Zebrafish Xenotransplant Cancer Model for Drug Screening - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 219

Figure 17.2 HumanColo320 colon cancer cells after xenotransplant in 2dpf zebrafish. One thousand five
hundred CM-DiI-labeled Colo320 cells in 25 nL HBSS were injected into the zebrafish yolk sac and cancer
cell proliferation and migration were tracked. Images are from the same Xt zebrafish
at 0, 1, and 4 dpx. E¼eye, H¼heart, Y¼yolk sac, and T¼ tumor.

cells migrated and formed masses. Since Colo320 and SW620 cells showed different
cancer characteristics, they represented good cell lines for model development.
Human lymphoblastoid TK6 cells were used as a noncancer control cell line.
Although we observed that both 2 and 3dpf zebrafish were suitable for xeno-
transplant, 3dpf zebrafish tolerated a greater number of cells. To optimize model
development, after xenotransplant, we tracked CM-DiI-labeled cancer cell progres-
sion in live zebrafish for 7 days using fluorescence microscopy. Although Xt zebrafish
generally survived up to 3 dpx (

85% survival), death increased markedly at 4 dpx,
likely due to increased cancer cell burden and nutrient depletion. In contrast, human
TK6 lymphoblast control cells did not survive in zebrafish and by 5 dpx, few
fluorescently labeled TK6 cells were visible (data not shown). These studies showed
that early-stage zebrafish tolerate thousands of human cancer cells with no rejection
response, supporting use as an in vivo animal model for drug screening. Because
Colo320 cells have a short doubling time (

>

38 h) in Xt zebrafish, we selected this cell

line for drug screening.
In a previous study, we determined that zebrafish injected with 50 human
melanoma cells can be maintained at 35 C (Haldi et al., 2006) supporting normal
development of the host as well as proliferation of human cancer cells. In these
studies, we also established the yolk sac as the optimum injection site. For the first
7dpf, the yolk sac is the sole source of nutrition for the rapidly developing zebrafish.
After this stage, zebrafish can ingest food.

17.4.2 Development of Xt ELISA

To quantitate drug effects in Xt zebrafish, we initially visually counted cancer cells
labeled with CM-DiI tracking dye. However, in pilot experiments, we observed that
similar to mammalian models, after xenotransplant, some colon cancer cells formed
masses, impeding visual counting. Furthermore, dissociating zebrafish and counting
labeled cancer cells was time consuming and less reliable because some cancer cells
were destroyed during processing. Therefore, we decided to develop a quantitative Xt
ELISA using a human cell-specific antibody. For these studies, we assessed several