Abstract
Background: We investigated whether an antibody targeting N-cadherin facilitates the adhesion and spreading of N-cadherin-positive tumour cells under static conditions. Materials and Methods: Two human melanoma cell lines, HMB-2 and BLM, were selected for their presence and lack of expression of N-cadherin, respectively. In vitro adhesion experiments were performed in the presence of a monoclonal antibody targeting N-cadherin (GC-4) or a control (antibody to α-tubulin). Quantitative data from the spreading assays were calculated by converting the images obtained by fluorescence microscopy to binary images. Results: For HMB-2 cells, the average cell width was significantly larger in the presence of GC-4 vs. control at all measured time points, with the exception of the measurement at 70 minutes (p=0.051). No differences were observed between controls and GC-4 for BLM cells. Conclusion: The adhesion and spread of N-cadherin-positive tumour cells can be facilitated by the presence of an immobilized antibody to N-cadherin.
The development of metastases from solid tumours is a complex multistep process involving interactions between different ecosystems (1). One of the critical steps for hematogenous dissemination is the escape of tumour cells into the peripheral circulation. The correlation found between the number of circulating tumour cells (CTCs) and overall survival in metastatic breast, colorectal and prostate cancer supports the hypothesis of a critical role for these CTCs in tumour progression and metastasis (2, 3). Consequently, it is hypothesized that the hematogenous transport phase of CTCs offers an interesting therapeutic window to interrupt the metastatic process resulting in a reduction of disease progression and mortality (4). A specific approach of this strategy is the targeting of cell adhesion molecules expressed on the surface of CTCs. A few in vivo devices have been developed utilizing a vascular shunt continuously screening for CTC via selectin-mediated adhesions (5). This induces the CTCs to adhere and roll over the shunt's internal surface, which is equipped with apoptosis-inducing molecules (5). Another interesting cell adhesion molecule expressed on the surface of CTCs is N-cadherin (6), a key player in the transformation process of multiple solid tumours called epithelial-to-mesenchymal transition (EMT) involved in the dissemination of tumours (7). Consequently, N-cadherin could be an interesting alternative molecule for trapping CTCs. As a first proof-of-concept, the current study investigated whether an antibody targeting N-cadherin facilitates the adhesion and spreading of N-cadherin-positive tumour cells under static conditions. We opted to use melanoma cell lines because the progression from a predominantly E-cadherin phenotype as melanocytes to a predominantly N-cadherin phenotype plays an important role during the transition into melanoma and the acquisition of a vertical growth phase (8).
Materials and Methods
Cell lines. Two human melanoma cell lines, HMB-2 and BLM, were obtained from D. Rutherford (Rayne Institute, St Thomas Hospital, UK) and L. Van Kempen (University of Nijmegen, the Netherlands), respectively. The melanocytic origin of these cell lines has been previously documented by immunocytochemistry (9). HMB-2 cells express N-cadherin, but no P- nor E-cadherin, while BLM cells do not express any of these type I cadherins at all (10). Both cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) from Invitrogen, (Merelbeke, Belgium), supplemented with 10% heat-inactivated fetal bovine serum (Greiner Bio-One, Wemmel, Belgium), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B. They were incubated in 25 cm2 tissue culture flasks at 37°C in an atmosphere of 10% CO2 in air.
Reagents and plastic substrata. GC-4 is murine-purified monoclonal anti-N-cadherin immunoglobulin (C3865 from Sigma, St Louis, MO, USA) that was applied to target N-cadherin on HMB-2 cells. A murine monoclonal antibody to α-tubulin (T5168; from Sigma) was used as a non-relevant control antibody against an intracellular target. Phalloidin-FITC, used to visualize the F-actin cytoskeleton, was purchased from Invitrogen. Protein G-coated 96-well microtiter plates were from Pierce (Rockford, Il, USA).
Spreading experiments. Coating of substrata for cell spreading: Protein G-coated 96-well microtiter plates were rinsed twice with 200 μl sterile phosphate-buffered saline (PBS). Solutions containing 5 μg/ml or 10 μg/ml GC-4 and solutions containing 5 μg or 10 μg α-tubulin antibody as controls (denoted as 5NC and 10NC, respectively) were prepared in sterile PBS. To each well of the microtiter plates 100 μl of one of these 4 antibody solutions was added, and the plates were incubated at 4°C overnight. Next, the antibody solutions were aspirated, the wells were washed three times with 200 μl of sterile PBS and kept in this buffer solution at 4°C until the addition of cells. Seeding of suspended cells: HMB-2 and BLM cells were detached from their tissue culture plastic substrata as follows. After rinsing the cells twice with Moscona's solution, they were incubated with 1 ml trypsin-calcium at 37°C in an atmosphere of 10% CO2 in air for 2 to 5 min. Trypsin (0.01% w/v) – calcium (0.04 mM) was used for the preservation of the ectodomains of cadherins and integrins. The cells were then suspended in 5 mL of fresh DMEM, and counted with a Bürker chamber. After aspiration of PBS from the plates, 1×104 cells in 100 μl were added to each coated well.
Observation of cell spreading: The microtiter plates were incubated at 37°C in an atmosphere of 10% CO2 in air. Images were obtained with an inverted phase-contrast microscope (ocular ×10, objective ×20) at different time intervals, ranging from 5 min to 24 h. After 50, 70 or 120 min of incubation suspension fluids were removed from the microtiter plates by turning a number of them upside down and back. The wells were rinsed individually with 300 μl ice-cold PBS, and the adhering cells were fixed with 3% paraformaldehyde in PBS at room temperature for 20 min. After permeabilization with 0.2% Triton X-100 in PBS, cells were stained with 100 μl phalloidin-FITC solution in the dark at room temperature for 20 min. After removal of the staining solution, the plates were left to air-dry or Tris-buffered saline 0.01 M (TBS) was added to the wells. Images were obtained with an inverted fluorescence microscope using the Axiovision software (Axiovision 4.8, Carl Zeiss, Göttingen, Germany). Quantitative data from the spreading assays were calculated by converting the images obtained by fluorescence microscopy to binary images (black and white). The average cell width in pixels was then measured with the Image J program (open source, National Institute of Health, MD, USA) and used as the measure of cell spread.
Statistical analysis. Statistical analysis of data was carried out with SPSS 19.0. The distribution of data was assessed by performing a Shapiro-Wilk test and a Kolmogorov-Smirnov test. Data groups with a normal distribution were compared using an unpaired, two-tailed Student's t-test. Data groups with a non-Gaussian distribution were compared using a Mann-Whitney U-test. The level of significance (α) was set at 0.05. If p<0.05, differences in average cell width were considered significant.
Results
Figure 1 shows the results for the N-cadherin-negative BLM cells, comparing the average cell width between controls and N-cadherin antibody-exposed cells at different times. No significant differences were observed between groups (p>0.05). For the N-cadherin-positive HMB-2 cells, comparison under the same conditions at the same time intervals resulted in significant differences in average cell width between controls and the antibody exposed cells at all measured time points and for both antibody concentrations (Figure 2), with the exception of the comparison of 5 μg/ml anti-α-tubulin versus 5 μg/ml GC-4 at 70 min (p=0.051). We did not observe any influence of the concentration of the N-cadherin antibody on the average cell width of HMB-2 cells (Table I).
Conclusion
The adhesion and spread of N-cadherin-positive tumour cells can be facilitated by the presence of an immobilized anti-N-cadherin antibody. This static experiment holds promise that N-cadherin antibodies are potentially useful to stabilize N-cadherin-positive CTCs adhered to artificial substrates used as capture devices.
- Received August 23, 2012.
- Revision received October 9, 2012.
- Accepted October 12, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved