Abstract P1-03-04: Zebrafish xenografts as a fast screening platform for bevacizumab impact on tumor growth, angiogenesis and micrometastasis in breast cancer
Authors: Almeida Cátia , Mendes Raquel , Ribeiro Joana Mourato , Cardoso Maria João , Cardoso Fátima , Ferreira Miguel Godinho , Fior Rita 
Published: March 2020

Abstract
Background: VEGF-A is the most potent pro-angiogenic factor and is upregulated in a variety of tumors. Therapies were developed to neutralize VEGF signalling, such as Bevacizumab (Bev). Despite promising pre-clinical results, the average response rate to Bev is reduced for most cancers, with significant side effects. In breast cancer (BC) several studies and a meta-analysis have also confirmed a limited i efficacy in unselected populations. Lack of predictive biomarkers prevents a good patient selection. As such guidelines consider Bev only in a minority of cases. Additionally, depending on the individual tumor, VEGF-A can either block or promote metastasis. In this context, an assay able to predict individual responses to Bev, including its impact on metastasis would prove of great value.
Methods: We developed an assay, using zebrafish xenografts(zX), where tumor response and the impact on angiogenesis and metastasis (mets) can be readily analysed. By using Triple negative BC (TNBC) cell line models (Hs578T and MDA-MB-468) and zebrafish patient derived xenografts(zPDX) we evaluated the impact of Bev on proliferation, apoptotis, tumor size, angiogenesis and metastasis.
Results: a) Human TNBC cell lines display different tumoral features in zX: Hs578T presented the most vascularized tumors (vessel density ~32.4%) and the lowest metastatic capacity (2.6% of xenografts presented mets, n=77). In contrast, MDA-MB-468 that only recruits blood vessels to the base of the tumor, showed a higher metastatic potential (42.5%, n=87). No direct correlation between vessel density and metastasis could be seen. b) zX were able to reveal different individual tumor responses to Bev in 4 days. Analysis of cell death revealed higher apoptosis rate in Hs578T xenografts (1.6-fold, p&lt;0.0001) with a reduction in tumor size (40% tumor reduction, p&lt;0.0001). MDA-MB-468 displayed resistance to Bev with no changes in the analysed parameters. c) zX can reveal different impact of Bev in tumor micro-environment in 4 days. Hs578T tumors had the highest angiogenic potential being the most affected by Bev treatment (vessel density reduction, [20% p=0.0023] and vessel infiltration [32% reduction, P=0.0027]. In MDA-MB-468 tumors no significant impact on vessel network was observed. d) We identified the previously reported paradoxical responses of antiangiogenic therapies in metastasis in this fast time window of 4 days assay. In Hs578T tumors Bev monotherapy resulted in a 2-fold increase in the frequency of mets(p=0.03). In contrast MDA-MB-468 tumors, that exhibited the highest metastasis potential, showed a reduced frequency of mets upon Bev treatment (42,5% vs 19,7% in control vs Bev group; p=0.0013). e) Differential responses to Bev in zPDX were seen. We processed primary TNBC resected samples and treated the resulting zPDX with Bev. A variety of phenotypes were observed. zPDX1 and zPDX3 had a profile reminiscent to MDA-MB468, with Bev having no anti-tumor effect (p=1.0, p=0.42,respectively), but reducing the micrometastasis incidence (zPDX1-from 42% to 0%, p=0.072; zPDX3- from 20% to 0%, p=0.167). zPDX2 exhibited a profile like Hs578T, where we could detect a significant increase in apoptosis upon Bev treatment (6.5% to 18.7%, p=0.01), accompanied by a trend to tumor shrinkage (21% reduction, p=0.42) but increase mets frequency (12% to 53%, p=0.26).
Conclusions: Our results show that the zebrafish larvae xenografts have enough resolution to display differential tumor responses to Bev in TNBC models and zPDX in just 4 days. We propose zebrafish cancer xenografts as a promising fast in vivo platform for Bev screening, allowing analysis of personalized responses.
Citation Format: Cátia Almeida, Raquel Mendes, Joana Mourato Ribeiro, Maria João Cardoso, Fátima Cardoso, Miguel Godinho Ferreira, Rita Fior. Zebrafish xenografts as a fast screening platform for bevacizumab impact on tumor growth, angiogenesis and micrometastasis in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-04.