An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Bone marrow cells were obtained from patients with acute leukemia who had marrow study done at the time of diagnosis. Patients gave informed consent for additional aliquot of the marrow aspirate to be used for research purposes in accordance with the ethical guidelines of Singapore General Hospital. Patients with AML were diagnosed using the French-American-British (FAB) classification system; subtype M1 (patients Leu 32 and BMI 1786), M4 (patient BMI 1808), M5 (patient BMI 1690), and M5a (patients Leu 14, Leu 29, and Leu 33). Bone marrow cells were processed using ficoll density gradient centrifugation to isolate mononuclear cells. Cells were frozen and stored in liquid nitrogen until use.

NOD-scid Il2rγ ^null (NSG) mice were purchased from The Jackson Laboratory. All mice were bred and kept under specific pathogen-free conditions in Biological Resource Centre, Agency for Science, Technology and Research, Singapore. All experiments and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Agency for Science, Technology and Research, Singapore, in accordance with the guidelines of the Agri-Food and Veterinary Authority and the National Advisory Committee for Laboratory Animal Research of Singapore.

For primary xenotransplantation, BM mononuclear cells were depleted of CD3⁺ cells using PE selection kit (STEMCELL Technologies) upon labeling with PE-conjugated mouse anti-human CD3 antibody (Biolegend) according to manufacturer’s instructions. One to 3-day-old NSG pups were sub-lethally irradiated at 1Gy and engrafted with 8.7 × 10⁴–7.9 × 10⁵ of CD3-depleted AML mononuclear cells from seven AML patients (Leu 14, Leu 29, Leu 32, Leu 33, BMI 1690, BMI 1786, and BMI 1808) via intrahepatic injection route. As described previously [17], intrahepatic inoculation was performed by maintaining the irradiated NSG pup in posterior position (face-up) between the thumb and index finger to expose the abdomen and the liver, which is visible on the right flank. An insulin syringe loaded with 50 μl of cell mixture was then held perpendicular to the pup body by the other hand and inserted straight into the pup liver with bezel facing upwards to release the contents. Mice were bled submandibularly to evaluate the engraftment of AML cells in peripheral blood at 2–4-week intervals after week 6 post-engraftment using flow cytometry. Engraftment of AML cells in BM and spleen was evaluated at endpoint (week 12–20 post-engraftment) using flow cytometry. Cells from BM and spleen were pooled and used for serial xenotransplantation.

Secondary and tertiary xenotransplantation were evaluated on pooled BM and spleen cells from Leu 14. For serial xenotransplantation, CD34⁺ cells from pooled BM and spleen cells were purified with either fluorescence-activated cell sorting (FACS) using FACSAria (BD Biosciences) after labeling with fluorochrome-conjugated mouse anti-human CD45 (Biolegend) and anti-human CD34 (BD Biosciences) monoclonal antibodies or by magnetic-sorting using CD34 positive selection kit (STEMCELL Technologies) according to manufacturer’s instructions. The purity of human CD34⁺ cells was > 95% after FACs or magnetic-sorting. Cell number range from 1 × 10⁴–5 × 10⁵ cells were injected into irradiated NSG recipients. Xenotransplantation in NSG adult mice (6-week-old) was performed via tail-vein intravenous injection after sublethal irradiation at 2.5Gy.

Peripheral blood was collected submandibularly from mice in EDTA tube (Greiner Bio-One). Red blood cells (RBCs) were lyzed using RBC lysis buffer (Life Technologies) prior to flow cytometry analysis. For spleen and BM, tissues were meshed and cell contents from femur and tibia were flushed using a syringe, respectively. Cell debris was removed by passing contents through 70 μm cell strainer (Thermo Fisher Scientific). RBCs were further lyzed and contents passed through 70 μm cell strainer prior to flow cytometry analysis and storage.

Live immune cells from peripheral blood, spleen, and BM were determined by staining with live/dead fixable blue dead cell stain kit (Life Technologies) for 30 min prior to cell-specific marker labeling. Cells were labeled with anti-human CD34 (clone 581; BD Biosciences), anti-human CD3 (UCHT1; Biolegend), anti-human CD56 (MEM-188, Biolegend), anti-human CD14 (63D3; Biolegend), anti-human CD19 (SJ25C1; BD Biosciences), anti-human CD117 (104D2; Biolegend), anti-human CD38 (HB-7; Biolegend), anti-human CD33 (WM53; BD Biosciences), mouse CD45.1 (A20; BD Biosciences), anti-human CD8 (SK1; Biolegend), anti-human CD4 (SK3; BD Biosciences), and anti-human CD45 (HI30; Biolegend) monoclonal antibodies for 30 min at room temperature. After incubation, cells were washed and resuspended in FACs buffer containing phosphate buffered saline (PBS), 0.2% bovine serum albumin (GE Healthcare Life Sciences), and 0.05% sodium azide (Merck) for flow cytometry data acquisition. Data was acquired using a LSR II flow cytometer (BD Biosciences) with FACSDiva software, and analysis was performed using FlowJo software (version 10; Tree Star Inc). Absolute count of cells in peripheral blood was determined using CountBright™ Absolute Counting Beads (Thermo Fisher Scientific).

Multiple organs including brain, heart, lungs, liver, kidneys, forelimbs/hind limbs, and spleen were removed from sacrificed mice at endpoint. The organs were fixed in 10% formalin, embedded in paraffin wax, processed to obtain 5 μm sections, and subjected to Hematoxylin & Eosin (H&E) (Thermo Fisher Scientific) or immunohistochemistry staining following established protocols. Primary antibodies including anti-human CD45 (cat# ab781), anti-human MPO (cat# ab134132), and anti-human c-kit (cat# ab32363) monoclonal antibodies were purchased from Abcam and used for immunohistochemistry. The primary antibody was detected using Rabbit specific IHC polymer detection kit HRP/DAB (AbCam) or Mouse on Mouse Polymer IHC Kit (AbCam) following manufacturer’s instructions. Histopathological images were acquired using Axio Scan. Z1 slide scanner (Zeiss) and analyzed using Zen 2 (blue edition; Zeiss) software.

Sorafenib tosylate (Nexavar®; Bayer Healthcare Pharmaceuticals Inc) and Regorafenib (Stivarga®; Bayer HealthCare Pharmaceuticals Inc.) tablets were crushed and dissolved in isotonic saline water (B. Braun Medical Inc). Successfully engrafted mice with more than 30 human CD45 cells per microliter of blood (between week 12 and 16 post-engraftment) were randomly assigned to either untreated Regorafenib or Sorafenib treatment groups. Mice were given a daily dose of Regorafenib (5 mg/kg body weight) or Sorafenib (10 mg/kg body weight) via oral gavage and monitored for 1 month.

Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software Inc). Pairwise comparison was performed using two-tailed Mann Whitney U test. P value less than 0.05 is considered statistically significant. All data are represented as mean ± standard error of mean (SEM).

It is well documented that flow cytometric analysis using CD45/SSC gating can distinguish leukemic blast cells (low CD45 expression) from normal hematopoietic cell types (high CD45 expression) [18]. As shown by previous publications, CD33 (myeloid cell marker), CD34 (primitive stem cell marker), CD117 (c-kit receptor), and CD38 (cell activation marker) are key AML leukemic blasts-associated markers [19‐21]. To confirm the presence of AML leukemic blasts in seven individuals with AML, BM mononuclear cells were isolated, labeled with anti-human CD34, CD117, CD38, CD33, and CD45 monoclonal antibodies, and analyzed using flow cytometry (Additional file 1: Figure S1a and Table 1). Out of the seven individuals with AML, samples from five individuals contained more than 80% leukemic blasts based on CD45^lo expression (Table 1). Although all samples expressed CD33, only three samples (Leu 14, BMI 1690, and BMI 1808) expressed CD33 within the CD45^lo compartment, suggesting that Leu 14, BMI 1690, and BMI 1808 are positive for AML leukemic blasts (Additional file 1: Figure S1a). In line with previous studies, these CD45^lo AML leukemic blasts from Leu 14, BMI 1690, and BMI 1808 expressed varying levels of CD34, CD38, and CD117 [19‐21].

Delivery of fetal liver hematopoietic stem cells via intrahepatic injection into NSG newborn pups is routinely used in the humanization of NSG mice but have yet to be described in AML xenotransplantation [16, 22]. To evaluate the engraftment of AML in NSG mice using intrahepatic injection, CD3-depleted mononuclear cells derived from seven AML patients were injected intrahepatically into sublethally irradiated NSG newborn pups (Fig. 1, Additional file 1: Figure S1b and c, and Table 1). The number of cells injected range from 8.7 × 10⁴–7.9 × 10⁵ per NSG newborn pup (Table 1). The mice were monitored for survival, and peripheral blood was collected submandibularly at 2–4 weeks intervals after week 6 post-engraftment to determine the engraftment level using flow cytometry. The levels of AML engraftment were calculated based on the proportion of human CD45⁺ cells relative to total CD45⁺ cells (human and mouse CD45). Phenotypic analysis revealed that NSG recipient mice injected with Leu 14, BMI 1690, or BMI 1808 were successfully engrafted, with more than 10% AML engraftment detected in the peripheral blood (Fig. 1a and Additional file 1: Figure S1b).

Despite injected with similar number of cells, engraftment of Leu 14 (range 6.0–10.4%) was detected as early as week 6 post-engraftment as compared to BMI 1690 (0.1–0.2%; week 6 post-engraftment) and BMI 1808 (0.1–0.3%; week 8 post-engraftment; data not shown). The frequency of periphery AML cells in Leu 14 and BMI 1690 recipient mice increased with time, with more than 50% AML cells detected at endpoint (week 12–14 post-engraftment; Fig. 1a). In contrast, the frequency of periphery AML cells in BMI 1808 recipient mice persisted around 13–16% at endpoint (week 18 post-engraftment; Fig. 1a).

Consistent with peripheral blood, high frequency of AML cells were detected in spleen (range 16.6–99.1%) and BM (range 63.4–99.9%) of Leu 14, BMI 1690, and BMI 1808 recipient mice at endpoint (Fig. 1b, c, Additional file 1: Figure S1b). These primary engrafted cells expressed similar AML-associated markers to that of primary cells (Additional file 1: Figure S1a). Expression of normal hematopoietic cell markers such as CD3, CD4, CD8, CD19, and CD56 was absent (data not shown). The expression profile of AML cells differs among BM, spleen, and peripheral blood (Additional file 2: Figure S2). In comparison, BM harbors the highest frequency of human CD45⁺ cells, accompanied by the greatest level of CD117 expression, as opposed to spleen and peripheral blood (Additional file 2: Figure S2).

Except for mild enlargement of spleen, no solid tumors were observed in the recipient mice at endpoint (data not shown). Histological assessment by H&E and immunohistochemical staining of human CD45 revealed that AML cells infiltrated into multiple organs such as kidneys, lungs, liver, spleen, and BM, but not the brain (Fig. 1c). Collectively, these results demonstrated that the construction of AML mouse model via intrahepatic injection of patient-derived mononuclear cells into NSG newborn pups was successful and recapitulated human leukemogenesis.

Acute myeloid leukemia initiating cells or stem cells are believed to be restricted in the CD34⁺ compartment [2, 8]. To examine if CD34⁺ cells have greater engraftment capacity in our model, cells pooled from the spleen and BM of primary engrafted mice were sorted for CD34⁺ and CD34⁻ using flow cytometry. Proliferative capacity and colony-forming ability of CD34⁺ cells were assessed in vitro using colony-forming assay. Sorted CD34⁺ fraction gave rise to significantly more colonies (~ 10 fold more) compared to CD34⁻ fraction at 3-weeks post-assay (Fig. 2a), indicating that CD34⁺ cells are more proliferative than CD34⁻ cells. In vivo, secondary NSG recipient mice engrafted with CD34⁺ cells exhibited a more severe pathological outcome compared to mice engrafted with CD34⁻ cells at endpoint (week 10 post-engraftment). While no solid tumors were detected, spleen enlargement and massive accumulation of green soft tissue resembling soft tissue sarcoma with increased vascularization at the trunk, abdomen, limbs, and kidneys were observed in CD34⁺ secondary engrafted NSG mice (Fig. 2b and Additional file 3: Figure S3a). Further histological assessment using H&E and an immunohistochemical panel staining for myeloid sarcoma [23] which included human CD45, myeloperoxidase (MPO), and CD117 revealed massive infiltration of AML cells into multiple organs and confirmed that the soft tissue mass was myeloid sarcoma composed of AML leukemic blasts (Additional file 3: Figure S3b).

Although AML cell engraftment was detected in all secondary NSG recipient mice, engraftment levels were significantly higher in mice engrafted with CD34⁺ than CD34⁻ cells in peripheral blood, spleen, and BM (Fig. 2c–e). The frequency of AML cells was ~ 80 fold, 25 fold, and 100 fold greater in CD34⁺ engrafted mice compared to CD34⁻ engrafted mice in the peripheral blood, spleen, and BM, respectively (Fig. 2c, d). This translated to ~ 18,000 fold, 22,000 fold, and 9000 fold greater in absolute AML count in peripheral blood, spleen, and BM, respectively (Fig. 2e). Phenotypically, except for the frequency of human CD45⁺ cells, there was no significant difference in the frequency of CD34⁺ in the spleen and BM between CD34⁻ and CD34⁺ engrafted mice (Fig. 2f). Taken together, these results indicate that CD34⁺ cell has greater proliferative and engraftment capacity as compared to CD34⁻ cells.

It is not known if AML cells delivered intrahepatically populate peripheral blood, spleen, or BM first. To address this, we sacrificed secondary NSG recipient mice engrafted with CD34⁺ cells early at 4 weeks post-engraftment. Immunophenotypic analysis revealed that AML cells repopulated the BM at greater frequency and absolute count followed by spleen and peripheral blood in descending order (Additional file 4: Figure S4). Taken together, these results suggest that AML cells delivered intrahepatically homed to the site of leukemogenesis (BM) first before progressing to spleen and peripheral blood.

Next, we compared the engraftment efficiency of AML cells between our model and existing model utilizing NSG adult mice and intravenous injection. Newborn NSG pups and adult mice (6-week-old) were irradiated sublethally before injection with 1 × 10⁵ sorted CD34⁺ cells from secondary engrafted mice via intrahepatic and tail-vein intravenous route, respectively. The mice were monitored for 20 weeks. Engrafted NSG newborn pups began to exhibit weakness at week 15 post-engraftment and were sacrificed when moribund, while all NSG recipient adult mice survived at week 20 post-engraftment (Fig. 3a). Across multiple time points, significantly greater (~ 27 fold greater at endpoint) AML cell engraftment was detected in the peripheral blood of tertiary NSG recipient newborn pups (Fig. 3b) and this translated to pronounced accumulation of abdominal soft tissue mass and significant spleen enlargement as compared to recipient adult mice (Fig. 3c). The absolute number of human CD45⁺ and CD34⁺ AML cells was significantly greater in the peripheral blood and spleen but not in the BM of NSG newborn pups compared to adult mice (Fig. 3d). Phenotypically, except for the frequency of human CD34⁺ cells in the peripheral blood, there was no significant difference in the frequency of human CD45⁺, CD34⁺, or CD117⁺ cells in the peripheral blood, spleen, and BM between NSG recipient newborn pups and adult mice (Additional file 5: Figure S5). Taken together, these results demonstrate that AML engraftment is more efficient in NSG newborn pups as compared to NSG adult mice.

Stem cell factor receptor (CD117) and its ligand (stem cell factor) have been implicated to play a key role in hematopoiesis and leukemogenesis [24, 25]. Given that CD117 is expressed on leukemic blasts of Leu 14, BMI 1690, and BMI 1808 among the seven AML patients (Additional file 1: Figure S1), we hypothesized that CD117 is essential for successful engraftment (Fig. 1 and Additional file 2: Figure S2). To test this notion, cells pooled from the spleen and BM of primary engrafted mice were magnetically sorted to CD34⁺CD117⁺ and CD34⁺CD117⁻ fractions and evaluated for their proliferative and engraftment capacity (Fig. 4). Colony forming assay in vitro demonstrated that CD34⁺CD117⁺ cells were more proliferative and formed significantly more colonies than CD34⁺CD117⁻ cells after 3 weeks (Fig. 4a). Although peripheral blood engraftment was detected in both CD34⁺CD117⁺ and CD34⁺CD117⁻ NSG recipient mice at week 12 post-engraftment, the frequency and absolute count of AML cells were significantly greater in CD34⁺CD117⁺ NSG recipient mice in a dose-dependent manner (Fig. 4b, c). At week 16 post-engraftment, NSG recipient mice engrafted with 1 × 10⁵ CD34⁺CD117⁺ or CD34⁺CD117⁻ cells were sacrificed to evaluate their engraftment levels and pathological outcomes. Significantly greater engraftment was observed in the peripheral blood, spleen, and BM of CD34⁺CD117⁺ NSG recipient mice (Fig. 4d). Although either CD34⁺CD117⁺ or CD34⁺CD117⁻ cells were used for engraftment, CD34⁻CD117⁻, CD34⁻CD117⁺, CD34⁺CD117⁺, and CD34⁺CD117⁻ subsets were present in all NSG recipient mice (Fig. 4e). Interestingly, significantly greater frequency of CD34⁺CD117⁺ subset (but not absolute count) was detected in mice engrafted with CD34⁺CD117⁻ (Fig. 4e, f). Despite significant increase in engraftment, CD34⁺CD117⁺ NSG recipient mice did not exhibit more severe pathological outcome than CD34⁺CD117⁻ NSG recipient mice (data not shown). Collectively, these results indicated that AML initiating cells are enriched but do not reside exclusively in the CD117⁺ fraction.

Sorafenib (Nexavar®) and Regorafenib (Stivarga®) are FDA-approved multi-kinase inhibitors that have shown effective anti-tumor activity in patients with solid tumors [26, 27]. More recently, the potential usefulness of Sorafenib and Regorafenib in AML treatment has emerged in numerous in vitro, pre-clinical studies and phase I/II clinical trials [28‐33]. To evaluate the potential use of our model as a pre-clinical tool for therapeutics assessment in vivo, successfully engrafted NSG recipient mice were gavage-fed a daily dose of Sorafenib (10 mg/kg body weight) or Regorafenib (5 mg/kg body weight) and monitored for 1 month. Peripheral blood engraftment was significantly reduced in Sorafenib- and Regorafenib-treated mice compared to untreated mice at week 2 and 4 post-treatment (Fig. 5a). The fold change of reduction was more drastic in Sorafenib treatment, as the level of engraftment after 2 weeks of treatment fell below the engraftment level before treatment. In contrast, Regorafenib treatment suppressed the rate of increase in engraftment level (Fig. 5a). At the endpoint, Sorafenib and Regorafenib treatment did not result in complete resolution of myeloid sarcoma. The extent of myeloid sarcoma was reduced, accompanied by significant reduction in spleen size (Fig. 5b). While the frequency of CD34⁺ and CD117⁺ cells remain unchanged between untreated and treated mice (Additional file 6: Figure S6), significant reduction of absolute AML count was observed in the peripheral blood, spleen but not BM upon Sorafenib and Regorafenib treatment (Fig. 5c). This suggests that AML cells in the peripheral blood and spleen are sensitive to Sorafenib and Regorafenib, whereas those in the BM are protected from the drugs. Overall, the favorable response of Sorafenib and Regorafenib in AML treatment can be recapitulated in our model.