Abstract P5-05-09: Autophagy in three dimensional cultures provides survival advantage against trastuzumab in HER2+ mammary adenocarcinoma cells
Authors: Rodríguez Cristina E , Reidel Sara , Bal de Kier Joffé Elisa D , Jasnis María A , Fiszman Gabriel L 
Published: May 2015

Abstract
HER2 is overexpressed in 20-25% 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 them are resistant or acquire resistance during treatment. Autophagy has been proposed as a tumoral escape mechanism. Multicellular tumor spheroids (MCS) are a model of cell growth in 3D that mimics the structure of in vivo avascular tumors. We have previously demonstrated that MCS present different subpopulations, with a gradient of proliferative, quiescent and apoptotic cells towards the center of the spheroid and these characteristics makes it more resistant to Tz than monolayers. The aim of this study was to analyze the role of autophagy in MCS growth and its relevance on the resistance of breast cancer cells to Tz. MCS of overexpressing HER2+ human mammary tumor cells (BT474 cell line) were cultured as cell suspensions on agar, one per well, and experiments were conducted when MCS reached 550 um initial size. When the autophagy marker LC3 was analyzed in MCS by Western blot, both LC3-I and LC3-II were significantly up-regulated compared with cells cultured as monolayers. The functional autophagic flux was confirmed by immunoblots of LC3 and p62 (SQSTM1/sequestosome1) in cells treated with Bafilomycin A1 (5 nM). MCS were fixed and included in paraffin to analyze the expression of LC3 and observed a differential localization of autophagic cells, increasing towards the center of the spheroid, correlating with the hypoxic population previously described. Uppon Tz adittion at a concentration of 50 ug/ml, a higher and uniform expression of LC3 was found in all the living cells. These observations were further supported by the finding that p62 was down-regulated in Tz treated spheroids in opposition to controls (commercial IgG). In 2D, Tz (1 ug/ml) also exerted LC3-II conversion and increase in autophagosomes formation. Autophagy inhibition by 3-methiladenine (3-MA) used at 1 mM, in combination with Tz decreased two fold vs Tz alone in monolayers (49% vs 76% cell viability respectively, p&lt;0.05). In 3D, the reduction in size induced by the autophagy inhibition plus Tz was 13% vs 10% Tz alone. 3-MA alone did not elicited citotoxicity in 2D or 3D. To investigate the link between apoptosis and autophagy, we exposure monolayers and MCS to Tz during 6h with 1h of pre-incubation with 3-MA and analyze by Annexin V/propidim iodide. In monolayers, the combination of 3-MA with Tz enhanced Tz sensitivity since they induced an increase in total cell apoptosis 35% compared to Tz alone (15% vs 11% early + 21% vs 13% late apoptosis, p&lt;0.05). Surprisingly in 3D, Tz had an opposite effect and decreased by 30% late apoptosis; however, this Tz-protection against apoptosis was fully reversed by the inhibition of autophagy. We conclude that Tz exerted a differential effect in BT474 breast cancer cells cultured as MCS, inhibiting apoptosis and generating a smaller spheroid composed only of remaining Tz-resistant living cells which are autophagic addicted. We propose that this model could be useful to study the mechanisms involved in resistance to Tz.
Citation Format: Cristina E Rodríguez, Sara Reidel, Elisa D Bal de Kier Joffé, María A Jasnis, Gabriel L Fiszman. Autophagy in three dimensional cultures provides survival advantage against trastuzumab in HER2+ mammary adenocarcinoma cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-09.