Abstract 349: Autophagy, cancer stem cells, and trastuzumab resistance in three dimensional cultures of HER2+ breast cancer cells
Authors: Rodríguez Cristina E. , Reidel Sara , Jasnis Maria Adela , Bal de Kier joffe Elisa , Fiszman Gabriel L. 
Published: August 2015

Abstract
HER2 is overexpressed in 20% invasive breast tumors and correlates with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used to treat HER2+ tumors; however more than half of the patients are resistant or acquire resistance during treatment. Multicellular tumor spheroids (MS) are a 3D growth model that mimics the structure of in vivo avascular tumors. Autophagy has been proposed as a tumoral escape mechanism. The cancer stem cell (CSC) hypothesis postulates that tumor growth is maintained by a small fraction of cells with unlimited proliferative potential that are also able to differentiate into cell populations that become the bulk of the tumor. CSCs are thought to be more resistant to chemotherapy and radiotherapy and related with tumor recurrence. We have previously demonstrated that MS present different subpopulations showing basal autophagy and apoptosis that increase towards the center of the spheroid, and these characteristics make them more resistant to Tz than monolayers. We also observed that Tz inhibited basal apoptosis in MS and was capable of inducing autophagy. Our aim was to analyze alternative mechanisms of resistance of HER2+ breast cancer cells against Tz. For this purpose, we used BT474 (HER2+) human breast cancer cell line, from which we developed a resistant subline (BT474-MR). MS were cultured one per well in agar covered plates. We observed that cells from BT474 MS treated with Tz for 15 days upregulated the autophagy marker LC3 and showed 100% Tz resistance when compared with cells from MS treated with non-related human IgG as control. BT474 cells were treated with the autophagy inhibitor 3-metyiladenine that was dose dependently cytotoxic both in 2D and 3D cultures. Interestingly, the higher concentration used was critical for spheroids survival but not for monolayers. BT474-MR cells were more sensitive to autophagy inhibition than parental cells (75.7 vs 100% cell viability, p&lt;0.05). In order to investigate if the increase in autophagy after Tz treatment could favor a CSCs propagation, we used a mammosphere assay, which enriches for mammary progenitor cells and mammary CSCs. BT474 cells were cultured in low-attachment plates without serum and supplemented with B27; formed mammospheres were treated with Tz for 7 days, after which they were disaggregated and cultured again as secondary mammospheres. We found that Tz, rather than favor CSC growth, was able to effectively target this cell population, significantly decreasing secondary mammosphere-forming efficiency compare to IgG control (227 vs 164 respectively, p&lt;0.05). In conclusion, Tz exerted a differential effect in cells cultured as MS, inducing autophagy related to resistance. Since BT474 CSCs were a specifically aim of this immunotherapy, it is valid to propose that autophagy addiction of resistant cells could be associated with an increase in CSCs that are no longer eliminated by Tz.
Citation Format: Cristina E. Rodríguez, Sara Reidel, Maria Adela Jasnis, Elisa Bal de Kier joffe, Gabriel L. Fiszman. Autophagy, cancer stem cells, and trastuzumab resistance in three dimensional cultures of HER2+ breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 349. doi:10.1158/1538-7445.AM2015-349