PMC

SerpinE2 drives melanoma invasiveness. (a) Boyden chamber invasion assay of sorted label-retaining cells (LRC) or non-LRC in presence (blue columns) or absence (gray columns) of human recombinant SerpinE2. Bars show fold change in invasion (mean±s.e.m.) of samples with SerpinE2 over untreated cells. (b) Invasion assay in presence of an anti-human SerpinE2 neutralizing antibody or Isotype control (ctrl). The percentage of invading cells after neutralization is shown. Data represent mean±s.e.m. of three independent experiments. (c) Invasion assay after SerpinE2 knockdown. Data shown are percentage of invading cells found after silencing with 5 different shRNA (Sh_13-17) relative to Sh_ctrl (ctrl). Bars represent mean±s.e.m.

Label-retaining, disseminated melanoma cells express high levels of SerpinE2. (a) Table lists proteins identified after whole-cell proteomic analyses of LRC and non-LRC. The last column lists the cell lines with differential expression of the indicated protein. (b) Violin plots (left panels) illustrate quantification of SerpinE2 (white spot, top) and BMP1 (white spots, bottom) as determined by mRNA FISH. mRNA counts for single cells are shown on the Y-axes. Note differences in Y-axes. Representative photomicrographs are shown on the right (total magnification: 100 ×). (c) Protein expression was analyzed by immunofluorescence and quantified at single-cell levels (scatter plots on left and representative images on right). LRC (red), non-LRC (orange), total cells (gray). (d) Flow cytometry of disseminated WM989 melanoma cells detected in three mouse organs (top panel). The percentage of cells double positive for CD146 and SerpinE2 is shown in the upper right quadrants; the lower right quadrants indicate the percentage of cells singly positive for CD146. Micrographs show examples of disseminated SerpinE2-positive cells (arrows, middle and right panels) in mouse lungs. Left panel: negative control.

SerpinE2 expression is increased in malignant cells and correlates with tumor progression. (a) Western blot analysis of SerpinE2 secretion by melanoma cells (n=21) and human melanocytes (n=5) into culture supernatants. Recombinant SerpinE2 protein (500 ng) was used as a positive control and Ponceau’s staining is shown for loading control. Graph shows quantification (right panel). (b) SerpinE2 staining of 3D skin reconstructs with melanocytes (left) and melanoma cells (right). (c) GEM_1375 data set analysis of SerpinE2 mRNA expression in melanomas, non-malignant nevi and normal skin. (d) Examples of immune histochemistry at two different magnifications of: normal skin, benign nevi, in situ, vertical growth phase (VGP) and lymph node metastatic melanomas (n=15 for each group). SerpinE2 protein expression is indicated by purple staining. Arrows indicate the dotted SerpinE2 expression pattern found in benign nevi only. (e) Intensity score of SerpinE2 expression in normal skin, benign nevi and malignant melanomas (radial growth phase (RGP), VGP and metastatic). Bars represent mean±SEM of tissue sections of 15 specimens from each group; P-value after t-student test are given.

Melanoma cells identified as slow-cycling, LRC are predominantly in the G2/M cell cycle phase and are more invasive in vitro. (a) Six melanoma cell lines were labeled with CellTrace Dye and analyzed at different time points (days 0–4–8–12). Day 0 indicates how the cells appear directly after CellTrace loading. LRC are defined and gated as shown in the dot plots; numbers indicate % of LRC detected on day 12 in culture; standard error of the mean (s.e.m.) in parenthesis. (b) Relative dilution of the CellTrace Dye for LRC (red circles), non-LRC (yellow inverted triangles) and total cells (gray squares). Slopes indicate mean fluorescence intensity ratio between CellTrace-stained and unstained cells. Mean±s.e.m. are reported. (c) Cells were loaded with CellTrace Dye and stained with propidium iodide for cell cycle analyses after 7 days in culture. Percentage of cells in each cell cycle stage is shown in the Y-axes. Data represent mean±s.e.m. Three representative cell lines are shown. (d) LRC (red), non-LRC (orange) and total (gray) were sorted and analyzed by mRNA FISH for JARID1B expression in single cells, with representative images below (total magnification 100 ×). Data indicate that some but not all LRC are JARID1B-positive. Violin plots depict mRNA counts (Y-axes); ns=not significant. (e) Invasive capacity of LRC (red bars) and non-LRC (orange) in Boyden chamber assays after sorting, for three representative cell lines. Data represent mean±s.e.m. of three independent experiments of the fold change of LRC over non-LRC. All reported P-values were determined using a 2-sided t-student test.

Melanoma LRC disseminate in vivo early from the primary tumor site. (a) Schematic for detecting LRC in vivo. Melanoma cells (WM989 and 451Lu) were labeled with PKH26 and subcutaneously injected into SCID Hairless Outbred mice. Mice were imaged weekly using IVIS (dorsal, ventral and lateral scanning). Between days 0 and 20, the fluorescent signal was detected in the primary tumor at the injection site; after day 20, proliferating tumor cells lost the dye at the primary site. Instead, fluorescent signals indicating the presence of LRC were detected in distant sites. (b) Mice on left: dorsal view of mice scanned 7 days post-tumor injection. For the left two mice of each melanoma, palpable tumors overlapped with fluorescence signals. Graphs on right: tumor growth (black line, left Y-axis) and dilution of PKH26 signal (red line, right Y-axis) at the primary tumor site (n=5). Arrows indicate the cross point of the curves. After reaching ~500 mm³ tumor size, PKH26 signal is rapidly lost. (c) Dorsal (left), ventral (middle), and lateral (right) views of mice scanned when tumors reached ~700 mm³ size. (d) Scatter plots showing quantification of the fluorescent signals for individual mice at days 0–20 (left plots) and 20–60 (right plots) post injection (n=5), for the same two melanoma cell lines as in c. Primary tumors (blue), peritoneal cavities (red), and left flank (black). (e) Bars represent flow cytometry quantification of melanoma LRC (CD146+/PKH26+ positive cells) in primary tumor (gray), mouse lymph nodes (LN, yellow), lungs (blue), liver (green) and spleen (purple); Zebra plot (below) are representative examples from lungs. Upper right quadrant: melanoma LRC double positive for CD146 and PKH26; lower right quadrant: melanoma non-LRC cells (CD146+ positive). Numbers are % of positive cells after subtraction of the percentage detected in the isotype control (left plot). (f) Representative photomicrographs of immunofluorescence performed on frozen tissue from lungs and spleen for a combination of melanoma markers (CD146, β3 integrin, MSCP-1). Cells can be detected as clusters (white square in spleen) or disseminated (white arrows). Bottom panel is the negative antibody control staining.