nobenzaldehyde (DEAB) (a specific ALDH inhibitor) for 10 minutes before treatment with the Aldefluor substrate. Test cells did not receive DEAB treatment. FACS for ALDH expression was carried out following incubation with the Aldefluor substrate using a FACSARIA (Becton Dickinson, Oxford, UK). ALDH$^{+}$ cells were identified as those cells emitting fluorescence clearly greater than control inhibitor-treated cells. ALDH$^{+}$ and ALDH$^{-}$ cell populations were seeded into proliferation and clonogenic assays immediately after sorting. To determine whether ALDH expression is a fixed phenotype in CM, sorted ALDH$^{+}$ cells and ALDH$^{-}$ cells were cultured for 14 days and subsequently reassessed for ALDH activity using a FACSARIA (Becton Dickinson).

\section{Expression of Stem Cell Surface Markers CD44 and CD133}

To determine expression of the stem cell surface markers CD44 and CD133, cells were stained with anti-CD44 (PE-conjugated) and anti-CD133 (APC-conjugated) antibodies (Miltenyi, Bergisch Gladbach, Germany). The anti-CD44 antibody detected all isoforms of CD44. An antibody against the AC133 epitope of the CD133 antigen was used because this specific epitope is expressed exclusively on stem and progenitor cells and represents the glycosylation-dependent epitope of CD133. Fluorophore-conjugated isotype control antibodies were used in all experiments.

Briefly, two tubes containing $5 \times 10^{5}$ cells were centrifuged and cell pellets were resuspended in blocking buffer before antibody incubation. After incubation, cells were washed in blocking buffer and fluorescent staining was visualized via flow cytometry on a FACSCALIBUR (Becton Dickinson).

\section{Results}

\subsection{ALDH Activity in CM Cell Lines Does Not Select for Increased Proliferative or Clonogenic Capacity}

In the present study, both CM cell lines A375 and WM793 were assessed for ALDH activity and the clonogenic and proliferative capacity of subsequent ALDH$^{+}$ and ALDH$^{-}$ populations tested. Both CM cell lines were seen to express ALDH in a high percentage of cells in culture (Figs. \ref{fig:figure_1}). The mean percentage of ALDH$^{+}$ A375 cells was $45.9\% \pm 13.4\%$ ($n = 3 \pm$ SD) and $21.8\% \pm 9.7\%$ ($n = 3 \pm$ SD) in WM793 cells. Following identification of ALDH$^{+}$ cells in both CM cell lines, cells were sorted into ALDH$^{+}$ and ALDH$^{-}$ subpopulations and subsequently seeded into proliferation and clonogenic assays. In both CM cell lines, ALDH$^{+}$ cells and ALDH$^{-}$ sorted cells were found to proliferate at an equivalent rate (Fig. \ref{fig:figure_1}). When seeded out at clonal density, ALDH$^{+}$ and ALDH$^{-}$ cells both readily formed colonies. There was no significant difference in proliferative or clonogenic capacity between ALDH$^{+}$ and ALDH$^{-}$ cells and no difference in colony morphology observed (Fig. \ref{fig:figure_1}).

ALDH$^{+}$ and ALDH$^{-}$ populations were reassessed for ALDH expression 14 days post sort. Both A375 cells and WM793 cells demonstrated that ALDH activity is a fluid process and mixed populations of ALDH$^{+}$ and ALDH$^{-}$ cells were present in both populations of previously sorted cells, summarized in the Table. In the A375 cell line, the number of ALDH$^{+}$ cells arising from previously sorted ALDH$^{+}$ cells was comparable to the number of ALDH$^{+}$ cells in unsorted populations ($44.6\% \pm 1.7\%$). ALDH$^{-}$ sorted populations were able to give rise to daughter cells that expressed ALDH, although these ALDH$^{+}$ cells represented only $2.0\% \pm 0.1\%$ of the population ($n = 3 \pm$ SD). In the WM793 cell line, previously sorted ALDH$^{+}$ cells largely retained their ALDH$^{+}$ phenotype when grown in culture, containing $85.8\% \pm 2.9\%$ ALDH$^{+}$ cells following 14 days in culture. Sorted ALDH$^{-}$ WM793 cells also tended to retain their original phenotype in culture, giving rise to only $2.6\% \pm 0.2\%$ ALDH$^{+}$ daughter cells.

\subsection{ALDH Activity in UM Cell Lines and STCs Is Not Associated With a Stem Cell Phenotype}

Two UM cell lines and seven UM STCs were tested for expression of ALDH. Interestingly, ALDH expression was absent in both UM cell lines ($n = 3$; data not shown). In contrast to observations in UM cell lines, all UM STCs were found to contain a subpopulation of ALDH$^{+}$ cells ranging from $0.8\%$ to $16.0\%$ (mean $= 6\%$) (Fig. \ref{fig:figure_1}). Where possible, repeats were performed; however, due to limited material, this was not possible in all cases. To determine whether expression of ALDH was related to proliferation capacity, sorted populations were seeded into proliferation assays. Four of seven UM STCs tested for ALDH expression were taken forward for growth assays. We show that there was no difference in the proliferative capacity of ALDH$^{+}$ cells versus ALDH$^{-}$ cells in any of the four UM cases studied (Fig. \ref{fig:figure_1}; data represent SOM 577).

\subsection{CD133 Is Present in a Small Subpopulation of CM Cells and Select UM STCs}

In the present study, the two CM cell lines were seen to contain small subpopulations of CD133$^{+}$ cells (A375: mean of $0.6\%$ (Fig. \ref{fig:figure_2}B)). WM793: mean of $0.3\%$ (Fig. \ref{fig:figure_2}D); $n = 3$).

UM cell lines were found not to contain a subpopulation of CD133$^{+}$ cells ($n = 3$; Figs. \ref{fig:figure_2}). Of the six UM primary cultures tested, only one (OMM 588) contained a subpopulation of CD133$^{+}$ cells ($0.3\%$ of cells) (Fig. \ref{fig:figure_2}J). The remaining five cultures were negative for CD133 expression (data not shown). Therefore, it was concluded that CD133 is not likely to represent a CSC population in UM and was not studied further.

\subsection{CD44 Is Highly Expressed in All Melanoma Cells and Not Just a Minor Subpopulation}

The present study found that in the two CM cell lines ($>95\%$ CD44$^{+}$), and also the two UM cell lines ($>95\%$ CD44$^{+}$), CD44 was expressed in most cells in culture and not just a subset of cells (Figs. \ref{fig:figure_2}). Furthermore, CD44 was found to be expressed in most cells in the six UM cultures tested, ranging from $77\%$ to $92\%$ CD44$^{+}$ cells (Fig. \ref{fig:figure_2}; data represent SOM 588).

\subsection{A Cellular Hierarchy Is Absent in Established UM and CM Cell Lines}

To determine whether UM cell lines SOM 157d and SOM 196b were organized in a hierarchical manner, cells were seeded out into clonogenic assays. Colonies were observed after 14 days. The clonal efficiency for SOM 157d cells was $16.0\% \pm 0.4\%$ and $13.0\% \pm 1.0\%$ for SOM 196b (data represent the average colony count and clonal efficiency of three repeat triplicate experiments $\pm$ SEM). In both UM cell lines, colony morphologies remained consistent with no variation in cell type or proliferative capacity (Fig. \ref{fig:figure_3}). When isolated from culture using clonal discs, all colonies picked and reseeded were able to reestablish further colonies which resembled the parent colony. This observation could be seen in all subsequent generations of colonies picked, with $100\%$ efficiency. There appears to be only one clone type present in UM cell lines whereby every cell within the population has the capacity to drive tumor progression.

To determine whether the CM cell lines, A375 and WM793, reflect the clonal behavior of UM cell lines, cells were seeded