The CD44 isoform V6 was particularly limited to melanospheres, remaining not expressed in differentiated cells, nor in tumor cells freshly isolated from melanosphere derived xenografts nor in melanocytes. selleckchem Melanospheres may be expanded in vitro for many months and their proliferation price was not lost with time. They have been composed by a large fraction of self renewing sphere reforming cells. Lastly, secondary and tertiary spheres have been formed having a very similar frequency and tertiary spheres were capable to proliferate indefinitely, indicating the fraction of self renewing cells didn’t lower with passages. The clonogenic action was increased in melanospheres than inside their differentiated counterpart.
Under ideal conditions, melanospheres generated a progeny of cells with morph ology selleck Afatinib and phenotype of melanocytic, adipogenic or osteogenic cells, demonstrating multiple differentiation capacity and recapitulating the plasticity of neural crest cells. Melanospheres have been remarkably tumorigenic when injected subcutaneously in NOD Scid or Nude mice and all samples displayed tumor take of 100% down to 25000 cells. For 1 sample we carried out a limiting dilution experiment and also as very low as 5 cells readily created a tumor within 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 created a gradually rising tumor which has a ten week delay publish injection.
Immunohistochemical evaluation of melanosphere derived xenografts, carried out for all samples, revealed a large similarity among the xenograft plus the unique patient tumor regarding morphology and expression with the melanoma associated diagnostic antigens MART1 and S100. Following xenograft dissociation and re injection we quickly obtained secondary and tertiary tumors, suggesting that tumorigenic possible was not lost with passages in mice, in actual fact these effects proved the ability of tumorigenic cells to self renew in vivo. Based mostly on these in vitro and in vivo effects, 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 now applied inside the treatment method of melanoma individuals. Melanospheres had been exposed to cisplatin, temozolomide, dacarbazine and paclitaxel for 48 hrs and cell viability was assessed by MTT assay. All round a weak cytotoxic result was observed without any therapeutic win dow as in contrast to regular melanocytes. Conversely, differentiated cells have been exceptionally delicate to cisplatin, in 3 out of three samples assessed.