Cancer Res. 2008 68:???-???.

In vivo switching of human melanoma cells between proliferative and invasive states.

Hoek KS, Eichhoff OM, Schlegel NC, Doebbeling U, Kobert N, Schaerer L, Hemmi S, Dummer R.

Department of Dermatology, University Hospital of Zürich, Gloriastrasse 31, 8091 Zürich, Switzerland. keith.hoek@usz.ch

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 subcutaneous injection into immunocompromised mice. Tumor growth rates were measured and post-excision 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 +/- 3 days post injection. By comparison, invasive signature cells required a significantly longer (p < 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 margine 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 which evoke one-way changes in gene expression. We present a new model for melanoma progression which accounts for transcription signature plasticity and provides a more rational context for explaining observed melanoma biology.

Submitted version copies of this manuscript are available on request.

Figures

Figure 1A 		A gene expression heatmap, generated by clustering samples based on the normalized expression of 105 metastatic potential genes highlights subtype-specific signatures.

Figure 1B 		In vitro growth experiments correlate with subtype-specific signatures.

Figure 1C 		In vitro motility experiments correlate with subtype-specific signatures.

Figure 2 		Immunohistochemical analysis of paraffin-embedded cell lines shows that proliferative and invasive signature lines have differential staining for Mitf (93% and 0%, respectively) and Ki67 antigen (94% and 45%, respectively).

Figure 3A 		siRNA-mediated knockdown of Mitf in a proliferative signature melanoma cell line (M000921) was confirmed by Western blot analysis.

Figure 3B 		The ratio of TGFß-mediated inhibition of growth in cells treated with siRNA targeting Mitf over cells treated with a control siRNA is compared between proliferative (M000921) and invasive (M991121) signature melanoma cell lines. This shows that Mitf knockdown promotes resistance to TGFß-mediated growth inhibition in a proliferative signature melanoma cell line while identical treatment does not change susceptibility in an invasive signature line. TGFß-treatment of proliferative signature lines (M980513, M000907) results in reduction of Mitf mRNA.

Figure 4 		Human melanoma cell lines (M980513, M000907, M991121, M010308) were injected into both flanks of immunocompromised nude mice. Proliferation of melanoma cells led to tumor growth which was monitored daily. Proliferative melanoma cells (M980513, M000907) formed tumors rapidly, while invasive melanoma cells (M991121, M010308) took weeks longer to initiate tumor growth.

Figure 5A-E 		A day 75 tumor resulting from an invasive signature melanoma (M010308) with Mitf and Ki67 stains.

Figure 5F-J 		A day 22 tumor resulting from a proliferative signature melanoma (M980513) with Mitf and Ki67 stains.

Figure 6 		Early phase melanoma cells expressing the “proliferative signature” gene set proliferate to form the primary lesion. Following this an unknown signal switch, likely brought about by altered microenvironmental conditions (e.g. hypoxia or inflammation), gives rise to cells with a significantly different “invasive signature” gene set. Invasive signature cells escape and, upon reaching a suitable distal site, revert to the proliferative state and nucleate a new metastasis where the cycle is repeated. Each switch in phenotype (state change) is accompanied by an exchange in expressed gene sets from proliferative to invasive and vice versa.