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Intravital Imaging Reveals Transient Changes in Pigment Production and Brn2 Expression during Metastatic Melanoma Dissemination

¹ Tumour Cell Biology Laboratory, ² Light Microscopy Laboratory, ³ FACS Laboratory, and ⁴ Electron Microscopy Laboratory, Cancer Research UK LRI; ⁵ Signal Transduction Laboratory, Institute of Cancer Research, London, United Kingdom; and ⁶ Ludwig Institute for Cancer Research Ltd., Headington, Oxford, United Kingdom

Requests for reprints: Erik Sahai, Cancer Research UK, 44 Lincoln's Inn Fields, London, UK, WC2A 3PX, United Kingdom. Phone: 44-20-726-93165; Fax: 44-20-726-93168; E-mail: erik.sahai{at}cancer.org.uk.

Key Words: Intravital imaging • Melanoma • Metastasis

How melanoma acquire a metastatic phenotype is a key issue. One possible mechanism is that metastasis is driven by microenvironment-induced switching between noninvasive and invasive states. However, whether switching is a reversible or hierarchical process is not known and is difficult to assess by comparison of primary and metastatic tumors. We address this issue in a model of melanoma metastasis using a novel intravital imaging method for melanosomes combined with a reporter construct in which the Brn-2 promoter drives green fluorescent protein (GFP) expression. A subpopulation of cells containing little or no pigment and high levels of Brn2::GFP expression are motile in the primary tumor and enter the vasculature. Significantly, the less differentiated state of motile and intravasated cells is not maintained at secondary sites, implying switching between states as melanoma cells metastasize. We show that melanoma cells can switch in both directions between high- and low-pigment states. However, switching from Brn2::GFP high to low was greatly favored over the reverse direction. Microarray analysis of high- and low-pigment populations revealed that transforming growth factor (TGF)β2 was up-regulated in the poorly pigmented cells. Furthermore, TGFβ signaling induced hypopigmentation and increased cell motility. Thus, a subset of less differentiated cells exits the primary tumor but subsequently give rise to metastases that include a range of more differentiated and pigment-producing cells. These data show reversible phenotype switching during melanoma metastasis. [Cancer Res 2009;69(20):7969–77]