Invasiveness-triggered state transition in malignant melanoma cells

2.1 Human tissue specimens

Studies using tissues from human subjects were approved by the Research Ethics Committee of Tangdu Hospital, Fourth Military Medical University. Formalin-fixed paraffin-embedded tumor blocks from patients with melanoma were retrieved from the Department of Dermatology in Tangdu Hospital. Hematoxylin- and eosin-stained sections (5 μm thickness) were reviewed in each case by two pathologists blinded to the study, along with the pathology reports and case histories. The number of mitosis per 40 high-power fields in the primary skin lesion areas was assessed.

2.2 Melanoma cell lines and culture

The melanoma cell lines A2058, WM793B, and 451Lu were obtained from the American Type Culture Collection (ATCC) and cultured according to a previous report (Wang et al., 2009). Briefly, melanoma cells were cultured in RPMI-1640 medium (GIBCO, NY) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 U/ml streptomycin. Cells were maintained at 37°C in 5% CO₂.

2.3 Scratch assays

Scratch assays were performed by plating cells in slide chambers coated with 10 μg/ml collagen (Wang et al., 2009). Different cell suspensions in serum-free RPMI 1640 with 10% FBS were applied to chambers and incubated at 37°C in 5% CO₂. After cells reached confluency, a scratch was made through the collagen with a plastic pipette tip. After washing with phosphate-buffered saline (PBS), the scratched monolayers of the cells were allowed to heal for 12 hr in RPMI 1640 containing 10% FBS. Photographs of cells invading the scratch were obtained by an IX50 microscope (Olympus, Tokyo, Japan) at different time points.

2.4 Transwell migration assay

Transwell migration assays were performed using an 8 µm pore polycarbonate membrane (3422; Corning). The lower-chamber side filter was coated for 1 hr with 0.1% gelatin B. Typically, 1 × 10⁵ cells were seeded in the upper chamber, which was placed over the lower chamber containing HT1080-conditioned media. Migration was allowed to proceed for different time points (4 hr, 6 hr, 12 hr, 24 hr, 48 hr, 72 hr and 96 hr) at 37°C, and cells remaining on the upper-chamber side of the filter were removed with cotton swabs. The lower surface of the filter was fixed with 70% methanol and stained with 1% crystal violet solution. The number of migrated cells was determined by counting stained cells from multiple randomly selected microscopic visual fields.

2.5 Small interfering RNA (siRNA)-mediated knockdown of M3 mAChR

The sequences of M3 mAChR siRNA were as follows: siRNA 1, 5′-CCUCGCCUUUGUUUCCAAATT-3′, 5′-UUUGGAAACAAAGGCGAGGTT-3′; siRNA 2, 5′-CCUGGUAAUUGUGUCAUUUTT-3′, 5′-AAAUGACACAAUUACCAGGTT-3′; siRNA 3, 5′-GGUCAUCUCCUUUGUCCUUTT-3′, 5′-AAGGACAAAGGAGAUGACCTT-3′. SiRNA were chemically synthesized by Sangon Biotech (Shanghai, China) using standard phosphoramidate chemistry, annealed, and purified by HPLC to a purity of >97%. Lyophilized siRNAs were dissolved in diethyl pyrocarbonate (DEPC)-treated water to a final concentration of 20 nM. Six-well plates were seeded with 1 × 10⁵ cells/well. CHRM3 or control siRNA was added to each well in the presence of OptiMEM medium with a final concentration of 250 nmol/l. The medium was replaced with full medium 4 hr after transfection and the cells were incubated overnight.

2.6 Western blot analysis

A2058 melanoma cells were incubated for 2 days at 37°C in RPMI-1640 medium containing 10% fetal calf serum. Cells were washed with PBS and scraped into ice-cold lysis buffer (20 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM sodium vanadate, 10 mM NaF, 1 mM EDTA, 1 mM Pefabloc) and incubated on ice for 30 min. Lysates were clarified by centrifugation at 10,000g for 20 min at 4°C. Total proteins (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12.5% polyacrylamide gel. The proteins in the gel were transferred to a 0.45-μm nitrocellulose membrane with a Trans-Blot SD semidry electrophoretic transfer cell (BIO-RAD, CA). Membranes were blocked with 10% nonfat dry milk and were probed with the rabbit anti-mAChR M3 antibody (sc-9108; 1:200; Santa Cruz Biotechnology, CA). β-Actin was used as a loading control. After washing the membranes in TBS-T, blots were incubated with anti-rabbit immunoglobulin G (IgG; 7074; 1:2,000; Cell Signaling Technology, MA). The immunoreaction was visualized using the Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Millipore, MA) on an X-ray film (Agfa, Antwerp, Belgium).

2.7 Immunofluorescence and confocal microscopy

Immunofluorescent staining was performed in A2058 melanoma cells. The cells were fixed with ethanol/acetic acid. After blocking with 3% bovine serum albumin (BSA; Sigma-Aldrich, MO) for 30 min, coverslips with the cultured A2058 cells were incubated with different primary antibodies, including mouse anti-p75 nerve growth factor (NGF) receptor (ab8877; 1:200; Abcam), rabbit anti-mAChR M3 (sc-9108; 1:200; Santa Cruz Technology, MA), mouse anti-E-cadherin (sc-21791; 1:200; Santa Cruz Technology, CA), mouse anti-desmoplakin (sc-390975; 1:200; Santa Cruz Technology, CA), mouse anti-N-cadherin (sc-393933; 1:200; Santa Cruz Technology, CA), and mouse anti-Vimentin (sc-6260; 1:200; Santa Cruz Technology, CA). Secondary antibodies were Alexa Fluor 488-labeled donkey F(ab′)2 anti-mouse IgG (ab150101; 1:200; Abcam, MA) and Alexa Fluor 647-labeled donkey anti-rabbit IgG (ab150063; 1:200; Abcam, MA). Nuclei were stained with DAPI (1:2,000; Invitrogen, CA). Coverslips were mounted in antifading embedding medium (BioMeda, CA) and the distribution of the signal was studied using a fluorescence microscope.

2.8 Co-immunoprecipitation

Cell extracts were prepared via the lysis of A2058 cells in RIPA buffer (P0013; Beyotime, Shanghai, China) containing certain protease inhibitors for 15 min on ice and clarified via centrifugation. Protein extracts were diluted fourfold with immunoprecipitation (IP) buffer (50 mM Tris–HCl pH 8.0, 0.01% sodium deoxycholate, 150 mM NaCl, 1% Triton X-100, and a complete protease inhibitor mixture). In all, 2 μg of mouse anti-p75NTR antibody (ab8877; 1:500; Abcam) was incubated with the protein extract for 2 hr at 4°C with rotation before the addition of 20 μl of protein G Plus agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. The immunoprecipitates were incubated for 1 hr at 4°C with rotation to capture immune complexes, and then the beads were pelleted via brief centrifugation and washed three times with WIP buffer. Bound proteins were eluted by boiling with 50 μl of SDS-PAGE loading buffer. Protein content was analyzed via western blot analysis and the HRP-linked secondary antibodies were anti-rabbit IgG (7074; 1:2,000; Cell Signaling Technology, MA) and anti-mouse IgG (7076; 1:2,000; Cell Signaling Technology, MA).

2.9 Statistical analysis

Statistical analyses were performed on the mean values of biological replicates in each group using an unpaired two-tailed Student's t test or one-way ANOVA with Tukey's multiple comparisons test using GraphPad Prism (GraphPad, CA), as described in the figure legends. p < 0.05 were considered significant. Error bars depict SEM.

3.1 Epithelial cells rather than mesenchymal cells dominate human melanoma tissue

Melanoma cells are highly heterogeneous in tissue sections of melanoma patients in the different stages, including in situ melanoma confined to the epidermis (Figure 1a), superficial spreading melanoma (SSM; Figure 1b), nodular melanomas in the vertical growth phase (VGP; Figure 1c), and highly aggressive melanoma in the lymph node metastases phase (Figure 1d). The cancer cells have high morphological heterogeneity from pagetoid cells in the epidermis to epithelioid melanocytes with abundant cytoplasm, vesicular nuclei often a brisk mitotic activity to spindle-shaped, hyperchromatic melanocytes that may lack pigment production, and to multinucleated melanoma cells with dense nuclear chromatin and unapparent nucleoli. From the primary tumor to distant metastatic stages, however, more than 97% of melanoma cells are epithelial tumor cells, whereas mesenchymal tumor cells are less than 3% (Figure 1e). These results demonstrate that epithelial rather than mesenchymal tumor cells dominate in the development of human melanoma.

3.2 Melanoma cell transwell invasion, but not superficial migration, triggers morphological transition

One possibility of human melanoma in the state of epithelial phenotypes is that cancer cells maintain their original morphology and rarely change during melanoma development. We first compare the cellular morphology of human melanoma tissues (Supporting Information Figure S1a) and cultured cell lines (Supporting Information Figure S1b). The WM793B cell line is established from skin primary SSM. The 451Lu cell line is established from skin nodular melanoma in VGP. These two cell lines are in epithelial states and well mimicked the pathological properties in melanoma patients. The similar morphological states both in vitro and in vivo indicate the poor ability of morphological transition in WM793B and 451Lu melanoma cells. Differently, highly invasive A2058 cell lines derived from lymph node metastases from skin melanoma are present in the spindle-shaped mesenchymal cells. The morphology of this cancer cell line is quite different from the human melanoma tissue, which has a predominantly epithelial phenotype (Figure 1). The results suggest the state transitions during malignant melanoma cells invasion and metastasis in vivo. We further investigate this hypothesis in A2058 cells. In the scratch assay, superficial migration of A2058 cells into the scratch maintained spindle-shaped mesenchymal tumor cells (Figure 2a). However, the original spindle-shaped A2058 melanoma cells showed an epithelioid morphology after vertical invasion through 8 μm-diameter micropores (Figure 2b). The next state transition from epithelial cells to mesenchymal cells lasts for more than 3 days (Figure 2c). Our results strongly indicate that melanoma cell vertical invasion, but not superficial migration, can cause morphological changes between mesenchymal and epithelial cells. ITST might maintain cancer cells in the epithelial state during the early stage of invasion and metastasis.

To confirm that the morphological changes were melanoma state transition at the molecular level, we further used the immunofluorescent staining method to detect the expressions of EMT biomarkers, including E-cadherin, desmoplakin, N-cadherin, and vimentin, in A2058 melanoma cells (Supporting Information Figure S2). EMT-related protein E-cadherin is highly expressed in epithelial cells and vimentin is highly expressed in mesenchymal cells. Although desmoplakin and N-cadherin are detectable in A2058 melanoma cells by immunofluorescent staining, the dynamic changes are minimal from epithelial to mesenchymal cells. These results confirm that the morphological transitions during melanoma cancer cells invasion are associated with the dynamic changes of EMT-related proteins.

3.3 Uncoupling of M3 mAChR and p75NTR in the morphological transition of melanoma cells

Our previous study indicated the muscarinic receptors could modulate the functions of different stages of melanoma cells (Wang et al., 2009). To reveal the ITST mechanisms of A2058 melanoma cells, the interactions of M3 mAChR and p75NTR are further investigated. Double immunofluorescence staining showed that M3 mAChR and p75NTR were intensively coexpressed in the cytoplasm and the cell membrane of spindle-shaped mesenchymal cells. However, M3 mAChR not overlapping with p75NTR was present in the cell membrane of epithelial tumor cells (Figure 3a and Supporting Information Figure S3). The results indicate the coupling of M3 mAChR and p75NTR. Therefore, co-IP experiments were conducted to test the interaction. We found three blot bands at about 55, 100, and 250 kD (Supporting Information Figure S4). Except for the known dimers of M3 mAChR (McMillin, Heusel, Liu, Costanzi, & Wess, 2011), the 250 kD band might indicate potential forms other than oligomers and dimers of M3 mAChR. Nevertheless, these results indicate the uncoupling of M3 mAChR and p75NTR in the state transition of melanoma cells.

To further confirm the contribution of M3 mAChR in melanoma ITST, we designed siRNAs of CHRM3 gene encoding M3 mAChR (Figure 4a). Transwell invasion assay results (Figure 4b) showed that M3 mAChR knockdown markedly decreased the total number of invaded melanoma cells and epithelial cells (Figure 4c). In contrast, the cell number (Figure 4d) and percentage (Figure 4e) of spindle-shaped mesenchymal cell were increased after siRNA treatment. These results support the contribution of M3 mAChR toward the state maintenance of epithelial melanoma cells.