zPDX-Analysis of Invasiveness: Investigating Invasive Behavior of Metastatic Cancer Cells in Zebrafish Embryo Xenografts

1. Image and Analyze the Metastatic Process

1. Collect several anesthetized embryos with a wide-tip Pasteur pipette and transfer them to the glass bottom of a polystyrene dish.
2. Remove excess water and keep a limited amount of egg water. Manipulate the embryo into position with a hair loop tool and place a cover on top of the glass.
3. Use an inverted confocal microscope in combination with water-immersion or long-distance dry objectives. Position the embryo such that the region of interest is as close to the objective as possible.
4. Perform imaging immediately after anesthesia to reduce the risk of death due to liquid evaporation.
   1. Capture signals from EGFP-labeled vasculature and mCherry-labeled tumor cells at the same position on the embryos to co-register injected cells with blood vessels by merging the two imaging channels.
   2. For each zebrafish embryo, collect two different sets of images from the head region and tail region.
5. Quantify the number of disseminated cells.
   1. For perivitelline space injections, count the number of cells in each fish that have disseminated from the cell mass towards the embryonic fish body within the head and tail regions 4,15; the regions are beyond the boundaries of the heart cavity frontally, on top of the swim bladder dorsally, and beyond the urogenital opening caudally.
   2. For the Doc injection, count the number of individual cells that have invaded the collagen fibers of the tailfin from circulation (MDA-MB-231) or the number of clusters formed by cells collectively (M2) in the caudal hematopoietic tissue (CHT) of each zebrafish19.
6. Study invasion and metastasis in more detail by using confocal microscopy (highly recommended).
   1. Use low magnification (4X objective) to image the whole body and to obtain an overview of the tumor cell dissemination pattern.
      ​NOTE: Higher magnification (20X and 40X objectives) is suitable for studying intra- and peri-tumoral angiogenesis and the precise localization of disseminated cells in the embryo body.
   2. Use a 488 nm laser to scan the zebrafish embryo vasculature and a 543 nm laser to scan implanted tumor cells labeled with red fluorescence. Obtain a high-quality image by scanning each embryo in eight to ten steps. Scan and average each step six times.
7. Carefully place the embryo back into the egg water if it is required for further experiments.