Breast cancer is the 2nd leading cause of cancer-related death among women; however, the majority of deaths arise as complications from metastasis, rather than the primary tumor. The cancer stem cell (CSC) hypothesis provides an explanation for the limited success of current therapies for metastatic breast cancer. CSCs are defined as a subpopulation of tumor-initiating cells with the stem cell-like characteristics of self-renewal and multipotency. CSCs have been shown to enter circulation and reach distal tissues, where they may remain cell-cycle arrested, and therefore resistant to conventional chemotherapeutics. Recent work in our lab has shown that circulating tumor cells use dynamic tubulin-based microtentacles (McTNs) to reattach to distant tissues, a critical step in metastasis. McTNs are novel cellular structures formed by epithelial cells when detached from the extracellular matrix and are increased in more metastatic breast cancer cell lines. Given the proposed metastatic efficiency of CSCs, we examined McTN incidence and function in mammary stem cells and breast cancer stem cells. Flow cytometry for the CSC markers CD44 and CD24 showed that breast tumor cell lines with increased CSC characteristics display higher McTN frequencies. Given this correlation, CD44 and CD24 immunofluorescence was used to separate human mammary epithelial (HMLE) cells into CSC and non-CSC subpopulations with flow cytometry. Stem-like HMLE cells (CD44hi/CD24lo) have increased McTN levels compared to non-CSC cells (CD44lo/CD24hi) from the same HMLE cell line. The CSC subpopulation also demonstrated increased cytoskeletal modifications that promote McTN formation, such as elevated vimentin expression and an increase in both the amount and the bundling of stabilized, detyrosinated tubulin. Vimentin and detyrosinated tubulin localize to McTNs in suspended stem-like HMLEs. The increased McTNs in stem-like HMLEs promote faster initial reattachment of suspended cells that is inhibited by the tubulin-directed drug, Colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live cell confocal microscopy demonstrates that McTNs participate in the structure of breast stem cell mammopsheres, extending between adjacent cells along cell-cell junctions. McTNs can be reduced by the breast CSC targeting agent Curcumin. These studies show that McTNs contribute to the metastatic potential of breast CSCs as well as the ability of mammary stem cells to form multicellular mammospheres. We anticipate that this work will clarify the molecular mechanisms underlying tubulin alterations in breast cancer stem cells and identify how these tubulin modifications may be targeted more specifically to reduce McTNs and the metastatic reattachment efficiency of breast cancer CSCs.

Citation Format: Monica S. Charpentier, Rebecca A. Bettes, Michele I. Vitolo, Amanda E. Boggs, Jana Slovic, Keyata Thompson, Lekhana Bhandary, Jennifer Yoon, Stuart S. Martin. The role of microtentacles in the metastatic potential of breast tumor stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3772. doi:10.1158/1538-7445.AM2013-3772