Following prolonged culture, we obtained exponentially growing “m

Following prolonged culture, we obtained exponentially growing “melanospheres” with efficiency of 80% (Figure 1A left). The same cells cultured in conditions specific for the growth of melanocytes generated monolayers of tumor cells whose morphology resembled differentiated cells, suggesting the capacity of melanospheres to differentiate in vitro (Figure 1A right). Figure 1 Melanosphere isolation and validation. A) Image of melanospheres (left) and their differentiated progeny (right). B) Tumor volumes of xenografts generated by spheres or differentiated (diff)

melanoma cells injected subcutaneously in Nude mice at the indicated cell doses. Mean ± SD of 3 independent experiments is shown. ** p < 0,01. AZD1480 C) Table of melanospheres tumorigenicity in dose response experiments. Cell numbers, number of mice injected and percentage of tumor engraftment is indicated for each condition. Tumors were monitored for 8 weeks post-injection. D) Hematoxylin and eosin (H&E) or immunohistochemistry for the indicated antigens performed on patient tumor or xenograft generated

by melanospheres. The original magnification of each image is indicated. We next investigated the expression of antigens that have been previously associated with MIC. Melanospheres did not express CD133, CD20, CD24, ABCB5 or CD271 (Additional file 1: Figure

S1A-B), while p-glycoprotein was detectable at low levels. They expressed stem cell-related markers as c-Kit, Cripto, CD146, CD44 and CD166 (Additional file 1: Figure S1A) in agreement with previous reports on cell line-derived melanospheres [38]. Finally, embryonic stem cell markers Nanog and Oct-4 were detected at the RNA level in all samples analyzed (Additional file 1: Figure S1C). The CD44 isoform V6 was specifically restricted to melanospheres, being not expressed in differentiated cells, nor enough in tumor cells freshly isolated from melanosphere-derived xenografts nor in melanocytes (Additional file 1: Figure S1D). Melanospheres could be expanded in vitro for several months and their proliferation rate was not lost with time (Additional file 2: Figure S2A). They were composed by a large (mean 42% ± 8 in all examined samples) fraction of self-renewing sphere-reforming cells (Additional file 2: Figure S2B upper left). Finally, secondary and tertiary spheres were formed with a similar frequency and tertiary spheres were able to proliferate indefinitely, indicating that the fraction of self-renewing cells did not decrease with passages (Additional file 2: Figure S2B upper right panel). The clonogenic activity was higher in melanospheres than in their differentiated counterpart (Additional file 2: Figure S2B lower panels).

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