OP222: Stem-like populations of salivary adenoid cystic carcinoma and tumor-sphere formation

Abstract

The morphologic heterogeneity of human salivary gland tumors belies the traditional concept of tumor progression through stepwise alterations in multiple molecular and cellular pathways. The cancer stem-like cell (CSC) hypothesis states that a subpopulation of intratumoral cells is uniquely capable of propagating the tumor, relying on hierarchical model to explain tumor heterogeneity and behavior. CSCs are a subset of self-sustaining cancer cells with exclusive ability to maintain the tumor. Self-renewal, multipotency, and increased proliferative capacity allow CSC populations to maintain a pluripotent phenotype, while also producing a heterogeneous tumor. CSCs have been reported in solid tumors: breast, brain, prostate, lung, colon, pancreas, liver, and skin. The CSC concept – if applicable to adenoid cystic carcinoma (AdCC) – allows for a better understanding of aggressive tumor cell biologic traits, such as chemoresistance and metastasis. We hypothesize that salivary AdCC contains CSC-like cells. The present project was designed to test the ability of salivary adenoid cystic cancer stem-like cells derived from primary tumors to form tumor spheres. ACC cell lines: SACC-83 cells. Clinical samples: 2 solid tumor samples were collected from patients with AdCC of salivary glands. Sphere formation: we adapted the protocol published for mammospheres formation. Identification and enrichment of stem-like cells. Cell clusters could be identified within 24 h, and after 48 h, spheres ⩾ 60 μm were evident as determined with an eyepiece reticle and stage micrometer. Tumor spheroids. To characterize the tumorsphere cells, cytospin cell preparations were immunostained for markers of differentiated luminal epithelial cells (cytokeratin cocktail, CK7), differentiated myoepithelial cells (cytokeratin, CK 14, p63), and progenitor cells (p63, NOXA). Tumorspheres from both passages were positive for cytokeratin cocktail and cytokeratin 7 was present in smaller procentage of cells. Both keratins were decorating the cells in a cytoplasmic and membranous pattern of staining. p63, a myoepithelial nuclear marker was detected in spheroids from both passages although the number of p63 positive cells in the second spheroid passage was lower compared to the first passage. ALDH activity. As an alternative, the Aldefluor assay is used to identify and isolate stem-like cells from primary adenoid cystic carcinoma samples. In our attempts with the Aldefluor assay, the second passage showed a small procentage of ALDH + CD133 + stem cells present in the SACC-83 cell line and tumorspheres derived from solid tumor. Cancer stem cells (CSCs) are believed to be responsible for ACC formation and recurrence. These cells can be identified in established cell lines and primary patient samples. CSCs can be identified on the basis on functional activity (self-renewal, serial tumor propagation) and phenotypic markers.