Page 165 - Haematologica March 2020

Modeling chronic myeloid leukemia in zebrafish
the PB cell count compared with approximately 0.01±0.00% in WT adult zebrafish (n=55) (Online Supplementary Table S1). This was similar to the “Ph-posi- tive eosinophilic/basophilic CML” described by Goh et al.24 Large numbers of lymphocytes accumulated in the PB in 8 of 77 (10.39%) CML-like Tg(hsp70:p210BCR/ABL1) adult zebrafish, accounting for >5% of the PB cell count com- pared with around 1.80±0.23% in WT adult zebrafish (n=55) (Online Supplementary Table S1), similar to the lym- phocytosis25 observed in CML patients. Thrombocytosis26 is present in approximately half of all newly diagnosed CML patients. Thirteen of the 77 (16.88%) CML-like Tg(hsp70:p210BCR/ABL1) adult zebrafish presented with thrombocytosis, with platelets accounting for >0.5% of the PB cell count compared with around 0.11±0.04% in WT adult zebrafish (n=55) (Online Supplementary Table S1). Histological examination of the spleen in CML-like Tg(hsp70:p210BCR/ABL1) demonstrated expansion of the splenic red pulp, predominantly by granulocytic myeloid cells (Figure 3D). In addition, the morbidity of CML-like disease in heat-shock-treated Tg(hsp70:p210BCR/ABL1) adult zebrafish was higher than in non-induced Tg(hsp70:p210BCR/ABL1) adult zebrafish. The ratios of indi- viduals in CML-CP2 and CML-AP were increased among heat-shock-treated Tg(hsp70:p210BCR/ABL1) compared with untreated Tg(hsp70:p210BCR/ABL1) adult zebrafish (Figure 3E). This result suggests that overexpression of BCR/ABL1 is an important factor in accelerating the course of CML.
Tg(hsp70:p210BCR/ABL1) transgenic fish displayed abnormal myeloid cell expansion resulting from increased proliferation and inhibition of apoptosis
The above results indicated that myeloid cells accumu- lated in Tg(hsp70:p210BCR/ABL1) fish from the embryonic stage to the adult, which could be caused by accelerated proliferation or reduced apoptosis. To clarify the cellular mechanisms responsible for myeloid cell expansion in Tg(hsp70:p210BCR/ABL1) fish, we monitored myeloid cell pro- liferation and death by BrdU incorporation and TUNEL assay, respectively. BrdU incorporation was significantly increased in Tg(hsp70:p210BCR/ABL1) larvae and adult KM compared with WT controls, indicating that myeloid cell expansion in Tg(hsp70:p210BCR/ABL1) fish was the result of increased proliferation (Figure 4A, B, E and F). However,
myeloid cell apoptosis was also significantly decreased in Tg(hsp70:p210BCR/ABL1) larvae and adult KM compared with WT controls, suggesting that the expansion of myeloid cells Tg(hsp70:p210BCR/ABL1) was also caused by reduced apoptosis (Figure 4C, D, G and H).
Tg(hsp70:p210BCR/ABL1) transgenic cells with induced CML-like disease were transplantable
To determine the aggressiveness of the leukemia induced by Tg(hsp70:p210BCR/ABL1) activity, whole KM blood cells from Tg(hsp70:p210BCR/ABL1) fish were trans- planted into γ-irradiated WT adult hosts and the resulting fish were tested to determine if the CML-like phenotype developed in the Tg(hsp70:p210BCR/ABL1) fish could be trans- planted into the WT fish. We used 1-year old Tg(hsp70:p210BCR/ABL1-lyz:DsRed) CML-like donors and Tg(lyz:DsRed) control donors, in which the granulocytes were marked by red fluorescence. Each irradiated WT fish received 0.2 million KM blood cells from donors and were then raised under normal conditions. All four sur- viving recipients of Tg(hsp70:p210BCR/ABL1-lyz:DsRed) CML- like donor cells developed CML-like disease within 2-3 weeks of transplantation with whole KM blood cells, indicated by infiltration of DsRed+ granulocytes into the periphery (Figure 5A) and the robust expansion of myeloid cells in both the PB and KM (Figure 5B). In con- trast, no control fish showed signs of a CML-like pheno- type. We collected leukemia cells from the PB and KM and showed that these inflated cells were BCR/ABL1+ by PCR (Figure 5C). We concluded that the myeloid cells that accumulated in Tg(hsp70:p210BCR/ABL1) fish could pro- liferate autonomously and could cause CML-like disease in a WT host.
Tg(hsp70:p210BCR/ABL1) transgenic leukemic model responded to chemotherapeutic drug treatment
Recent studies demonstrated that zebrafish shares 82% of disease-associated targets and numerous drug metabo- lism pathways with humans.12 To determine if the phar- macological mechanism in Tg(hsp70:p210BCR/ABL1) trans- genic zebrafish was also conserved compared with CML patients, we treated the WT and Tg(hsp70:p210BCR/ABL1) embryos with the widely used anti-CML drugs, imatinib, dasatinib, and bosutinib, to the maximum teratogenic doses, with DMSO as a placebo (Online Supplementary
Table 1. Hemogram and classification of Tg(hsp70:p210BCR/ABL1) fish at 6-12 months
Classification
WT
Tg(hsp70:p210BCR/ABL1)
Group Number
PercentagesinPB(%) PercentagesinKM(%)
Leukemiacell types
Neutrophils and myeloid precursors
Myeloid precursors and blasts
Blasts
Blasts
0.06 ± 0.04
0.40 ± 0.12
1.75 ± 0.82
99.28§
Myeloid Myelocytes Lymphocytes Blasts precursors
Myeloid precursors
12.56 ± 0.76
12.16 ± 0.92
18.54 ± 1.51†
1.96
Myelocytes Lymphocytes
Normal
CML-CP
CML-AP
CML-BP
55
68
8
1
1.68 ± 0.39
2.99 ± 0.59
19.00 ± 3.95†
0.26
8.09 ± 1.45
8.88 ± 1.24
14.20 ± 3.06
0.13
90.17 ± 1.55
87.73 ± 1.45
65.05 ± 5.86
0.33
4.41 ± 0.56
3.37 ± 0.51
10.36 ± 1.32‡
90.25§
46.89 ± 1.29
50.48 ± 1.39*
36.29 ± 2.98
4.56
36.14 ± 1.21
34.00 ± 0.98
34.82 ± 2.47
3.23
The White blood cell counts were obtained by identifying at least 500 cells per kidney marrow (KM) preparation and at least 1500 cells per peripheral blood (PB) preparation.The percentages were indicated by mean ± SEM. Adult Tg(hsp70:p210BCR/ABL1) fish characterized by myelocytes > 15% in PB or > 50% in KM were divided into CML-CP, blasts usually account for < 2% in PB or < 5% in KM.Adult Tg(hsp70:p210BCR/ABL1) fish characterized by blasts > 10% in PB or KM were divided into CML-AP.Adult Tg(hsp70:p210BCR/ABL1) fish characterized by blasts > 90% in both PB and KM were divided into CML-BP. * Indicates myelocytes in PB or KM increased > 15% or 50%. † Indicates myeloid precursors in PB or KM increased > 10% or >15%. ‡ Indicates blasts in PB or KM increased > 10%. § Indicates blasts in PB or KM increased > 90%.
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