The failure to eradicate most cancers and in particular melanoma may be as fundamental as a misidentification of the target. The identification of cancer stem/initiating cells within the tumour population with a crucial role for tumour formation may open new pharmacological perspectives. Our data show three main novelties for human melanoma: firstly, melanoma biopsy contains a subset of cells expressing CD133 (CD133+) and the latter is able to develop a Mart-1 positive tumour in NOD-SCID mice. Secondly, the WM115, a human melanoma cell line, has been found to express both CD133 and ABCG2 markers. This cell line grows as floating spheroids, expresses typical progenitors and mature neuronal/oligodendrocyte markers and is able to transdifferentiate into astrocytes or mesenchymal lineages under specific growth conditions. As in xenografts generated with CD133+ biopsy melanoma cells, those produced by the cell line displayed lower levels of CD133 and ABCG2. Thirdly, the WM115 cells express the most important angiogenic and lymphoangiogenic factors such as notch 4, prox1 and podoplanin which can cooperate in the development of the tumourigenic capability of melanoma in vivo. Therefore, in this study, we demonstrate the presence of stem/initiating subsets in melanoma both in biopsy and in an established melanoma cell line grown in vitro and in xenografts. Interestingly, considering that melanoma gives metastasis primarily through lymphatic vessels, herein, we demonstrated that a melanoma cell line expresses typical lymphoangiogenic factors.

Plasticity of cancer invasion and metastasis depends on the ability of cancer cells to switch between collective and single cell dissemination, controlled by cadherinmediated cell-cell junctions. In clinical samples, E-cadherin expressing and deficient tumors both invade collectively and metastasize equally, implicating additional mechanisms controlling cell-cell cooperation and individualization. Using spatially defined organotypic culture, intravital microscopy of mammary tumors in mice and in silico modeling, we here identify cell density regulation by 3D tissue boundaries to physically control collective movement irrespective of the composition and stability of cell-cell junctions. Deregulation of adherens junctions, including E-cadherin and p120-catenin, resulted in a transition from coordinated to uncoordinated collective movement along extracellular boundaries, whereas singlecell escape depended on locally free tissue space. These data indicate that cadherins and ECM confinement cooperate to determine unjamming transitions towards step-wise epithelial fluidization and, ultimately, cell individualization.

The transition between epithelial and mesenchymal states has fundamental importance for embryonic development, stem cell reprogramming, and cancer progression. Here, we construct a topographic map underlying epithelial-mesenchymal transitions using a combination of numerical simulations of a Boolean network model and the analysis of bulk and single-cell gene expression data. The map reveals a multitude of metastable hybrid phenotypic states, separating stable epithelial and mesenchymal states, and is reminiscent of the free energy measured in glassy materials and disordered solids. Our work not only elucidates the nature of hybrid mesenchymal/epithelial states but also provides a general strategy to construct a topographic representation of phenotypic plasticity from gene expression data using statistical physics methods.