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.
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.
A retroviral vector-mediated gene transfer system was used to introduce m gamma-IFN and h gamma-IFN genes into mouse and human tumor cells, respectively. Murine tumor cell lines and primary human melanoma tumor cells were successfully transduced with gamma-IFN vector, and these transduced cells secreted measurable levels of biologically active m gamma-IFN and h gamma-IFN, respectively. Both murine and human tumor cell lines that expressed gamma-IFN exhibited increased surface expression of HLA class I antigens when tested by Western blot and FACS analysis. gamma-IFN--transduced human melanoma cells were more active in stimulating tumor-specific cytolytic activity of CTLs from melanoma patients in vitro. m gamma-IFN--transduced tumor cells were substantially less tumorigenic than the corresponding parent tumor cell lines in immune-competent mice. In addition, injection of m gamma-IFN--transduced tumor cells resulted in activation of tumor-specific CTL in vivo. We plan to use gamma-IFN--transduced autologous tumor cells to boost host immune responses as a potential therapy for human melanoma.
Background and Objectives: Dendritic cells (DC) pulsed in vitro with a variety of antigens have proved effective in producing specific antitumor effects in vivo. Experimental evidence from other laboratories has confirmed that shared antigens can be encountered in histologically distinct tumors. In our experiments, we set out to evaluate the immunotherapeutic potential of vaccines consisting of DC pulsed with MCA‐106 fibrosarcoma or B16 melanoma cell lysates and to determine whether a cross‐reactivity exists between the two tumors. Methods DC were prepared from the bone marrow of C57BL/6 (B6) mice by culturing progenitor cells in murine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). They were separated into three equal groups and were either pulsed with B16 melanoma cell lysates (BDC), pulsed with tumor extract from the syngeneic fibrosarcoma MCA‐106 (MDC), or left unpulsed (UDC). DC were then used to immunize three groups of mice, with all mice receiving two weekly intravenous (IV) doses of 1 × 106 DC from their respective preparations on days −14 and −7. A fourth group of control mice were left untreated. On day 0, all mice were challenged with subcutaneous injections of 1 × 105 B16 and 1 × 105 MCA tumor cells, administered in the left and right thighs, respectively. After the inoculations, the mice were monitored closely with respect to tumor growth and survival. Results The MDC mice developed specific cellular immunity directed against not only MCA‐106 tumor cells, but also against B16 melanoma, as measured through chromium‐release assays of splenocyte preparations, while remaining ineffective at killing both L929 fibroblasts and CT26 tumor cells. By day 30 after tumor inoculations, control mice manifested the largest B16 tumor volumes at a mean of 2185 mm3, followed by the UDC, MDC, and BDC groups at 92 mm3 (P = 0.00008), 3 mm3 (P = 0.000002), and 2 mm3 (P = 0.00004), respectively. The survival data mirrored this pattern, with control animals displaying the shortest mean survival time (37.1 ± 4.0 days), followed by UDC (44.8 ± 6.6), MDC (56.2 ± 14.7), and BDC (56.4 ± 18.3) animals. No significant differences were noted between MCA‐106 and B16 cell lysate‐pulsed DC vaccines with respect to their abilities to inhibit B16 tumor growth and to prolong survival. These findings were confirmed using a B16 pulmonary metastasis model. Likewise, vaccination with interferon‐γ gene‐modified MCA‐106 tumor cells was shown to be effective at protecting against a subsequent subcutaneous B16 tumor challenge in 3 of 4 mice observed. Conclusions These results demonstrate that immunization with antigen‐pulsed DC confers cellular immunity, retards tumor growth, and prolongs the survival of tumor‐challenged mice. The ability of MCA‐106 cell lysate‐pulsed DC vaccines to inhibit the growth of subcutaneous B16 tumors also suggests the presence of shared tumor‐associated antigens between these two histologically distinct tumors. J. Surg. Oncol. 1998;68:79–91. © 1998 Wiley‐Liss, Inc.
consisted of 2 million cells, followed by 6 million for the third and fourth injections, 2 Chiron Viagene Inc., San Diego, California.and then 18 million for the fifth and sixth injections. The humoral immune responses of the patients were assessed by enzyme-linked immunoadsorbent assay, radioimmunoassay, and radioimmunoprecipitation. RESULTS.Thirteen of the 20 patients completed the immunization protocol. Eight of these 13 patients showed a humoral immunoglobulin (Ig)G response against autologous and allogeneic melanoma cells. The other five patients either had no detectable antimelanoma antibodies or showed a weak IgG response that did not rise significantly above the preimmune level. All the sera contained low or undetectable levels of antimelanoma IgM antibodies. The IgG response increased progressively in titer during the course of immunization. The positive sera showed preferentially strong binding to melanoma cell lines and some cross-reactivity to nonmelanoma tumors. A 75-80 kD antigen on melanoma cells was immunoprecipitated by postimmune sera of 3 of the responding patients. Preimmune sera from Presented in abstract form at a meeting of the these three patients and sera from other patients immunized with a standard American Association for Cancer Research, nontransduced melanoma cell vaccine failed to precipitate this antigen. Two paWashington, DC, April 1996.tients with significant increases in serum IgG had clinical tumor regression, and two additional patients with low serum IgG response had transient shrinkage of Supported in part by National Cancer Institute nodular disease during therapy. grant no. 1RO1-CA-64959-01 and by Depart-CONCLUSIONS. These data suggest that gene therapy with IFN-g-transduced melament of Veterans Affairs grant no. Seigler 0001.noma cells is safe and worthy of further investigation in patients with less advanced Address for reprints: Zeinab Abdel-Wahab, stage malignant melanoma. The ability to monitor changes in the humoral responses M.B., Ch.B., Ph.D., Department of Surgery, Box of the immunized patients has been demonstrated.
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