Fig 1.
In vitro experiments yielded inconclusive results for predicting in vivo metastatic potential of L1CAM knockdown melanoma cells.
Statistics: All numbers n represent the total number of individual samples (each from one individual animal) of the respective experiment. All values of P are derived from linear models. If PGroup X Line is below 0.05 (i.e. if L1CAM knockdown displays different effects for cells from the two lines), a separate PLuc vs. L1kd is given for each cell line. Flow Cytometry: Staining of MeWo Luc and L1CAM knockdown (L1 kd) (left panel) and MV3 Luc and L1 kd (right panel) for L1CAM. In contrast to the Luc controls, surface L1CAM1 is reduced by more than 85% (MeWo) and apporoximately 75% (MV3) on the L1 kd cells (A). Proliferation assay: 5 X 104 cells seeded per well and incubation for 48 h: Significant increase in proliferation for MeWo with L1CAM knockdown (L1 kd) compared with the MeWo Luc controls with unchanged surface L1CAM and significant decrease for MV3 L1 kd compared with MV3 Luc (B). Invasion assay: 1 X 105 cells were seeded per well and incubated for 24 h: No significant change in the L1 kd knockdown cells’ ability for invasion (C). Colony forming assay: Cells were seeded in matrigel / culture medium in a 96-well plate; colonies were counted and evaluated using a light microscope after 14 days: Significant increase in colony numbers for MeWo L1 kd cells against MeWo Luc and no significant difference in colony numbers between MV3 L1 kd cells and MV3 Luc controls was observed (n = 48, each) (D). Laminar flow adhesion assay on activated endothelium: Interactions of melanoma cells suspended in culture medium with a HUVEC monolayer under laminar flow. The number of events (only adherence and tethering, no rolling events) increased significantly for MeWo L1 kd against MeWo Luc cells and decreased significantly for MV3 L1 kd against MV3 Luc cells (E).
Fig 2.
Xenograft spontaneous metastasis model of human melanoma cells with high L1CAM expression (MeWo) in scid mice.
L1CAM knockdown reduces metastasis by more than 80%. Statistics: All numbers n represent the total number of individual samples of the respective experiment. Survival of mice was analyzed using a linear model which was moreover adjusted for the type of death (ulcerated vs. not ulcerated). Tumor weight was analyzed in an analogous model. Number of metastases was analyzed and evaluated/quantified using a negative binomial regression which was adjusted for survival time and tumor weight and included furthermore CTCs as variable. CTCs as outcome variable were logarithmized and analyzed using a linear model, which was adjusted for survival time and tumor weight. If PGroup X Line is below 0.05 (i.e. if L1CAM knockdown displays different effects for the two cell lines), a separate PLuc vs. L1kd is given for each cell line. Animals’ survival after subcutaneous injection of 106 melanoma cells: L1CAM knockdown did not significantly change the animals’ survival time (A). The weight of the MeWo L1 kd tumors was not significantly different from the MeWo Luc control tumors, the weight of the MV3 L1 kd tumors was significantly higher than the tumor weight of the MV3 Luc control tumors (B). The number of lung metastases was significantly lower (reduced 7.26 fold) in the L1 kd than in the Luc control groups both for MeWo and MV3 (C). The numbers of circulating tumor cells (CTC) in the animals’ blood were not significantly altered by L1CAM knockdown both for MeWo and for MV3 (D).
Fig 3.
L1CAM expression in lung metastases from human melanoma xenograft as detected by immunohistochemical analysis.
Immunohistochemical staining for L1CAM expression (red) in lung metastases of melanoma cells MeWo (upper panels) and MV3 (lower panels) with unchanged L1CAM expression (Luc, right panels) and L1CAM knockdown (L1 kd, left panels). All scale bars: 50 μm. Please note that staining was amplified for MV3.
Fig 4.
Comparison of the gene expression of subcutaneous MeWo tumors with L1CAM knockdown with expression of MeWo Luc control tumors.
Analysis revealed a central role for TGFβ. Analysis of the regulation profile in L1CAM knockdown tumors compared with Luc control tumors using the Pathway Studio software including all genes which display a respective 1.5 fold up- or downregulation in expression in the L1 kd tumors. Gene expression upregulated in the L1kd tumors is given in red, downregulated expression in blue.
Fig 5.
Verification of gene expression array data by immunohistochemical analysis of Jagged 1 and Notch 1 expression in subcutaneous tumors and lung metastases from a human melanoma (MeWo) xenograft experiment.
Immunohistochemical staining for Jagged 1 (JAG1) expression (red) in subcutaneous (s.c.) tumors (upper left and upper right panel) and lung metastases (lower left and lower right panel) of melanoma cells (MeWo) with unchanged L1CAM expression (Luc, upper and lower left panels) and L1CAM knockdown (L1 kd, upper and lower right panels). All scale bars: 50 μm. Stainings show a tendency towards increased Jagged 1 for L1 kd tumors and metastases (A). Immunohistochemical staining for Notch1 expression (red) in subcutaneous tumors (upper left and upper right panel) and lung metastases (lower left and lower right panel) of melanoma cells (MeWo) with unchanged L1CAM expression (Luc, upper and lower left panels) and L1CAM knockdown (L1 kd, upper and lower right panels). All scale bars: 50 μm. Stainings show a tendency towards increased Notch 1 for L1 kd tumors and metastases (B). Immunohistochemical staining for Jagged 1 (JAG1) expression (red) in subcutaneous tumors (big panel) and lung metastases (smaller panel, lower left) of MV3 melanoma cells with unchanged L1CAM expression (Luc, left panels) and L1CAM knockdown (L1 kd, right panels). All scale bars: 50 μm. Stainings show no tendency towards altered Jagged 1 for L1 kd tumors and metastases (C).
Fig 6.
Gene expression with the same regulation (up or down respectively) in MeWo and MV3 subcutaneous tumors with L1CAM knockdown compared with their respective Luc control counterparts with unaltered L1CAM expression.
Analysis using Pathway Studio software: Gene expression upregulated in L1CAM knockdown tumors compared with Luc control tumors with unaltered L1CAM; upregulated expression is given in red, downregulated expression in blue, potentially affected factors / processes in grey.
Table 1.
Genes with the same regulation (up or down respectively) in MV3 and MeWo subcutaneous tumors with L1CAM knockdown (L1 kd) compared with their respective Luc control counterparts with unaltered L1CAM (Luc) expression.
Included are only fold changes at least +/- 1.5; ANOVA and adjusted p-values <0.05, both for MV3 and MeWo tumors.