New Xenograft Method For Studying Leukemia and Drug Response In Turkey Embryos
Authors: Deutsch Varda R , Kay Sigi , Farnoushi Yona , Matalon Erez , Ohayon Tal , Baron Shoshana , Jan Hila , Naparstek Elizabeth , Cipok Michal 
Published: October 2019

Abstract

Background
A widely accepted in vivo model for studying leukemia and its treatment is the highly immune-deficient mice NOD/SCID (b2M-/- or rag-/-). While this model is powerful and recapitulates the phenotypes of blood malignancies in vivo. it is costly and complex, requiring 1-2 months to establish engraftment and the mice are susceptible to spontaneous neoplasms. For these and other reasons the testing of new drugs on leukemia is primarily performed in vitro. The development of antileukemia therapies could be facilitated by a rapid and cost-effective in vivo system for evaluating human leukemia growth and its response to new drugs. Additionally, the treatment of relapsed or refractory disease could be individually tailored by this rapid and cost-effective in vivo system by evaluating patient's cells response to new agents. Turkey embryos are inexpensive, require no maintenance, are larger than chicks are more easily manipulated and have a more robust engraftment (Grinberg I, et al, Leuk Res, 2009; 33:1417-26). We recently described this new in-vivo system for studying multiple myeloma in the pre-immune turkey embryo (Farnoushi, Y., et al.,Br J Cancer, 2011; 105:1708-18). We now demonstrate application of this rapid alternative xenograft system for the preclinical assessment of leukemia growth and therapy.

Methods
BCR/Abl+ human leukemia lines K562 or LAMA-84 c-Kit+ CHRF 4288 and fresh patient cells were injected into turkey egg chorioallantoic membrane (CAM) veins. Cell injections were performed on day embryonic day E11as previously optimized (Farnoushi, Y., et al.,as above). To determine the engraftment of human AML cells on E19-23, in hematopoietic tissue, the engraftment of human AML cells in the BM was detected in BM by flow cytometry (FC) using anti-human CD71 for LAMA and K562, anti- human CD33 for CHRF and fresh leukemia samples. Engraftment in bone marrow (BM) and other organs was also monitored using Quantitive real time PCR (Q-PCR) comparing the amount of genomic human to the amount of avian DNA and number of human cells / avian cells in BM. Drug response was tested by IV injection of therapeutic range doses of Imatinib (Glivec ®) and Doxorubicin, 48H after cell grafting, at drug levels precalibrated to be non-toxic to the developing embryo by LD50 and BM cell viability compared to control. Six days later (E19) the embryos were sacrificed and the BM collected for FC and hematopoietic and non-hematopoietic tissues for molecular analysis.

Results
The optimal treatment and readout times were resolved by injecting cells on E11 and determining the kinetics of leukemia cell engraftment in the BM on E15, E18, and E23 in BM and liver. The highest engraftment level in the BM bone marrow (BM) and liver of lines tested was detected at E18 by Q-PCR, and FC in more than 90% of the injected embryos. The average engraftment (±s.d.) in the BM after one week was 4.6%+0.75 K562, 5.16%+2.15 LAMA-84, 7.65%+1.15 CHRF-4288 ( n=7-12 per group) and 2.5% fresh leukemia cells was detected by FC. Q-PCR results were similar to those of FC. Imatinib toxicity testing revealed 100% survival of embryos with no BM toxicity on embryos treated on E13 with doses similar to a human therapeutic dose, up to 0.75 mg/egg. Treatment of embryos with 100 ug Doxorubicin was previously shown to be not toxic to the embryos (Taizi M et al. Exp Hematol 2006; 34:1698–708). A single dose of 0.75 mg Imatinib/embryo dramatically reduced engraftment in BM and several other organs of all 3 AML cell lines or fresh patient leukemia cells. A similar effect was also obtained by a single dose Rx 100ug Doxorubicin. Treatment of a single dose of 0.75 Imatinib mg/embryo 48H after injecting ARH-77 (multiple myeloma) had no effect on cell engraftment. Treatment with a single non toxic dose of Revlimid as previously described (Farnoushi, Y., et al. as above) eliminated engraftment of ARH77 cells, clearly demonstrating the specificity of the drug treatments.

Conclusions
The results presented demonstrate the potential utility of a practical avian embryo model for testing drug activity in vivo. With further work the turkey embryo may provide a new xenograft in vivo method for studying the biology of leukemia engraftment, and for rapidly and affordably testing leukemia therapies. This system may provide a new platform for developing individualized patient screening for response or resistance to particular therapeutic agents.

Disclosures:
No relevant conflicts of interest to declare.