Stem cells play a critical role in normal tissue maintenance, and mutations in these stem cells may give rise to cancer. We hypothesize that melanoma develops from a mutated stem cell and therefore residual stem cell characteristics should be able to be identified in melanoma cell lines. We studied three metastatic melanoma cell lines that exhibited multiple morphologic forms in culture and demonstrated the capacity to pigment. We used the ability to efflux Hoechst 33342 dye, a technique known to enrich for stem cells in many tissues, to segregate cell populations. The cells with the greatest ability to efflux the dye were (1) small in size, (2) had the capacity to give rise to larger cell forms, and (3) had the greatest ability to expand in culture. The small cells were found to have a decreased proliferative rate and were less melanized. Large dendritic cells that appeared to be nonproliferative were identified in cultures. Treatment with cytosine beta-D-arabinofuranoside hydrochloride (Ara-C) expanded the large cell population but the residual proliferative capacity, both in vitro and in vivo, remained concentrated in the smaller cell fraction. Antigenic staining patterns were variable and heterogeneous. Nestin (a neural stem cell marker) and gp100 (premelanosomal marker) favored the smaller cell population, while nerve growth factor receptor often labeled larger cells. Morphologic and antigenic heterogeneity remained intact after clonal purification. These findings are consistent with the behavior expected for a tumor based on stem cell biology; this finding has diagnostic and therapeutic implications for melanocytic neoplasias.

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To investigate the ability of human dendritic cells (DC) to process and present multiple epitopes from the gp100 melanoma tumor-associated Ags (TAA), DC from melanoma patients expressing HLA-A2 and HLA-A3 were pulsed with gp100-derived peptides G9154, G9209, or G9280 or were infected with a vaccinia vector (Vac-Pmel/gp100) containing the gene for gp100 and used to elicit CTL from autologous PBL. CTL were also generated after stimulation of PBL with autologous tumor. CTL induced with autologous tumor stimulation demonstrated HLA-A2-restricted, gp100-specific lysis of autologous and allogeneic tumors and no lysis of HLA-A3-expressing, gp100+ target cells. CTL generated by G9154, G9209, or G9280 peptide-pulsed, DC-lysed, HLA-A2-matched EBV transformed B cells pulsed with the corresponding peptide. CTL generated by Vac-Pmel/gp100-infected DC (DC/Pmel) lysed HLA-A2- or HLA-A3-matched B cell lines pulsed with the HLA-A2-restricted G9154, G9209, or G9280 or with the HLA-A3-restricted G917 peptide derived from gp100. Furthermore, these DC/Pmel-induced CTL demonstrated potent cytotoxicity against allogeneic HLA-A2- or HLA-A3-matched gp100+ melanoma cells and autologous tumor. We conclude that DC-expressing TAA present multiple gp100 epitopes in the context of multiple HLA class I-restricting alleles and elicit CTL that recognize multiple gp100-derived peptides in the context of multiple HLA class I alleles. The data suggest that for tumor immunotherapy, genetically modified DC that express an entire TAA may present the full array of possible CTL epitopes in the context of all possible HLA alleles and may be superior to DC pulsed with limited numbers of defined peptides.