Abstract 4950: Targeting a breast cancer xenograft by modulation of the microenvironment with zoledronic acid.
Authors: Nienhuis Hilde H. , Arjaans Marlous , Timmer-Bosscha Hetty , de Vries Elisabeth G.E. , Schröder Carolina P. 
Published: August 2014

Abstract
Introduction - Targeting breast cancer (BC) via its microenvironment is a way to avoid tumor plasticity and development of resistance towards therapy. Bisphosphonates (BPs) are used to treat BC bone metastases. Prior studies suggest that BPs also exert an anti tumor effect by interacting with the microenvironment. However, the precise nature of this interaction is unclear. The chorioallantoic membrane (CAM) assay allows assessment of both human BC and microenvironment cells, circumventing issues of species specificity.
Aim - 1) evaluate whether an indirect anti-BC effect by BPs, can be determined using the CAM assay 2) Assess potential biomarkers for this intervention.
Methods - MTT survival assays were performed to assess a differential effect of BP zoledronic acid (ZA) on survival of human BC cell lines (MCF-7, SUM149 and MDA-MB-231 progeny cell line SCP2) and human stromal cell line Hs27a. For biomarker analysis, excretion of vascular endothelial growth factor (VEGF)-A and Transforming Growth Factor (TGF) β was measured in the supernatant of Hs27a and MCF-7 cultures and co-cultures treated with ZA by Enzyme-Linked Immuno Sorbent Assays (ELISA). The CAM assay was used to study the effect of ZA on a co-culture system of BC- and stromal cells. SCP2 cells were inoculated on the CAM with and without Hs27a cells on day 6 of embryonic development. ZA (200 μM) or PBS was added on the CAM on day 10 and tumors were harvested at day 14. Immunohistochemistry (IHC) for pan-cytokeratin was performed to identify BC cells in the tumors.
Results - ZA (4 days continuous incubation) did not induce a direct anti BC effect in vitro (IC50&gt;100 μM), but strongly affected stromal cell survival (IC50 8 μM). ZA treatment decreased TGFβ excretion by Hs27a cells dose dependently (ZA 10, 50, 100 or 500 μM for 48 hours) from 538±63 pg/ml (control) to 446±57 (ns), 285±98 (p&lt;.01), 198±47 (p&lt;.01) and 186±60 pg/ml (p&lt;.001). In the co-culture, ZA (10, 50, 100 or 500 μM for 48 hours) also decreased TGFβ excretion dose dependently from 625±60 pg/ml (control) to 613±33 (ns), 535±83 (ns), 389±48 (p&lt;.01) and 244±55 (p&lt;.001) pg/ml. No TGFβ secretion by sole MCF-7 cells was detectable (&lt;0±60 pg/ml). ZA had no effect on VEGF-A excretion by BC or stromal cells. On the CAM, co-cultures were sensitive to ZA treatment (73±8.27 vs 32±13.15 mm3 (p&lt;.001)), whereas BC cells without stromal cells were resistant to ZA (tumor size: 22±14.35 vs 33±15 mm3 (ns)). In the co-cultures, tumor cell percentage evaluated by IHC tended to be smaller in the ZA treated group compared to control (31±15 vs 12±5 (ns)).
Conclusion - Effects of therapeutic interventions on tumors by microenvironment modulation can be studied using the CAM assay. Our data suggest an indirect anti-BC effect of ZA in vivo via stromal cells in the BC microenvironment. This is accompanied by decreased TGFβ excretion, which may therefore function as biomarker for ZA treatment effect.
Supported by Dutch Cancer Society grant RUG 2010-4739
Citation Format: Hilde H. Nienhuis, Marlous Arjaans, Hetty Timmer-Bosscha, Elisabeth G.E. de Vries, Carolina P. Schröder. Targeting a breast cancer xenograft by modulation of the microenvironment with zoledronic acid. [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 4950. doi:10.1158/1538-7445.AM2013-4950