We upcoming investigated the expression of antigens that have been previously associated with MIC. Melanospheres did not express CD133, CD20, CD24, ABCB5 or CD271, when p glycoprotein was detectable at lower levels. They expressed stem cell associated markers as c Kit, Cripto, CD146, CD44 and CD166 in agreement with former reports on cell line derived melanospheres. Lastly, embryonic stem cell markers Nanog and Oct 4 were detected with the RNA level in all samples analyzed. The CD44 isoform V6 was specifically restricted to melanospheres, being not expressed in differentiated cells, nor in tumor cells freshly isolated from melanosphere derived xenografts nor in melanocytes. Melanospheres may be expanded in vitro for various months and their proliferation fee was not lost with time.
They have been composed by a considerable fraction of self renewing sphere reforming selleck cells. Eventually, secondary and tertiary spheres were formed with a very similar frequency and tertiary spheres had been capable of proliferate indefinitely, indicating the fraction of self renewing cells did not lessen with passages. The clonogenic exercise was greater in melanospheres than inside their differentiated counterpart. Under suitable circumstances, melanospheres created a progeny of cells with morph ology and phenotype of melanocytic, adipogenic or osteogenic cells, demonstrating multiple differentiation potential and recapitulating the plasticity of neural crest cells. Melanospheres had been highly tumorigenic when injected subcutaneously in NOD Scid or Nude mice and all samples displayed tumor take of 100% right down to 25000 cells.
For one sample we performed a limiting dilution experiment and even as minimal as five cells readily produced Honokiol a tumor inside of 8 weeks. In contrast, melanosphere derived differentiated cells displayed a decreased and delayed tumor development in vivo, and as several as 5×104 differentiated cells generated a slowly growing tumor using a 10 week delay publish injection. Immunohistochemical evaluation of melanosphere derived xenografts, carried out for all samples, revealed a higher similarity among the xenograft along with the original patient tumor regarding morphology and expression in the melanoma connected diagnostic antigens MART1 and S100. Following xenograft dissociation and re injection we easily obtained secondary and tertiary tumors, suggesting that tumorigenic possible was not misplaced with passages in mice, in truth these benefits proved the capability of tumorigenic cells to self renew in vivo.
Based on these in vitro and in vivo results, we considered melanospheres as surrogate of melanoma initiating cells exploitable for pre clinical experimentation. Melanospheres are resistant to chemotherapeutic drugs and also to most pathway inhibitors We investigated the response of melaospheres to chemotherapeutic agents currently used in the therapy of melanoma patients.