Identification of E3.5 chick dorsal root ganglia tissue and nerve growth factor (NGF) drive Mart-1:GFP expression in human C8161 melanoma cells. (A) Schematic of co-culture assays using plated human C8161 human melanoma cells with either varying ages and regions of chick embryonic tissues from the head or trunk (top) or soluble factors (bottom). (B) Percentage of Mart-1:GFP-positive C8161 melanoma cells after co-culture with different ages and regions of chick embryonic tissues (E1.5 cranial nt, 0.5±0.5%, s.d.; E1.5 trunk nt, 2.1±0.7%, s.d.; E2.5 ba2, 3.9±5.5%, s.d.; E2.5 trunk, 2.6±0.8%, s.d.; E3.5 trunk, 4.7±2.2%, s.d.; E3.5 DRG, 5.68±9.5%, s.d.). One section/piece of tissue was added per well of cells (8-well chamber slide format). Experiment was repeated in triplicate. (C) Typical images of C8161 melanoma cells (pre-labeled with H2B-mCherry, red) co-cultured with successive ages of trunk tissues, E1.5 (left), E2.5 (middle), and E3.5 (right) show an increase in Mart-1:GFP re-expression (green) with age. (D) Percentage of Mart-1:GFP-positive melanoma cells versus distance from the tissue (in contact with tissue 7.7±2.1 μm, s.d.; ≤100 μm, 10.5±3.3 μm, s.d.; 100-200 μm, 8.8±3.4 μm, s.d.; 200-300 μm, 3.1±2.7 μm, s.d. and >300 μm, 0.8±0.7 μm, s.d.). Experiment was repeated in triplicate. (E) A typical image of C8161 melanoma cells (pre-labeled with H2B-mCherry, red) co-cultured with E3.5 DRG (bottom left in E) show higher Mart-1:GFP re-expression closer to transplanted DRG tissue (100 μm increments from the edge of the tissue are labeled). (F) Percentage of Mart-1:GFP-positive C8161 melanoma cells in the presence of individual soluble factors (Control, no factor 0.2±0.2%, s.d.; NGF, 5.46±1.65%, s.d., P<0.01; BDNF, 0.05±0.04%, s.d., P=0.2; CXCL12, 0.5±0.4%, s.d., P=0.3; NT3, 0.9±0.8%, s.d., P=0.3; BMP4, 0.93±0.9%, s.d., P=0.1; BMP7, 0.8±0.58%, s.d., P=0.1; FGF8, 1.4±0.86%, s.d., P=0.7; NGF+BDNF, 3.6±0.9%, s.d.; NGF+NT3, 4.17±0.8%, s.d.) typically found in E3.5 trunk tissue. Experiment was repeated in triplicate. (G) A typical image of C8161 melanoma cells (pre-labeled with H2B-mCherry, red) in the presence of no factor (left), NGF (middle) and Cxcl12 (right) and Mart-1:GFP re-expression (green). (H) Percentage of Mart-1:GFP-positive C8161 melanoma cells exposed to different concentrations of NGF (500 ng/ml, 10.3±4.03%, s.d., P<0.01; 100 ng/ml, 6.3±3.1%, s.d., P=0.2; 50 ng/ml, 3.4±1.5%, s.d., P=0.02; 10 ng/ml, 1.1±0.8%, s.d., P=0.1; 0 ng/ml, 0.1±0.2%, s.d.). Experiment was repeated in triplicate. Scale bars: 50 μm. All calulations performed on fixed samples. Statistical analysis was performed using Student's t-test.

NGF does not affect proliferation or migration of C8161 melanoma cells. (A) Percentage of BrdU-positive C8161 melanoma cells under different co-culture conditions with various soluble factors (Control, no factor 26.9±7.86 cells, s.d.; NGF, 28±8.6 cells, s.d., P=0.5; BDNF, 24.4±4.17 cells, s.d., P=0.6; CXCL12, 31.5±4.42 cells, s.d., P=0.4; NT3, 38.4±2.5, s.d., P=0.07). Experiment was repeated in triplicate. (B) C8161 melanoma cells seeded into a modified Boyden chamber show no preference for BSA control (1.15±0.1 s.d., RFU) or NGF (200 mg/ml with 1.08±0.1 s.d., RFU, P=0.4 and 100 ng/ml with 0.97±0.09 s.d., RFU, P=0.2), but migrate in response to a known chemokine, CXCL12 (2.06±0.2 s.d., RFU, P<0.01). Experiment was repeated in duplicate. (C) PBS- or NGF-soaked beads co-cultured in vitro with C8161 melanoma cells (labeled with H2B-mCherry, red) and observed at t=0 and 12 h show no directed cell migration towards either the PBS- or NGF-soaked beads, n=4 bead cultures per condition, repeated in triplicate. Scale bars: 100 μm (t=0) and 50 μm (t=12 h). All calculations performed on living samples. Statistically analysis was performed using Student's t-test.

NGF induces stable Mart-1:GFP-positive C8161 melanoma cell population. (A) Schematic of the NGF time course experiment. At day 0, all plates of C8161 cells received NGF (circles shaded red) and cultured for at least 3 days before removal of NGF (white circle). On successive days (x-axis) the NGF was removed. (B) Percentage of Mart-1:GFP melanoma cells in the presence of NGF for at least 3 days and then removal on day3 (blue), day 5 (red), day 7 (green). Within each day, no data set was significantly different from the others (Day 3: NGF 3 days=4.6±1.3%, s.d.; NGF 5 days, 4.7±0.8%, s.d.; NGF 7 days, 5.9±1.2%, s.d.; Day 4: NGF 3 days, 1.8±0.6%, s.d.; NGF 5 days, 3±0.7%, s.d.; NGF 7 days, 3.1±0.8%, s.d.; Day 5: NGF 3 days, 1.6±0.4%, s.d.; NGF 5 days, 2.5±0.3%, s.d.; NGF 7 days, 2.8±0.3%, s.d.; Day 6: NGF 3 days, 1.2±0.3%, s.d.; NGF 5 days, 1.2±0.1%, s.d.; NGF 7 days, 1.3±0.1% s.d.; Day 7: NGF 3 days, 1.37±0.3%, s.d.; NGF 5 days, 1.2±0.3%, s.d.; NGF 7 days, 1.1±0.3%, s.d.; at least 1000 cells counted per condition; each condition repeated in duplicate). (C) (Top Panel) C8161 melanoma cells after co-culture with NGF for 3 days (72 h) show ∼5% Mart-1:GFP-positive melanoma cells, then removal on subsequent days. A decrease in Mart-1:GFP-positive melanoma cells is seen on subsequent days (4-6). (Bottom Panel) C8161 melanoma cells maintained in culture with constant supply of NGF show same ∼5% Mart-1:GFP-positive melanoma cells at day 3 and similar decrease in Mart-1:GFP-positive cells at days 4-6. (D) Proliferation rate of C8161 melanoma cells changes after Mart-1:GFP expression. Percentage of BrdU incorporation in C8161 cells after 30 min BrdU pulse: Mart-1:GFP-positive melanoma cells, 10±2.3% (s.d.) and Mart-1:GFP-negative melanoma cells, 45.5±3.4% (s.d.), P<0.01 (left graph; n>500 cells counted per condition, experiment run in duplicate). (E) Time-lapse culture analysis shows Mart-1 positive cells proliferate less (1.1±0.4 cell divisions per hour) than Mart-1-negative cells [2.56±0.3 (s.d.) cell divisions per hour], P<0.01. n=4 time lapses per condition. (F) Theoretical calculation of cell populations based on differences in proliferation dynamics. After NGF exposure, ∼5% of C8161 melanoma cells re-express Mart-1:GFP and the cell proliferation rate decreases. A doubling rate of 2 (cell cycles in 24 h) is applied to the Mart-1:GFP-positive cell population starting with 5 out of 100 cells and a rate of 4 (cell cycles in 24 h) is applied to the Mart-1-negative population starting at 95 out of 100 cells. Using a 24-h doubling time, the projected changes in cell population distribution are seen by a decrease in blue cell (Mart-1:GFP-positive) and increase in yellow cell (Mart-1:GFP-negative) populations. Scale bars: 50 μm. All calculations performed on living samples. Statistically analysis was performed using Student's t-test.

NGF receptor expression on C8161 melanoma cells. (A) qPCR analysis of relative quantity of mRNA differences of NGF (verticle striped bars), trkA (verticle dashed bars) and p75 (solid black bars) on C81-61 and C8161 melanoma cells. C81-61: NGF=1.00±0.06 s.d., trkA=1.00±.05 s.d., p75=170±8.7 s.d.; C8161: NGF=23.3±3.1 s.d., trkA=3.99±0.25 s.d., p75=1.00±0.15 s.d.; calibrated normalized relative quantities. (B) Protein expression of trkA (green, top panel) and p75 (blue, bottom panel) on H2B:mCherry labeled C8161 cells (red). (C) Percent of Mart-1:GFP-positive C8161 cells in control (no NGF) or in the presence of NGF and either p75 or trkA inhibitors individually or in combination (Control, 0.17±0.15%, s.d.; p75 inhibitor, 0.7±0.3%, s.d, P=0.05; trkA inhibitor, 1.8±0.2%, s.d., P<0.01; p75/trkA inhibitors, 0.2± 0.2%, s.d., P=0.8). At least 2000 cells counted per condition. Scale bars: 50 μm. Statistical analysis was performed using Student's t-test.