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    • An important step towards further ACC CSC characterization i

    2018-11-08

    An important step towards further ACC-CSC characterization is identification of CD markers which, in combination with CD133, may be used for FACS sorting and multiplexed CyTOF analysis of ACC-heterogeneity and their signaling states. Such studies may significantly advance understanding of ACC-CSC roles in tumor tissue and their relation to tumor growth, neural invasion, metastases, resistance to radiation, and recurrence. Association of ACC-CSC with the CD24 cell surface marker in this study provides a new insight into ACC cell of origin. Indeed, our hypothesis that ACC and basal-like breast cancers have similar CSC driven by SOX10 (Ivanov et al., 2013b) has been recently supported by a study that implicated SOX10 in stimulation of CD24hi fetal mammary stem cells (Dravis et al., 2015). Thus, CD24 appears to be a biologically and clinically relevant cell surface marker that can be used in combination with CD133 and CD44 in CyTOF studies and for more efficient ACC-CSC purification. As it was recently demonstrated, expression of CD24, CD44, and CD133 may be linked with activation of distinct pro-survival signaling pathways and have different effects on radiation resistance and patient survival (Sahlberg et al., 2014). It remains to see if similar associations take place in ACC and if differential expression of CD markers may predict prognosis or resistance to therapy. Another important goal of this study was to purify ACC-CSC from mixed mouse/human cell cultures and grow them as long-term cell cultures. In this regard, we found that Accx11 spheroids are made entirely of human CD133+ ACC-CSC, which made them a straightforward tool for ACC-CSC purification (Fig. 3. and Suppl. Fig. 3). Similarly, generation of monoclonal cell populations completely purified ACC-CSC from mouse cells (Fig. 5B). We then used MACS with fluvoxamine maleate to CD24 and CD133 as an alternative approach to ACC-CSC purification. Even though this approach turned out to be not as efficient to rid cultures of mouse cell contamination, these markers are human-specific and can be used with cell cultures that don\'t produce spheroids as well as in analytical FACS/CyTOF and preparative FACS studies on unpurified primary ACC cultures at early passages. Finally, we concluded that the Miltenyi kit for mouse cell elimination is an effective means for ACC-CSC purification since this approach produced cell populations without detectable mouse cells (Fig. 5C).
    Conclusion Overall, we report in this study on the assays, tools, and markers for authentication, purification, and research of a novel population of CSC that we recently identified in ACC. Marked by expression of SOX10, similar CSC populations appear to drive breast basal-like carcinoma, melanoma, and other cancers that appear to arise from neural crest (Ivanov et al., 2013b; Panaccione et al., 2017). We confirmed that ACC cultures maintained MYB fusions found in xenografts or primary tumors and identified a novel MYB fusion to a non-coding RNA. We demonstrated that ACC-CSC that we characterized earlier as SOX10+/CD133+ (Panaccione et al., 2016; Yarbrough et al., 2016) also express CD24 and CD44 as well as signaling molecules commonly found in CSCs, STAT3 and β-catenin. To develop a new platform for drug screening with the goal to develop effective and specific therapies for ACC, we optimized ACC-CSC purification from mixed mouse/human cell cultures and created a novel ACC-CSC-initiated orthotopic model for pre-clinical studies. The following are the supplementary data related to this article.
    Conflict of interests
    Acknowledgements This study was supported by funds from the Adenoid Cystic Carcinoma Research Foundation and by grants 5R21DE023228 (WGY) and 5R21DE022641 (SVI) from the National Institute of Dental and Craniofacial Research. This work was also supported in part by funds from the Department of Surgery, the Division of Otolaryngology, Yale School of Medicine, by an endowment to the Barry Baker Laboratory for Head and Neck Oncology, by Laura and Isaac Perlmutter Cancer Center Support Grant NIH/NCI P30CA016087, and the National Institutes of Health S10 Grants NIH/ORIP S10OD01058 and S10OD018338 and by an NCI fellowshipF31 CA216915 (MR).