Primary melanoma cells express ErbB3 and ErbB4 as well as the EPO-R.

<p><i>A</i>, 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). <i>B</i>, 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. <i>C</i>, 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). <i>D</i>, 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.</p

Expression of cytokine- and growth factor receptors on melanoma cells.

<p>Score of reactivity of melanoma cells with antibodies: −,<5% positive cells; −/+, weakly expressed on a subset; +/−, 6–20%; +, 21–60%, and ++, 61–100% of cells positive.</p>a<p>Number of donors (n) analyzed.</p>b<p>Cells were obtained from tumors grown in NSG mice injected with unfractionated patient-derived melanoma cells.</p><p>Abbreviations: VEGF-R2, vascular endothelial growth factor receptor-2; EPO-R, erythropoietin receptor; IGF-I-R, insulin-like growth factor I receptor; G-CSF-R, granulocyte colony-stimulating factor receptor; GM-CSF-R, granulocyte/macrophage colony-stimulating factor receptor; M-CSF-R, macrophage colony-stimulating factor receptor; SCF, stem cell factor; IL-3-RA, interleukin-3 receptor alpha chain; FLT-3, FMS-like tyrosine kinase 3; n.t., not tested.</p

IC₅₀ values (µM) for the inhibition of the cell survival (MTT assay) in human melanoma cells by targeted drugs.

<p>Melanoma cells were cultured in complete medium at 37°C in the absence or presence of various concentrations of targeted drugs for 48 hours. Then, cell viability was examined by MTT assay as described in the text.</p>*<p>xenotransplanted patient-derived melanoma cells from patient #6 and #7; n.t., not tested.</p

Expression of KIT/CD117 on human melanocytes.

<p>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.</p

Phenotyping of Human Melanoma Cells Reveals a Unique Composition of Receptor Targets and a Subpopulation Co-Expressing ErbB4, EPO-R and NGF-R

<div><p>Malignant melanoma is a life-threatening skin cancer increasingly diagnosed in the western world. In advanced disease the prognosis is grave. Growth and metastasis formation in melanomas are regulated by a network of cytokines, cytokine-receptors, and adhesion molecules. However, little is known about surface antigens and target expression profiles in human melanomas. We examined the cell surface antigen profile of human skin melanoma cells by multicolor flow cytometry, and compared their phenotype with 4 melanoma cell lines (A375, 607B, Mel-Juso, SK-Mel28). Melanoma cells were defined as CD45−/CD31− cells co-expressing one or more melanoma-related antigens (CD63, CD146, CD166). In most patients, melanoma cells exhibited ErbB3/Her3, CD44/Pgp-1, ICAM-1/CD54 and IGF-1-R/CD221, but did not express CD20, ErbB2/Her2, KIT/CD117, AC133/CD133 or MDR-1/CD243. Melanoma cell lines were found to display a similar phenotype. In most patients, a distinct subpopulation of melanoma cells (4–40%) expressed the erythropoietin receptor (EPO-R) and ErbB4 together with PD-1 and NGF-R/CD271. Both the EPO-R+ and EPO-R− subpopulations produced melanoma lesions in NOD/SCID IL-2Rgamma^null (NSG) mice in first and secondary recipients. Normal skin melanocytes did not express ErbB4 or EPO-R, but expressed a functional KIT receptor (CD117) as well as NGF-R, ErbB3/Her3, IGF-1-R and CD44. In conclusion, melanoma cells display a unique composition of surface target antigens and cytokine receptors. Malignant transformation of melanomas is accompanied by loss of KIT and acquisition of EPO-R and ErbB4, both of which are co-expressed with NGF-R and PD-1 in distinct subfractions of melanoma cells. However, expression of EPO-R/ErbB4/PD-1 is not indicative of a selective melanoma-initiating potential.</p></div

Expression of adhesion-related molecules on melanoma cells.

<p>Score of reactivity of melanoma cells with antibodies: −, <5%; +/−, 6–20%; +, 21–60%, and ++, 61–100%.</p>a<p>Number of donors (n) analysed.</p>b<p>Cells were obtained from tumours grown in NSG mice injected with impure (non-sorted) patient-derived melanoma cells.</p><p>Abbreviations: ICAM-1, intercellular adhesion molecule-1; CEACAM-1, carcinoembryonic antigen-related cell adhesion molecule 1.</p

Specification of monoclonal antibodies (mAb).

<p>Abbreviations: PE, phycoerythrin; FITC, fluorescein isothiocyanate; PerCP, peridinin chlorophyll protein; APC, allophycocyanin; EPO-R, erythropoietin receptor; EGF-R, epidermal growth factor receptor; HGF-R, hepatocyte growth factor receptor; IGF-I-R, insulin-like growth factor I receptor; PD-1, programmed death-1; NGF-R, nerve growth factor receptor; HLA, human leukocyte antigens; PECAM-1, platelet/endothelial cell adhesion molecule-1; Siglec-3, sialic acid binding immunoglobulin-like lectin-3; HPCA-1, human progenitor cell antigen-1; LCA, leukocyte common antigen; ICAM-1, intercellular adhesion molecule-1; LAMP-3, lysosomal-associated membrane protein 3; CEACAM-1, carcinoembryonic antigen-related cell adhesion molecule 1; G-CSF-R, granulocyte colony-stimulating factor receptor; CSF-1-R, colony stimulating factor 1 receptor; GM-CSF-RA, granulocyte/macrophage colony-stimulating factor receptor alpha chain; SCF, stem cell factor; IL-3RA, Interleukin 3 receptor alpha chain; FLT-3, FMS-like tyrosine kinase 3; Mel-CAM (MCAM), melanoma cell adhesion molecule; ALCAM, activated leukocyte cell adhesion molecule; L1 Antigen/NCAM-1, leukocyte cell adhesion antigen-1/neural cell adhesion molecule-1); MDR-1, multidrug resistance protein-1; VEGF-R2/KDR, vascular endothelial growth factor receptor-2/kinase insert domain receptor; ABCG2, ATP-binding cassette (ABC) transporter subfamily G member 2; BD, Becton Dickinson.</p

Melanoma-formation in NSG mice.

<p>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. <i>A</i>, Results show the median tumor-volumes (mm³) in each group. <i>B</i>, A melanoma cell-containing tumor formed in an NSG mouse by EPO-R− cells obtained from patient #6.</p

Expression of potential stem cell markers and drug targets on melanoma cells.

<p>Score of reactivity of melanoma cells with antibodies: −, <5%; +/−, 6–20%; +, 21–60%, and ++, 61–100%.</p>a<p>Number of donors (n) analysed.</p>b<p>Cells were obtained from tumors grown in NSG mice injected with the non-sorted patient-derived melanoma cells.</p><p>Abbreviations: n.t., not tested.</p

Pelitinib blocks survival and induces apoptosis in melanoma cells.

<p><i>A</i>, 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. <i>B</i>, 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). <i>C</i>, 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.</p