A New Preclinical In-Vivo System for Studying and Testing Chemotherapy on Human Hematological Malignancies.

Abstract

Transplantation of cells and tissues to the embryonic chick, especially to its chorioallantoic membrane (CAM), has been used for many years to study the vascularization and growth of solid mammalian tumors. However, it has not previously been used to study mammalian hematological malignancies. We have now examined the potential of the pre-immunocompetent chick embryo as a host for human leukemias. Two human myeloid leukemia lines, K562 and DAMI, and the T-cell ALL line Jurkatt were engineered to stably express eGFP. K562 erythroleukemia cells introduced either by injection into the yolk sac or by layering onto the CAM, showed inconsistent engraftment. By contrast, K562 and DAMI cells engrafted in 100% of embryos following intravascular (iv) or intra-amniotic injection. The engraftment of these human leukemia cell lines following iv injection was rapid, appearing as soon as 7 days after injection into the embryos. This novel methodology is quite rapid compared to the standard xenografting model of SCID mice, which requires 6–8 weeks for engraftment of human leukemia after intravenous injection. Engraftment was visualized in-vivo as whitish tumor nodules in the avian chorioallantoic membrane (CAM). eGFP expression enhanced the detection of the tumors in terms of both speed and sensitivity. Leukemia cells were consistently detected by PCR amplification of human-specific α-satellite DNA sequences in the hematopoietic organs: bone marrow (BM), liver and spleen, as well as in the brain. DAMI cells also consistently engrafted in the embryos' hematopoietic organs, but at lower levels than K562. In contrast, Jurkatt cells did not show consistent engraftment. K562 cells were immunopositive for human CD45 and human mitochondrial and lysosomal antigens in histological sections of tumors in the CAM. K562 cells were also detected by immunostaining of peripheral blood smears from engrafted embryos. In one case, human cells were detected by PCR in the BM of the embryos one week after injection of a patient sample of fresh lymphoma cells. Engraftment levels of iv-injected K562 and DAMI were increased more than 100-fold (real-time qPCR) by using avian eggs with longer incubation periods than the chick to extend the graft period to 14 days. The potential use of this system for testing cytotoxic therapy was also investigated. Dramatic and consistent regression of tumors in the CAM was induced by a single dose of doxorubicin administered to K562-engrafted embryos. These results demonstrate for the first time that the chick embryo can be used for study of human hematological malignancies. This new platform has the advantages of significantly lower cost and the lack of requirement of sentient animals when compared to the other in-vivo models of transplantation to SCID mice and large mammalian embryos. In addition, grafting human blood malignancies into the chick embryo has the potential to provide a rapid assay for testing chemotherapies, which could significantly reduce drug development time and expense.