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Advancements in high-resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13 829 peptides were identified; 83-87% of these were 8-11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; m RNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA-type binding prediction for 10 078 9/10 mer peptides assigned 88-95% to a patient-specific HLA subtype, revealing a disparity in strength of predicted binding. HLA-B*27-specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells. [ABSTRACT FROM AUTHOR]

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1+) and basal/myoepithelial (CD10+) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally ‘enriching’ for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity. [ABSTRACT FROM AUTHOR]

Abstract: Ingenol-3-angelate is a new local chemotherapeutic agent in clinical trails that induces primary necrosis of tumour cells and transient local inflammation. Here we show that cure of subcutaneous tumours with ingenol-3-angelate (PEP005) resulted in the generation of anti-cancer CD8 T cells that could regress metastases. Furthermore, PEP005-mediated cure synergized with several CD8 T cell-based immunotherapies to regress further distant metastases. PEP005 was shown to have adjuvant properties, being able to upregulate CD80 and CD86 expression on dendritic cells in vivo, and to promote CD8 T cell induction when co-delivered with a protein antigen. PEP005 thus emerges as a unique local chemotherapeutic immunostimulatory debulking agent that could be used in conjunction with immunotherapies to promote regression of metastases. [Copyright &y& Elsevier]