Abstract 1398: Using zebrafish xenotransplantation to study the role of Y-Box binding protein (YB-1) in the metastasis of Ewing family tumors

Abstract

Sarcomas are malignant cancers of soft tissue or bone predominantly affecting children and adolescents. The most common subtypes are osteosarcoma and Ewing family tumours (EFTs). The most unfavorable prognostic factor is the presence of metastases, which accounts for 9 out of 10 sarcoma cancer deaths. Identifying factors and/or drugs that have an impact on metastatic spread have tremendous potential to affect outcome by reducing disease burden to the primary site, which can be more effectively treated by surgery and radiation. Pertinent animal models are critical for translating in vitro findings to clinical trials. Xenotransplantation of human cancer cells into transparent zebrafish embryos provides a novel in vivo platform for visualizing tumor micro-environment interactions contributing to sarcoma proliferation and spread in real time, which is not easily provided by other animal models. We recently demonstrated the effectiveness of the zebrafish xenotransplantation model for the study of specific drug-tumor interactions for both chronic myelogenous leukemia and acute promyelocytic leukemia and used a rapid and novel ex-vivo proliferation assay to quantify therapeutic responses (Corkery et al, BJH 2011). We have now applied this technology to EFTs. Human EFT TC-32 cells were fluorescently labeled with CmDiI, and microinjected into the yolk sac of two day old casper embryos, a double pigment mutant that prevents any auto-fluorescence that might interfere with image quality. EFT cells successfully engrafted, survived and proliferated over 96 hours post-injection (hpi). Migration of cells from the yolk sac to the tail occurred between 48 and 144 hpi with evidence of vascular extravasation and tissue infiltration. Y-box binding protein 1 (YB-1) is implicated in the metastatic spread of epithelial cancers due to its key role in promoting an epithelial-to-mesenchymal transition (EMT). In contrast to parental TC-32 cells, xenografted YB-1 knockdown (KD) TC-32 cells showed absent or significantly delayed migration, suggesting that YB-1 also regulates this process in zebrafish xenografts. Moreover, using transgenic fli-EGFP casper embryos that display fluorescent vasculature, we saw evidence of vascular recruitment into the tumor mass in WT TC-32 cells but not in YB-1 KD, potentially implicating angiogenesis as a mechanism that contributes to tumor spread in YB-1 expressing sarcomas. Exposure of TC-32 xenografted embryos to 5-40 Gy of ionizing radiation effectively reduced cell proliferation in a dose-dependent manner. These studies highlight the utility of the zebrafish xenograft model to elucidate the mechanisms underlying the metastatic behavior of EFTs and position this system as an in vivo tool for drug discovery to identify novel anti-proliferative and anti-metastatic agents to improve outcome in this disease. 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 1398. doi:1538-7445.AM2012-1398

Zebrafish xenografts as a tool for in vivo studies on human cancer

“…However, circulation of leukemic cells and migration of even larger human solid tumor cells, such as sarcoma cells, has been repeatedly demonstrated in zebrafish indicating that passage is possible (Fig. 2A and unpublished data) 68 . Moreover, we have used the nuclear stain, DRAQ5, to confirm that the human cells under observation are intact and not damaged due to injection or passage through the embryo 60 .…”

“…Recruitment of new blood vessels to the vicinity of the xenografted tumor mass can be visualized in real time (Fig. 2B and unpublished data), 68 similar to the more established chicken chorioallantoic membrane (CAM) angiogenesis model, but rivaling this system for more mechanistic studies due to the ease by which the zebrafish host can be genetically modified 89 . In another study, silencing of the two LIM domain kinases LIMK1 and LIMK2 was shown to reduce tumor cell‐induced neovascularization in human pancreatic tumor cell fish xenografts 75 .…”

“…Moreover, inhibition of retinoic acid (RA), an upstream regulator of miR‐10a, suppresses metastasis from pancreatic cancer cells in zebrafish xenografts. Additional genes implicated in metastasis, such as Y‐box binding protein 1 (YB‐1) 88 are also currently being examined using zebrafish xenograft approaches 68 …”

“…2A and unpublished data). 68 Moreover, we have used the nuclear stain, DRAQ5, to confirm that the human cells under observation are intact and not damaged due to injection or passage through the embryo. 60 Thus, zebrafish vessels, even though narrower in caliber, appear large enough to allow passage of most human cells.…”

“…Additional genes implicated in metastasis, such as Y-box binding protein 1 (YB-1) 88 are also currently being examined using zebrafish xenograft approaches. 68 By performing a siRNA kinome library screen Pardo et al identified ribosomal S6 kinase 1 (RSK1) as a key regulator of metastasis. 72 Downregulation of RSK1 in the lung adenocarcinoma cell line A549-enhanced cell migration in an in vitro cell motility assay.…”