Abstract 2930: The hostile microenvironment developed in 3D culture conditions induces Trastuzumab resistance involving cancer stem cells
Authors: Rodríguez Cristina E. , Fiszman Gabriel L. , joffe. Elisa D. Bal de Kier 
Published: July 2016

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 are a 3D cell growth model that mimics the structure of in vivo avascular tumors, with heterogenic cell subpopulations developed due to differential oxygen and nutrient supply through the spheroid. We have previously demonstrated that HER2+ cells cultured as spheroids are more resistant to Tz than monolayers. We also observed that in spheroids Tz inhibited basal apoptosis and was capable of inducing autophagy, leading to Tz resistance. In addition, cancer stem cells (CSC), widely associated with chemotherapy resistance, were specifically targeted by Tz.
The aim of this study was to analyze the resistance acquired in 3D and the impact of the CSC developed in these conditions.
Since Tz-treated BT474 (HER2+) spheroids overexpressed the autophagy marker LC3, correlating with resistance to the treatment, we first evaluated these cells viability after autophagy inhibition with 3MA. We found that these resistant cells became susceptible to Tz after autophagy inhibition, decreasing viability by 33% (p&lt;0.05). In an attempt to recreate the hostile microenvironment developed in 3D, we treated BT474 cells with CoCl2 to induce a pseudo hypoxia state, similar to that found on the inner core of spheroids. In these conditions, cells were resistant to Tz treatment and interestingly, when we inhibited autophagy, we also re-sensitize these cells to Tz, being indistinguishable from controls.
To further study this 3D-induced resistance, we analyzed the CSCs subpopulation in spheroids, studying the CD44+CD24low cell phenotype by flow cytometry. We found that 15 days Tz treatment increased CD44+CD24low subpopulation by 1.5 fold compared to controls (p&lt;0.05).
Then we analyzed HER2 expression in the spheroids and found 2 populations, HER2high and HER2low (37% vs 63%, respectively p&lt;0.05). Interestingly, after 15 days Tz treatment, HER2high subpopulation increased to 50%.
Thus, we decided to study the 3D architecture in MCF7 cells, with no HER2 amplification and unresponsive to Tz inhibition. We analyzed HER2 by flow cytometry, observing HER2low expression in 82% of the cells that was reduced to 61% in Tz treated spheroids (p&lt;0.05). Despite unresponsiveness of MCF7 cells to Tz as bulk spheroid, we detected a 30% reduction in the CD44+CD24low subpopulation after treatment.
In conclusion, the hostile microenvironment in 3D has a key role in the development of resistance to Tz and could be overcome by autophagy inhibition. These conditions could favor an increase of CSCs unresponsive to Tz despite HER2 expression. Moreover, MCF7 results suggest that Tz is able to target CSCs developed in 3D.
Citation Format: Cristina E. Rodríguez, Gabriel L. Fiszman, Elisa D. Bal de Kier joffe. The hostile microenvironment developed in 3D culture conditions induces Trastuzumab resistance involving cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2930.