Metastatic melanoma represents a complex and heterogeneous disease for which there are no therapies to improve patient survival. Recent expression profiling of melanoma cell lines identified two transcription signatures, respectively, corresponding with proliferative and invasive cellular phenotypes. A model derived from these findings predicts that in vivo melanoma cells may switch between these states. Here, DNA microarray-characterized cell lines were subjected to in vitro characterization before s.c. injection into immunocompromised mice. Tumor growth rates were measured and postexcision samples were assessed by immunohistochemistry to identify invasive and proliferative signature cells. In vitro tests showed that proliferative signature melanoma cells are faster growing but less motile than invasive signature cells. In vivo proliferative signature cells initiated tumor growth in 14 F 3 days postinjection. By comparison, invasive signature cells required a significantly longer (P < 0.001) period of 59 F 11 days. Immunohistochemistry showed that regardless of the seed cell signature, tumors showed evidence for both proliferative and invasive cell types. Furthermore, proliferative signature cell types were detected most frequently in the peripheral margin of growing tumors. These data indicate that melanoma cells undergo transcriptional signature switching in vivo likely regulated by local microenvironmental conditions. Our findings challenge previous models of melanoma progression that evoke one-way changes in gene expression. We present a new model for melanoma progression that accounts for transcription signature plasticity and provides a more rational context for explaining observed melanoma biology. [Cancer Res 2008;68(3):650-6]

Human melanoma is composed of distinct cell types reminiscent of neural crest derivatives and contains multipotent cells that express the neural crest stem cell markers CD271(p75 NTR ) and Sox10. When isolated from solid tumors by using a method that leaves intact cell surface epitopes, CD271-positive, but not CD271-negative, cells formed tumors on transplantation into nude or nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. These tumors fully mirrored the heterogeneity of the parental melanoma and could be passaged more than 5 times. In contrast, in more immunocompromised NOD/SCID/IL2rg null mice, or in natural killer cell-depleted nude or NOD/SCID mice, both CD271-positive and CD271-negative tumor cell fractions established tumors. However, tumors resulting from either fraction did not phenocopy the parental tumors, and tumors derived from the CD271-negative cell fraction could not be passaged multiple times. Together, our findings identify CD271-positive cells as melanoma stem cells. Our observation that a relatively high frequency of CD271/Sox10-positive cells correlates with higher metastatic potential and worse prognosis further supports that CD271-positive cells within human melanoma represent genuine cancer stem cells. Cancer Res; 71(8); 3098-109. Ó2011 AACR.

IntroductionPrimary cutaneous lymphomas (CLs) represent the second most common extranodal group of non-Hodgkin lymphomas and are characterized by an accumulation of clonal T or B lymphocytes preferentially homing to the skin (1). Most CLs are indolent diseases with good prognosis that display a chronic course, sometimes progressing over decades. This in turn creates an increasing need for treatment modalities that would effectively control the disease for a longer time period together with showing good tolerability. Immunotherapy is one such solution that currently belongs to the standard therapeutic armamentarium. The use of recombinant cytokines has found its rationale in the immunobiology of this disease group and aims to enhance antitumor immunity (1, 2). As an alternative to recombinant cytokines that are often associated with significant systemic side effects, we recently described adenovirusmediated (Ad-mediated) delivery of human IFN-γ gene directly into CL lesions, showing good tolerability together with promising efficacy (3). Adenoviral vectors belong to the most frequently used gene delivery systems due to their ability to transduce a broad spectrum of cells and package large sequences and their low production costs (4). The fact that they do not integrate into the host genome has made them attractive in cancer immunotherapy, where only

Supplementary Figure 1 from <i>In vivo</i> Switching of Human Melanoma Cells between Proliferative and Invasive States

<div>Abstract<p>Metastatic melanoma represents a complex and heterogeneous disease for which there are no therapies to improve patient survival. Recent expression profiling of melanoma cell lines identified two transcription signatures, respectively, corresponding with proliferative and invasive cellular phenotypes. A model derived from these findings predicts that <i>in vivo</i> melanoma cells may switch between these states. Here, DNA microarray–characterized cell lines were subjected to <i>in vitro</i> characterization before s.c. injection into immunocompromised mice. Tumor growth rates were measured and postexcision samples were assessed by immunohistochemistry to identify invasive and proliferative signature cells. <i>In vitro</i> tests showed that proliferative signature melanoma cells are faster growing but less motile than invasive signature cells. <i>In vivo</i> proliferative signature cells initiated tumor growth in 14 ± 3 days postinjection. By comparison, invasive signature cells required a significantly longer (<i>P</i> < 0.001) period of 59 ± 11 days. Immunohistochemistry showed that regardless of the seed cell signature, tumors showed evidence for both proliferative and invasive cell types. Furthermore, proliferative signature cell types were detected most frequently in the peripheral margin of growing tumors. These data indicate that melanoma cells undergo transcriptional signature switching <i>in vivo</i> likely regulated by local microenvironmental conditions. Our findings challenge previous models of melanoma progression that evoke one-way changes in gene expression. We present a new model for melanoma progression that accounts for transcription signature plasticity and provides a more rational context for explaining observed melanoma biology. [Cancer Res 2008;68(3):650–6]</p></div>