Zebrafish Xenotransplant Cancer Model for Drug Screening - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 220

human antibodies, includingHLA andHLA-DR (Invitrogen, Carlsbad, CA), survivin,
XIAP, and Bax (Cell Signaling Technology, Danvers, MA), and Keap1 (Santa Cruz
Technology, Santa Cruz, CA). Three dpf zebrafishwere microinjected with 1500 CM-
DiI-labeled Colo320 cells and fixed at 3 dpx. Whole mount immunostaining was
performed using human survivin antibody, followed by staining with FITC-conjugated
secondary antibody. Uninjected zebrafish and Xt zebrafish with no primary antibody
staining were used as negative controls. CM-DiI-labeled Xt cancer cells were
visualized in the rhodamine channel and antibody-stained cells were visualized in
the FITC channel using a Zeiss Stemi SV11 Apo upright fluorescence microscope
equipped with a Zeiss AxioCam. CM-DiI-labeled cells were visualized in the
rhodamine channel and human antibody-stained zebrafish were visualized in the
FITC channel using a Zeiss fluorescence microscope. Human survivin antibody
stained Colo320 colon cancer cells in zebrafish, but exhibited limited cross-reactivity
with whole zebrafish (data not shown).
We then performed the Xt ELISA as described in Section 17.3; signals from both
survivin and XIAP were adequate for assay development. Survivin and XIAP,
members of the inhibitors of apoptosis family of proteins (IAP), are constitutively
expressed in human cells and expression level has been shown to be elevated in many
human cancers (Deveraux et al., 1998; Altieri and Marchisio, 1999; Deveraux and
Reed, 1999; Tamm et al., 2000; Kasof and Gomes, 2001). In addition, survivin and
XIAP have previously been validated as cancer drug targets (Olie et al., 2000;
Grossman et al., 2001). Because survivin generated a higher signal/noise ratio than
XIAP, we used it for Xt ELISA development.

17.4.3 Linear Relationship Between
Chemiluminescence Signal and Number of Xt
Zebrafish in Microwells

We next established a linear relationship between chemiluminescence signal and
number of Xt zebrafish per well. Two dpf zebrafish were microinjected with 1500
Colo320 cells and fixed 3 days post injection for whole zebrafish ELISA processing.
Using relative chemiluminescence units (RCU), a linear relationship was observed
between chemiluminescence signal and number of Xt zebrafish per well (data not
shown).
Using same stage zebrafish processedwith andwithout primary antibody, we also
determined that S/N

2.3, which indicated that the assay discriminated Xt zebrafish
signal from background.

¼

17.4.4 Effects of FDA-Approved Drugs on
Xenotransplant Colon Cancer Cell Proliferation

To validate Xt ELISA for drug screening, we assessed effects of four FDA-approved
colorectal cancer drugs, 5-FU, oxaliplatin, camptothecin, and leucovorin, and one
drug combination, 5-FU þ leucovorin (Table 17.2), on human colon cancer cell in Xt