<p>Melanoma initiating cell frequency.</p>

Comparison of different methods previously used to test the CD271-based cancer stem cell model in human melanoma.

<p>Genomic copy number differences among sibling PDX melanomas.</p>

Typical sorting strategy used to isolate viable melanoma cells from cell suspensions derived by dissociation from patient tumors.

Description of additional methods and procedures used in the study.

Genomic copy number differences among CD271- and CD271+ cells from PDX melanomas.

Legend for Supplementary Figures S1-S8.

<p>Marker expression patterns in PDX tumors grown from CD271- and CD271+ cells.</p>

<p>Melanoma initiating cell frequency.</p>

Clinico-pathological characteristics of patients who donated melanomas.

Variable re-expression patterns in PDX tumors generated from CD271- and CD271+ cells.

Abstract The stability of markers that identify cancer cells that propagate disease is important to the outcomes of targeted therapy strategies. In human melanoma, conflicting data exist as to whether hierarchical expression of CD271/p75/NGFR (nerve growth factor receptor) marks cells with enriched tumorigenicity, which would compel their specific targeting in therapy. To test whether these discrepancies relate to differences among groups in assay approaches, we undertook side-by-side testing of published methods of patient-derived melanoma xenografting (PDX), including comparisons of tissue digestion procedures or coinjected Matrigel formulations. We found that CD271− and CD271+ melanoma cells from each of seven patients were similarly tumorigenic, regardless of assay variations. Surprisingly variable CD271 expression patterns were observed in the analyses of sibling PDX tumors (n = 68) grown in the same experiments from either CD271− or CD271+ cells obtained from patients. This indicates unstable intratumoral lineage relationships between CD271− and CD271+ melanoma cells that are inconsistent with classical, epigenetically based theories of disease progression, such as the cancer stem cell and plasticity models. SNP genotyping of pairs of sibling PDX tumors grown from phenotypically identical CD271− or CD271+ cells showed large pairwise differences in copy number (28%–48%). Differences were also apparent in the copy number profiles of CD271− and CD271+ cells purified directly from each of the four melanomas (1.4%–23%). Thus, CD271 expression in patient melanomas is unstable, not consistently linked to increased tumorigenicity and associated with genetic heterogeneity, undermining its use as a marker in clinical studies. Cancer Res; 76(13); 3965–77. ©2016 AACR.