Abstract 226: Trastuzumab effect on cell death and proliferation of HER2+ mammary adenocarcinoma cells using a tridimensional culture
Authors: Rodríguez Cristina E. , Reidel Sara , Moverer Luciana , Marino Lina , Bal de Kier Joffé Elisa , Jasnis Maria A. , Fiszman Gabriel L. 
Published: August 2014

Abstract
HER2 is overexpressed in 20-25% invasive breast tumors, and associated with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used as immunotherapy to treat HER2+ tumors; however more than half of them are resistant or acquire resistance during treatment. Cellular spheroids are a model of cell growth in 3D that mimic the structure of in vivo avascular tumors. We have previously demonstrated that tumor spheroids (TS) present different subpopulations, with a gradient of proliferative, quiescente and apoptotic cells from the outer rim towards the hypoxic core.
The aim of this study was to analyze the biological characteristics of the different cell populations that constitute the TS and their response to Tz.
TS of HER2+ human mammary tumor cells (cell line BT474) were cultured as cell suspensions on agar (hanging drop method) in 48-well plates (1 spheroid / well). Cell cycle at days 7, 14 and 21 of TS growth was analyzed by flow cytometry. A significant increase only in the G2/M phase (6.6%, 10.3% and 13.3% respectively) was detected. TS were incubated with Tz at a concentration of 50ug/ml; a commercial IgG (50ug/mL) was used as negative control. The effect on cell growth kinetics was evaluated with photographs taken at different times, observing a reduction of the spheroids volume until they reached a decrease by 71% at day 20 (p&lt;0.01 vs controls). Tz induced an increase in the% of cells arrested in G1 (80.8% vs 64.7% control IgG, p&lt;0.001) and a decrease in the proliferative subpopulation (7.4% vs 11.4%, p&lt;0.05). A decrease in the% of apoptotic cells was also detected (Annexin V/IP). When we analyzed cell viability at different time points, we observed that Tz elicited changes in live to dead cells ratio. A reversion of the inhibitory effect of Tz on TS growth was observed after antibody withdrawal.
Spheroids were fixed and included in paraffin to analyze the expression of HER2, pHER2, clived caspase 3, HIF-1a, p27, p21 and cyclinD1 by confocal microscopy. HER2 expression was not modulated by Tz; however, we observed lower expression of its activated receptor (pHER2). HIF-1a was expressed only in cells around the central core while clived caspase 3 was expressed in the central core. After Tz incubation, both markers were no longer detected.
We can conclude that Tz exerted a potent antiproliferative and direct cytotoxic effect on BT474 tumor cells growing in 3D; these activities might be due to the partial inhibition of HER2 activation. Tz inhibited the formation of the hypoxic core and therefore the apoptotic subpopulation, generating thereby a smaller TS composed only of remaining Tz-resistant living cells. We propose that this model could be useful to study the mechanisms involved in the effect and on the resistance to Tz.
Citation Format: Cristina E. Rodríguez, Sara Reidel, Luciana Moverer, Lina Marino, Elisa Bal de Kier Joffé, Maria A. Jasnis, Gabriel L. Fiszman. Trastuzumab effect on cell death and proliferation of HER2+ mammary adenocarcinoma cells using a tridimensional culture. [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 226. doi:10.1158/1538-7445.AM2013-226