Biomedical Engineering Reference
In-Depth Information
17.3.13 Vertebrate Animal Care and Safety
The Office of Laboratory Animal Welfare (OLAW), National Institutes of Health
(NIH), has approved our Animal Welfare Assurance effective through February 2012.
We euthanize zebrafish of all ages by overexposure to tricaine methanesulfonate.
These procedures are consistent with the American VeterinaryMedical Association's
(AVMA) Panel on Euthanasia.
17.4 RESULTS
In order to develop a quantitative zebrafish xenotransplant colon cancer model for
drug screening, we initially showed that two colon cancer cell lines, Colo320 and
SW620, proliferated in zebrafish. Next, we theorized that a human cell-specific
antibody that labels normal and cancer cells but has limited cross-reactivity with
zebrafish could be used to develop a whole animal ELISA for drug screening (Herlyn
and Koprowski, 1988; Seng et al., 2004; Li et al., 2008, 2009; McGrath and Li, 2008;
Haldi et al., 2009). Using whole mount staining, we identified two human cell-specific
antibodies, survivin and XIAP (Cell Signaling, Danvers, MA), for ELISA develop-
ment. Specificity was validated by (1) limited cross-reactivity with zebrafish tissue,
confirmed by whole mount immunostaining, and (2) a linear relationship between
signal and number of Xt zebrafish per well. This whole animal ELISA quantitated
signal from antigens constitutively expressed on xenotransplant human cells, fol-
lowed by reaction with a HRP-conjugated secondary antibody. Chemiluminescent
signal was then quantitated using a conventional microplate reader. Chemilumines-
cent light units produced in the reaction correlated with number of Xt cells. Next, we
used the Xt zebrafish ELISA to assess effects of four FDA-approved colorectal cancer
drugs, 5-FU, oxaliplatin, camptothecin, and leucovorin, and one drug combination,
5-FU
leucovorin. Inhibition of Xt cell proliferation by cancer drugs resulted in
decreased signal. The overall prediction success rate was 80% (4/5) and 60% (3/5) for
Colo320 and SW6320 cells, respectively, indicating good correlation with results in
humans. This novel approach is rapid, sensitive, quantitative, and amenable to
automation for drug screening. Because cancer patients are frequently treated with
multiple drugs, combination drug treatment by direct addition to fish water is a
significant advantage compared to drug treatment in mammalian models.
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17.4.1 Human Colon Cancer Cell Line Xt in Zebrafish
To generate a zebrafish xenotransplant colon cancer model, we initially injected 2 and
3dpf zebrafish with 1500 CM-DiI-labeled human colon cancer Colo320 and SW620
cells into the yolk sac. In pilot experiments, we observed that Colo320, a colon cancer
cell line derived from a moderately undifferentiated colon adenocarcinoma, metas-
tasized and proliferated in zebrafish and exhibited an infiltrative growth pattern
(Fig. 17.2). SW620 cell line, derived from a grade 3-4 colon adenocarcinoma that had
metastasized to lymph nodes, was tumorigenic in nude mice. In zebrafish, Xt SW620
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