Establishment of CD34 + hematopoietic stem cell-derived xenograft model of hyperleukocytic acute myeloid leukemia

Abstract

Background: Hyperleukocytic acute myeloid leukemia (HLL) is marked by high early mortality and presents significant therapeutic challenges. Research on HLL is still in its infancy, and comprehensive development of patient-derived xenograft (PDX) models, especially CD34 + hematopoietic stem cell-derived models, remains limited.

Methods: We evaluated the establishment of the HLL model through blood examinations, smear analysis, bone marrow biopsy, flow cytometry, and mutation analysis. Correlation between survival times in mice and patients was assessed using linear regression.

Results: In the HLL PDX mouse model, leukocyte counts could reach up to 37.35^10⁹/L, and immunophenotyping revealed the presence of hCD45+, hCD15+, and hCD33 + cells in both peripheral blood (PB) and bone marrow (BM) following inoculation with PB-derived cells for the establishment of the HLL PDX model. Similar results were observed with cells derived from the patient's BM. In the CD34 + hematopoietic stem cell-derived xenograft model, extensive infiltration of CD34 + cells into the BM, liver, and spleen was observed. Additionally, human WT1 and NRAS mutations were identified in the liver, spleen, and BM of the mice. A comparative analysis of multiple experiments revealed that shorter survival times were observed in mice receiving a higher irradiation dose of 2.5 Gy and a greater number of cells derived from PB. Additionally, shorter survival times were observed in model mice injected with cells carrying NRAS, DNMT3A, FLT3, or NPM1 gene mutations. Correlation analysis indicated that the survival times of the mice were significantly associated with the survival status of the patients.

Conclusions: We successfully established a CD34 + hematopoietic stem cell-derived xenograft model of HLL, providing a valuable tool for mechanistic research, drug screening, individualized therapy, and precision medicine.

Trial registration: Not application.

Keywords: B-NSG mice; CD34 + hematopoietic stem cell; Hyperleukocytic acute myeloid leukemia; Patient-derived xenograft model.

Fig. 1

Establishment of the model of HLL with PBMCs of patients. (a) The HLL cells were collected from HLL patients using a Fresenius COM.TEC machine, and then the PBMCs were separated with a Ficoll kit and cultured. After irradiation at day 0, the B-NSG mice were treated with 0 Gy (n = 5) or 2.5 Gy (n = 5) χ-rays for total body irradiation and intravenously injected with harvested from PBMCs of human HLL cells from Patient #1 mixed with granulocyte colony-stimulating factor. Every mouse was injected with 1.5 × 10⁷ cells within 24 h of irradiation at day 1. Blood samples were collected to test the WBC counts at D3, D9, D12, D17, D21 and D25. The immunophenotypes of leukemia cells, including hCD45⁺, hCD15⁺ and hCD33⁺, were detected by flow cytometry on days 9, 7, and 25. (b) The body weights were analyzed using GraphPad Prism software (version 8.0) with parametric unpaired t tests. ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. (c) Survival analysis. (d) Immunophenotype analysis of leukemia cells. ^*P < 0.05, ^**P < 0.01. (e-h) Routine blood tests with a Coulter STKS automated blood cell analyzer. e, WBC counts. f, RBC counts. g, PLT counts. h, HGB content. (i) Blood smear analysis were stained via Wright’s staining and observed with a microscope under an oil immersion lens to measure the ratio of leukemia cells. (j) Detection of the immunophenotype in peripheral blood by BD FACS Verse flow cytometry. HLL cells were stained with human anti-CD45-PerCPcy5.5, anti-CD15-FITC, and anti-CD33-PE antibodies. (k) Bone marrow smear analysis. (l) Detection of the immunophenotype in bone marrow by flow cytometry at day 25. (m) Histopathological analysis of leukemia cell infiltration into liver and spleen tissues. Magnification: ×400

Fig. 2

Construction of the HLL model with BM-derived cells of patients. (a) On day 0, B-NSG mice were treated with 0 Gy (n = 5) or 1.5 Gy (n = 6) χ-rays for total body irradiation and intravenously injected with harvested BM-derived cells from a human HLL patient (Patient #3) at day 1, then tested the change in body weight at intervals. (b) Survival analysis. (c) Blood smear analysis. PB, peripheral blood. (d) Bone marrow smear analysis. BM, bone marrow. (e) Immunophenotype analysis of leukemia cells in peripheral blood at day 30 by flow cytometry. ^**P < 0.01. (f) Detection of the immunophenotype in bone marrow by flow cytometry after mice were sacrificed

Fig. 3

Construction of a CD34⁺ hematopoietic stem cell-derived xenograft model of HLL. (a) The workflow of model construction. The B-NSG mice were treated with 1.5 Gy χ-rays for total body irradiation on day 0 and intravenously injected with isolated control cells (n = 5) or 1.5 × 10⁶ CD34⁺ cells using magnetic beads (n = 5) from Patient #2 (WT1 and NRAS mutation) mixed with G-CSF within 24 h of irradiation. (b) Detection of immunophenotype in peripheral blood by flow cytometry at day 9. (c) Blood smear analysis at day 9. (d) The analysis of body weight. (e-h) Routine blood tests. e, WBC. f, RBC. g, PLT. h, HGB. (i) Blood smear analysis at day 25. (j) Detection of immunophenotype in peripheral blood at day 25. The cells were stained with human anti-CD45-PerCPcy5.5 and anti-CD34-APC antibodies. (k) Bone marrow smear analysis at day 25. (l) Detection of immunophenotype in bone marrow at day 25. (m) Immunohistochemical analysis of leukemia cell infiltration into bone marrow, liver and spleen tissues. The tissues were stained with antibodies against human anti-CD45⁺. BM, bone marrow. (n) Detection of the positivity rate of the WT1 gene using a fluorescence quantitative PCR instrument in tissues of mice in the CD34⁺ group. (o) Detection of NRAS mutation in tissues of mice in the CD34⁺ group. Green represents base A, red represents base T, blue represents base C and black represents base G. The green arrow represents a mutation in BM. BM, bone marrow

Fig. 4

Summary of HLL models constructed with B-NSG mice. (a) List of experiments for HLL model establishment in B-NSG mice intravenously injected (i.v.) with patient-derived cells at D1. PB, peripheral blood. BM, bone marrow. The survival months were calculated from leukapheresis to presentation as of March 2021. (b) Analysis of the survival time in mice at different irradiation dosages. (c) Analysis of the survival time in mice with different injection cell counts from the PB of patients. (d) Analysis of the survival time in mice injected with cells derived from PB and BM with irradiation of 0–1.5 Gy. (e) Analysis of survival time in mice with different gene mutations. (f) Correlation analysis was performed to characterize the correlation between the survival time in mice and in patients by a linear regression model with variable selection “entered” with SPSS software (version 26.0). ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001. ns, no significance