Abstract 2881: Cytotoxic effect of trastuzumab on macrophage-infiltrated human mammary tumor spheroids

Abstract

Abstract The HER2 receptor, overexpressed in 20-25% of breast invasive tumors, is associated with low disease-free survival. Trastuzumab (Tz), monoclonal antibody anti HER2 is used as immunotherapy in HER2+ mammary tumors; however, 60% of patients are unresponsive to this therapy. The aim of our study is the analysis of the effect of Tz on the growth of human mammary tumor cells cultured in 3D (shperoids). The human adenocarcinoma cell line BT474 (HER2+) was used to generate spheroids grown in liquid RPMI1640 medium. One spheroid/well was seeded on a 1.5% agar layer, reaching 2.5 mm diameter at day 62 of culture. Differential expression of HER2, HIF-1, Bcl-2 and Ki67 were detected by immunohistochemistry. Proliferating, quiescent as well as apoptotic cells towards the hypoxic core of the tumor spheroids were identified. The effect of Tz (10 and 50 ug/ml/spheroid, 3 times/week) on the growth of 3D cultures of BT474 cells was evaluated, beginning with a volume of 0.23 ± 0.03 mm3 (day 0). Tz 10 ug/ml arrested spheroids growth since day 7 (3 doses) while Tz 50 ug/ml induced a significant reduction in the volume since the first dose (day 0) until day 27, when spheroids showed 71% of growth inhibition compared to untreated or human IgG controls (Tz 50= 0.12 ± 0.04 mm3 vs control= 0.45 ± 0.03 mm3, p<0.001). Spheroids showed lower sensitivity to the cytostatic effect of Tz 10 ug/ml when compared to the same cells cultured as monolayers. Tz exerted a direct and dose-dependent effect on BT474 tumor spheroids: while it was cytostatic at the lower dose (Tz10), it was cytotoxic at the higher (Tz50). Nitric oxide (NO) levels measured by Griess reaction in tumor spheroid supernatants were significantly higher upon Tz50 treatment compared to untreated controls (p<0.01), suggesting NO is involved in the direct cytotoxic mechanism of Tz. BT474 cells growing in monolayers responded to TZ with higher NO levels than 3D cultures. In order to assess whether immune system and tissue architecture have an impact on the Tz cytotoxic effect, we co-cultured single BT474 tumor spheroids with macrophages from human peripheral blood (ratio1:5). At 10 days co-culture, the macrophages had a type I response (M1) against tumor cells. In fact, we observed that the presence of macrophages in the culture increased the cytotoxicity of Tz. This period of time was not enough to switch macrophages to the M2 phenotype. Conditioned media from Tz50-treated BT474 spheroids decreased MMP9 production by macrophages cultures. We propose this 3D culture model as a useful tool to study susceptibility to trastuzumab therapy in HER2 + tumor cells, as well as to analyze the mechanisms of action and resistance of this treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2881. doi:1538-7445.AM2012-2881