Table 1.
Specification of monoclonal antibodies (mAb).
Figure 1.
Primary melanoma cells express ErbB3 and ErbB4 as well as the EPO-R.
A, Freshly isolated primary melanoma cells (patient #3) were stained with fluorochrome-conjugated monoclonal antibodies (mAb) against CD31, CD45, ErbB3, IGF-1-R, and CD146. Melanoma cells were gated as CD31−/CD45− cells and defined as CD146+ cells. Dot plots show expression of ErbB3 (middle panel) and IGF-1-R (right panel) on CD146+ melanoma cells. The isotype control is also shown (left panel). B, Melanoma cells of patient #10 were stained with mAb against CD31, CD45 and CD146 as well as EPO-R and CD24. The dot plot in the right panel shows co-expression of EPO-R and CD24 in a distinct subpopulation of (CD146+) melanoma cells. C, Xenotransplanted EPO-R+ melanoma cells of patient #6 were stained with biotinylated recombinant human EPO, an isotype-matched mouse IgG2b-PE antibody (left panel) and a mAb directed against the EPO-R (right panel). D, Melanoma cells of patient #10 were stained with mAb against CD146, an isotype-matched control antibody (left panel) and an antibody against ErbB4 (right panel). Dot plots show expression of ErbB4 in a distinct subpopulation of melanoma cells.
Figure 2.
Expression of KIT/CD117 on human melanocytes.
Normal epidermal foreskin melanocytes were stained with fluorochrome-conjugated monoclonal antibodies (mAb) directed against CD31, CD45 and CD146 for melanocyte detection, and mAb against KIT and EPO-R. Specificity of the staining reaction was controlled by applying an isotype-matched IgG2b antibody (left panel). As visible, melanocytes expressed KIT but did not express substantial amounts of EPO-R.
Table 2.
Expression of cytokine- and growth factor receptors on melanoma cells.
Table 3.
Expression of adhesion-related molecules on melanoma cells.
Figure 3.
Expression of NGF-R, CD33 and CD133 on distinct populations of melanoma cells.
A, Patient-derived melanoma cells (n = 3) were stained with a PE-labeled monoclonal antibody (mAb) against NGF-R (CD271) (left image) and a PE-labeled antibody against PD1 (right panel). Isotype-matched PE-labeled antibodies were used for determination of the cut-off values. B, Xenotransplanted melanoma cells from patient #6 were stained with mAb against CD33 and CD146. The dot plot shows expression of CD33 on a distinct subpopulation of CD146+ melanoma cells. C, Melanoma cells from patient #9 were stained with mAb against CD133 and CD146. The dot plot shows expression of CD133 on CD146+ melanoma cells. The reactivity of melanoma cells with isotype-matched control antibodies is shown in the left images of Fig. 3B and 3C.
Table 4.
Expression of potential stem cell markers and drug targets on melanoma cells.
Figure 4.
Melanoma-formation in NSG mice.
Unsorted (EPO-R+/−) and sorted EPO-R− and EPO-R+, patient-derived, melanoma cells (patients #6 and #7) were injected subcutaneously into NSG mice (4–6 mice in each group). After 15 weeks, tumors were visible, mice were sacrificed, and the tumor-volumes were determined. A, Results show the median tumor-volumes (mm3) in each group. B, A melanoma cell-containing tumor formed in an NSG mouse by EPO-R− cells obtained from patient #6.
Figure 5.
Pelitinib blocks survival and induces apoptosis in melanoma cells.
A, A375 melanoma cells were incubated in control medium (0) or with increasing concentrations of the ErbB blocker pelitinib at 37°C for 72 hours. Cell survival was measured by MTT assay. Results are expressed as percent of control and represent the mean±S.D. of three independent experiments. B, Evaluation of apoptosis of melanoma cells by TUNEL assay. A375 cells were incubated in control medium (left panel) or in medium containing 1 µM pelitinib for 48 hours. As visible, the ErbB blocker induced apoptosis in A375 cells. Nuclei were counterstained by TO-PRO3 dye (blue color). C, Measurement of pelitinib-induced apoptosis in A375 cells by AnnexinV-staining. A375 cells were incubated in control medium (left panel) or medium containing pelitinib at 1 µM (right panel) for 72 hours. Histogram plots show the mean fluorescence intensity of the staining reaction. The percentages of apoptotic (AnnexinV-positive) cells are also provided.
Table 5.
IC50 values (µM) for the inhibition of the cell survival (MTT assay) in human melanoma cells by targeted drugs.