracheal instillation of the TLR2/6 ligand, MALP-2. Syngeneic B16F10 murine melanoma cells strongly expressing VLA-4 (the major ligand of VCAM-1; Fig. 5A) were stably transfected with GFP and injected intravenously into syngeneic C57BL6/J mice. The lungs were analyzed after 1, 5, and 30 minutes (n = 3 mice in each group) by fluorescence microscopy.

Figure 5.

Pulmonary VCAM-1 expression in vivo is associated with morphogenesis of melanoma cells. A, Murine B16F10 melanoma cells stably transfected with GFP were analyzed for expression of VLA-4, the ligand of VCAM-1, by flow cytometry. B, C57BL6 mice were left untreated (light gray bars) or treated by intratracheal instillation of MALP-2. After 24 hours, the mice were injected intravenously with 106 GFP-labeled B16F10 melanoma cells. The lungs of the mice were harvested after 1, 5, or 30 minutes (n = 3 mice/group) and snap-frozen in liquid nitrogen. GFP-labeled melanoma cells as well as VCAM-1–expressing pulmonary blood vessels were analyzed in cryostat-cut sections by fluorescence microscopy. The left graph depicts the average numbers of VCAM-1–expressing pulmonary blood vessels, indicating induction by MALP-2. The middle and the right graphs depict the total numbers of melanoma cells and melanoma cells adjacent to VCAM-1–expressing blood vessels, respectively. Values shown represent mean counts from 41 microscopic fields (±SD). ***, P < 0.001. C, Representative examples of cryostat-cut sections showing areas with GFP-expressing melanoma cells (green) adjacent to (filled arrows) or distant from (open arrows) VCAM-1–expressing pulmonary blood vessels (red).

Figure 5.

Pulmonary VCAM-1 expression in vivo is associated with morphogenesis of melanoma cells. A, Murine B16F10 melanoma cells stably transfected with GFP were analyzed for expression of VLA-4, the ligand of VCAM-1, by flow cytometry. B, C57BL6 mice were left untreated (light gray bars) or treated by intratracheal instillation of MALP-2. After 24 hours, the mice were injected intravenously with 106 GFP-labeled B16F10 melanoma cells. The lungs of the mice were harvested after 1, 5, or 30 minutes (n = 3 mice/group) and snap-frozen in liquid nitrogen. GFP-labeled melanoma cells as well as VCAM-1–expressing pulmonary blood vessels were analyzed in cryostat-cut sections by fluorescence microscopy. The left graph depicts the average numbers of VCAM-1–expressing pulmonary blood vessels, indicating induction by MALP-2. The middle and the right graphs depict the total numbers of melanoma cells and melanoma cells adjacent to VCAM-1–expressing blood vessels, respectively. Values shown represent mean counts from 41 microscopic fields (±SD). ***, P < 0.001. C, Representative examples of cryostat-cut sections showing areas with GFP-expressing melanoma cells (green) adjacent to (filled arrows) or distant from (open arrows) VCAM-1–expressing pulmonary blood vessels (red).

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As expected (18), pretreatment with MALP-2 led to a significant 2.3-fold increase of the density of VCAM-1–expressing pulmonary blood vessels [3.65 (±0.69) vs. 8.39 (±1.73) vessels per ×20 microscopic field; n = 41 fields in each group; P < 0.0001; Fig. 5B, left graph]. The numbers of melanoma cells within the lungs were similar after all three time points, and the numbers of GFP-expressing B16F10 melanoma cells were similar in untreated mice and in mice pretreated with MALP-2 [4.02 (±1.39) cells per ×20 microscopic field vs. 3.61 cells (±1.00); n = 41 fields in each group; Fig. 5B, middle graph].

Surprisingly however, although similar proportions of melanoma cells were found in direct contact with pulmonary endothelial cells (7.89% in untreated vs. 7.42% in MALP-2–treated mice; Fig. 5B, right graph), in both models, virtually all melanoma cells contacting VCAM-1 in vivo appeared small and round shaped. In contrast, all other melanoma cells within the pulmonary parenchyma or contacting VCAM-1–negative endothelia were larger and of dendritic morphology (Fig. 5C), thus corroborating our in vitro results showing inhibition of melanoma cell spreading by VCAM-1.

The use of nanotechnology was essential for unraveling a hitherto unknown “dichotomic” function of VCAM-1 (Fig. 6), a long-known endothelial adhesion molecule implicated in tumor metastasis. First hints for this novel dichotomy, however, stemmed from our observations that inhibition of VCAM-1 facilitated spreading of melanoma cells on activated endothelial cells in vitro. This finding has been underscored in short-term experimental metastasis experiments in vivo, where stable expression of a fluorescent protein revealed that melanoma cells adjacent to VCAM-1–expressing pulmonary capillaries adopted a globular shape. Until now, the tumor-promoting effect of VCAM-1 has been attributed primarily to its adhesive function (11, 26), which is thought to enhance intravascular arrest of circulating tumor cells and, consequently, metastasis into distant organs (42–44). It is thought that tumor cells become physically entrapped and simultaneously interact with endothelial ligands in capillaries of metastatic end-organs prior to initiating the next steps of metastasis formation (45). However, our work now provides evidence that besides adhesion, VCAM-1 mediates a prolonged and density-dependent inhibition of melanoma cell spreading. Thus, the heterogeneous nanoscale decoration of VCAM-1 within the microvessels is likely a part of a regulatory network governing tumor cell adhesion on the one hand and spreading on tissue surfaces on the other (Fig. 6). Our findings might, therefore, shift the paradigm of the pathophysiologic function of VCAM-1 in melanoma progression. However, the net (patho)physiologic consequences of VCAM-1–mediated rounding of melanoma cells in vivo are not entirely clear at this time. Likewise, the actual activation patterns of Rac-1 and other GTPases in our system cannot be assessed. One may hypothesize, however, that smaller round cells may have advantages regarding extravasation, a notion that would be consistent with the long-known tumor-promoting activity of VCAM-1.

Figure 6.

Endothelial VCAM-1 tips the balance in melanoma cell plasticity. The schematic illustrates the cellular consequences following modulation of the VCAM-1 density in a putative (micro)environment that is permissive for cell spreading. At low densities of VCAM-1, RGD moieties facilitate spreading of melanoma cells. This morphologic phenotype is also characterized by numerous immature focal adhesions and delicate F-actin fibers. High densities of VCAM-1, in contrast, force the cell into a globular shape with fewer but mature focal adhesions and prominent F-actin stress fibers. This “morphogenic dictate” of VCAM-1 overrules the still-present signals by RGD moieties.

Figure 6.

Endothelial VCAM-1 tips the balance in melanoma cell plasticity. The schematic illustrates the cellular consequences following modulation of the VCAM-1 density in a putative (micro)environment that is permissive for cell spreading. At low densities of VCAM-1, RGD moieties facilitate spreading of melanoma cells. This morphologic phenotype is also characterized by numerous immature focal adhesions and delicate F-actin fibers. High densities of VCAM-1, in contrast, force the cell into a globular shape with fewer but mature focal adhesions and prominent F-actin stress fibers. This “morphogenic dictate” of VCAM-1 overrules the still-present signals by RGD moieties.

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The α4 integrin (CD49d, VLA-4) is involved in cell spreading and focal adhesion formation upon ligation (46, 47). However, although the intracellular interaction of paxillin with dephosphorylated α4 integrin and the downstream regulation of Rac-1 have been implicated in cellular morphogenesis (47–49), a plausible biophysical basis for the upstream VLA-4/VCAM-1 interaction on the ligand level has been lacking. We found that suppression of melanoma cell spreading on a “permissive” RGD matrix is most pronounced at a well-defined VCAM-1 site density of more than 280 ligands/μm² interspersed between RGD motifs. This bifunctional arrangement is a simplified model of an activated endothelium where VCAM-1 is highly expressed. It is conceivable, therefore, that VCAM-1 contains and controls the “permissive” environment created by RGD moieties on the respective surfaces. Considering that the regional, temporal, and stimulus-specific parameters of VCAM-1 expression in vivo are still not fully understood, our tunable bifunctional in vitro model with a stoichiometrically defined presentation of VCAM-1 provides the first handle on the biophysical regulation of the VCAM-1/VLA-4 interaction on the nanoscale with respect to tumor cell morphogenesis. Furthermore, the formation of more mature paxillin-related adhesions (of ≈1 μm2) on matrices with high densities of VCAM-1 (with VCAM-1 site distances in the range of 40–65 nm) might then be explained by dense clustering of its receptor, the VLA-4 integrin, on the melanoma cell side, which is predicted to physically facilitate the paxillin–VLA-4 interaction and subsequently inhibit cell spreading (47–49). It appears likely that the few large focal adhesions associated with high VCAM-1 densities mediated strong adhesion, while the smaller and more numerous focal adhesions together with the robust stress fiber formation seen on low VCAM-1 densities facilitated spreading of melanoma cells, thus providing a mechanistic explanation for the dichotomous function of VCAM-1. It is also in line with previous publications highlighting the relevance of VLA-4 for focal adhesion formation and melanoma cell motility (50–52).

In vivo, VCAM-1 is upregulated through paracrine cytokine activation during melanoma progression. Therefore, it is conceivable that melanoma cells themselves modulate their microenvironment toward higher VCAM-1 densities. It is tempting to speculate that melanoma cells within the capillary beds benefit from the morphologic changes effected by high densities of VCAM-1, for example, through favored extravasation of smaller cells or improved intravascular immune escape of tumor cells with smaller surfaces. In concordance with this hypothesis, a recent study has shown that the ability of A375 melanoma cells to migrate in confined VCAM-1–coated microchannels depends on α4/paxillin-mediated inhibition of Rac-1 activation (53). The dichotomic function of VCAM-1 identified here might thus facilitate two important steps of organ infiltration: first, firm interaction with the endothelium and second, morphologic preparation for penetration within small niches in capillary beds. Mechanistically, downregulation of Rac-1 has been identified as an important downstream event of enhanced VLA-4/paxillin interaction (53, 54). Accordingly, we have detected a significant downregulation of Rac-1 on high VCAM-1 densities, which is predicted to contribute to the inhibitory effect on melanoma cell spreading.

The regulation of VCAM-1 activity and its impact on tumor progression in vivo is not easy to fathom. In principle, regulation of endothelial VCAM-1 expression (modeled in our system by defined presentation at different densities) will in all likelihood influence tumor cell–endothelial cell interactions and modulate “permissive” microenvironmental conditions for melanoma cells. However, the local VCAM-1 density, although upregulated transcriptionally during inflammation, is also controlled through cleavage by leucocyte-derived proteases (27, 28, 55). As most, if not all, in vivo studies showing a tumor-promoting function of VCAM-1 have been performed in models that include at least some degree of inflammation (18, 23, 41, 56), it is difficult to gauge the regulation and net activity of VCAM-1 in vivo. We have, therefore, shown similar results in two complementary short-term mouse models, one without (untreated mice) and one with inflammation (MALP-2–treated mice with endothelial up-ragulation of VCAM-1 but concomitant influx of inflammatory cells). In any case, expression of VCAM-1 strong enough to be detectable by immunofluorescence microscopy appeared to suffice in our in vivo experiments to coerce melanoma cells into a globular shape at these microanatomic sites. In this light, nanotechnology provides a sophisticated tool to mimic, control, and fine-tune the presentation of functionally active VCAM-1 on the physiologically relevant nanometer scale. It allowed for the first time to demonstrate that tuning VCAM-1 densities decisively regulates cytoskeletal and focal adhesion formation of human melanoma cells. In comparison with other ligands [e.g., RGD motifs (37) or cadherins, unpublished data], the dichotomic role for tumor cell adhesion on the one hand and spreading on the other appears to be a rather unique feature of VCAM-1. Furthermore, it underlines the complexity of molecular interactions and implies a potential for unpredictable effects of blocking agents directed against VLA-4/VCAM-1 interactions. Finally, the bifunctional system with concomitant presentation of RGD shows that the remarkable capacity of VCAM-1 to modulate tumor cell spreading is relevant even when competing signals by “activating” integrins are involved.

No potential conflicts of interest were disclosed.

Conception and design: K. Amschler, S. Kruss, M.P. Schön

Development of methodology: K. Amschler, L. Erpenbeck, J.P. Spatz, M.P. Schön

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Amschler, E. Kossmann, L. Erpenbeck, S. Kruss, T. Schill, M. Schön, S.M.C. Möckel, M.P. Schön

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Amschler, E. Kossmann, S. Kruss, M. Schön, M.P. Schön

Writing, review, and/or revision of the manuscript: E. Kossmann, L. Erpenbeck, S. Kruss, M. Schön, M.P. Schön

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E. Kossmann, S.M.C. Möckel, J.P. Spatz, M.P. Schön

Study supervision: M.P. Schön

J.P. Spatz acknowledges financial support by the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Advanced grant agreement no. 294852. The authors gratefully acknowledge the excellent technical assistance of K. Zachmann, S. Köchy, and A. Bennemann and thank Y. Schön and R. Koelz for the preparation of nanostructured matrices and subsequent quality control by scanning electron microscopy. J.P. Spatz is the Weston Visiting Professor at the Weizmann Institute of Science and a member of the excellence cluster CellNetworks at the University of Heidelberg.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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