A Quantitative System for Studying Metastasis Using Transparent Zebrafish

Abstract

Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAFV600E;p53−/− fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, μ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (~1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.

CD271 Down-Regulation Promotes Melanoma Progression and Invasion in Three-Dimensional Models and in Zebrafish

“…The zebrafish has recently emerged as an important model in cancer research able to develop human tumors, with similar morphology and comparable M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 11 signaling pathways (Heilmann et al, 2015;Kaufman et al, 2016). In the present work, we demonstrate that the lack of CD271 is critical for melanoma progression in vivo.…”

Macrophage-Dependent Cytoplasmic Transfer during Melanoma Invasion In Vivo

“…Based on 3-dimensional renderings of confocal z-stacks, the macrophages and tumor cells overlap their fluorescent membranes and are in close proximity with one another (Figure S2A, B; Movie S3 and Movie S4). These interactions are not simply due to cross-species immune recognition because we also observed dynamic interactions between host macrophages and transplanted zebrafish melanoma cells (Heilmann et al, 2015) (Figure S2C, D). Furthermore, macrophage-tumor interactions were not due to the transplantation procedure because we observed similar prolonged interactions between macrophages and endogenous melanocytes that had been transformed by expression of a constitutively active NRas, NRasG12V, which is sufficient to drive early steps of transformation (Bos, 1989; Dovey et al, 2009; Feng et al, 2010) (Figure S2E, F).…”

“…20–40 melanoma cells were transplanted into larvae, and tumor cell dissemination was reported as the percent of fish with at least one disseminated tumor cell four days post-transplantation. We observed a range of larvae with tumor cell dissemination, between 15% to 70%, in the human melanoma cell lines we tested, and zebrafish mel-1 melanoma cells (Heilmann et al, 2015) exhibited a dissemination rate of 40% (Figure 1E). The metastatic potential of the melanoma cell lines was maintained in our xenotransplantation experiments: Cell lines derived from primary tumors disseminated at a lower frequency than cell lines established from the metastases of those primary tumors (Figure 1E, boxes).…”

A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation

“…Briefly, tumors were dissociated with a razor blade and trypsin, filtered, and plated on a fibronectin coated well and grown in rich media supplemented with FBS and zebrafish embryo extract as described (44). After several passages, the zcrest 1 line was sorted for EGFP + cells, which were continued as the line.…”