CD45dimCD34+CD38−CD133+ cells have the potential as leukemic stem cells in acute myeloid leukemia

Background Leukemia stem cells (LSCs) in play an important role in the initiation, relapse, and progression of acute myeloid leukemia (AML), and in the development of chemotherapeutic drug resistance in AML. Studies regarding the detection of LSCs and the development of novel therapies for targeting them are extensive. The identification of LSCs and targeting therapies for them has been continuously under investigation. Methods We examined the levels of CD45dimCD34+CD38−CD133+ cells in bone marrow samples from patients with hematological malignancies and healthy controls, using four-color flow cytometry. Results Interestingly, the CD45dimCD34+CD38−CD133+ cells were highly expressed in the bone marrow of patients with AML compared to that in healthy controls (HC). Moreover, the proportions of CD45dimCD34+CD38−CD133+ cells were also examined in diverse hematological malignancies, including AML, CML, DLBCL, MM, MDS, HL, ALL, and CLL. LSCs were prominently detected in the BMCs isolated from patients with AML and CML, but rarely in BMCs isolated from patients with DLBCL, MM, MDS, ALL, CLL, and HL. Additionally, the high CD45dimCD34+CD38−CD133+ cell counts in AML patients served as a significantly poor risk factor for overall and event free survival. Conclusions Therefore, our results suggest that CD45dimCD34+CD38−CD133+ cells in AML might potentially serve as LSCs. In addition, this cell population might represent a novel therapeutic target in AML.


Immunophenotyping
Background Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. It originates from a class of leukemic stem cells that are capable of self-renewal [1,2]. AML is a heterogeneous disease, with respect to the causative pathogenic mutations and clinical outcomes [3]. AML can progress aggressively within a short period of time and become lethal. Survival rates for adults with AML are very poor despite extensive chemotherapy and/or targeted therapies, provided along with supportive care [4].
The leukemia stem cells (LSCs) in AML play an important role in the development, relapse and progression of leukemia, and in the development of chemotherapeutic drug resistance in AML [5]. Recent studies have suggested that LSCs are capable of giving rise to identical daughter cells that can differentiate into other cells and maintain AML [6,7]. Rhenen et al. showed that a high percentage of CD34 + CD38 − stem cells at diagnosis significantly correlated with a high minimal residual disease frequency and subsequently to relapse in AML patients.

Isolation of bone marrow cells
The bone marrow cells (BMCs) were isolated by the density gradient method, as previously described [23]. In brief, BMCs were isolated via density gradient centrifugation at 400×g using Lymphoprep (Axis-Shield, Oslo, Norway; density, 1.077 g/mL). They were washed with phosphate-buffered saline (PBS).

Flow cytometric phenotypic analysis
The BMCs were collected and washed twice with FACS buffer (PBS containing 0.3% BSA and 0.1% NaN 3 ). The total bone marrow cell number used in the experiment was 4 × 10 6 cells. Cells were incubated with four antibodies against each cell surface antigen, including CD45, CD34, CD38, and CD133 on ice for 30 min. First, live BMCs were collected, and SSC low and CD45 dim cells were gated, as shown in Fig. 1a and b. And we always draw gates with the same criteria and select cells in the same section. The criteria are as follows: R1 Gate: live cells; R2 Gate: SSC-H, 100-500 and FL2-H, 10 1 -10 2 ; R3 Gate: FL2-H, 10 2 -10 4 , FL3-H, 10 0 -10 1 . The BMCs were incubated with three combinations of monoclonal antibodies (mAbs) on ice for 30 min; these included isotype control 1 (mouse anti-human CD45-FITC, mouse IgG-PE, mouse IgG-PE CY5, and mouse IgG-APC), isotype control 2 (mouse anti-human CD45-FITC, mouse antihuman CD34-PE, mouse anti-human CD38-PE CY5, and mouse IgG-APC), and sample (mouse anti-human CD45-FITC, mouse anti-human CD34-PE, mouse antihuman CD38-PE CY5, and mouse human CD133-APC), as shown in Fig. 1c and Fig. 1d. Cells were then washed twice with FACS buffer and analyzed using the FACSCalibur flow cytometer and CellQuest Pro software (BD Bioscience) as shown Fig. 1. Finally, the counts of CD45 dim CD34 + CD38 − CD133 + cells, CD133 positive cells among the R1, R2, R3-gated cells were measured, and the results were expressed as percentage change from the basal conditions including the isotype control 2. The 40,000 cells were used for flow cytometric acquisition in each sample tube.

ELISA for cytokine measurement
Cell-free plasma from bone marrow samples of patients with AML was collected and frozen at − 80°C. Plasma interleukin (IL)-1β, IL-6, IL-17, and IL-23 levels were measured using ELISA kits according to the manufacturer's introductions (R&D Systems).

Statistics
The data presented here represent the mean ± standard error of mean (SEM) of at least three independent experiments. All values were evaluated by one-way analysis of variance followed by Turkey range tests implemented in GraphPad Prism 7.0. Differences were considered significant at P < 0.05. For patients with AML, continuous variables were compared using the Student's t-test, whereas categorical variables were analyzed using the Pearson chi-square test or Fisher's exact test. Overall survival (OS) was calculated from the date of HCT to the date of death or last follow-up. Event-free survival (EFS) was defined from the date of HCT to the Fig. 1 The process of four-color staining flow cytometry using monoclonal antibodies. The BMCs were collected and washed twice with FACS buffer. Cells were incubated with four antibodies against cell surface antigens, including CD45, CD34, CD38, and CD133 on ice for 30 min. a, b The live BMCs were collected, and SSC low and CD45 dim cells were gated. c, d The BMCs were incubated with three types of combinations of monoclonal antibodies (mAbs) on ice for 30 min such as isotype control 1 (mouse anti-human CD45-FITC, mouse IgG-PE, mouse IgG-PE CY5 and mouse IgG-APC), isotype control 2 (mouse anti-human CD45-FITC, mouse anti-human CD34-PE, mouse anti-human CD38-PE CY5, and mouse IgG-APC), and sample (mouse anti-human CD45-FITC, mouse anti-human CD34-PE, mouse anti-human CD38-PE CY5, and mouse human CD133-APC). Cells were then washed twice with FACS buffer and analyzed using the FACSCalibur flow cytometer and CellQuest Pro software (BD Bioscience). Finally, the levels of CD45 dim CD34 + CD38 − CD133 + cells, CD133 positive cells among the R1, R2, R3-gated cells were measured and the results were expressed as percentage change from the baseline conditions including isotype control 2. The filled histogram represents the isotype control 2, and the empty histogram represents CD45 dim CD34 + CD38 − CD133 + cells date of relapse or death from any cause. Survival probabilities were estimated by the Kaplan-Meier method. Univariate and multivariate analyses for OS, EFS, and relapse probability were performed using the log rank test and Cox proportional hazards model, respectively. The following variables were included in univariate analyses: CD45 dim CD34 + CD38 − CD133 + cell proportion, age, white blood cell (WBC) count, platelet count, bone marrow blast percentage, cytogenetic risk groups, chemotherapeutic regimens, and immunophenotyping including CD7, CD33, CD34, and HLA-DR. Variables with a P-value < 0.1 in the univariate analyses were included in the multivariate analyses. The statistical analyses were performed with SPSS version 21.0 software (IBM Corp., Armonk, NY). For all analyses, the P-values were two-sided; a P-value of < 0.05 was considered statistically significant.

Results
CD45 dim CD34 + CD38 − CD133 + cells are present in high numbers in the bone marrow of patients with acute myeloid leukemia The work flow of the four-color flow cytometry experiments using monoclonal antibodies (mAbs) is shown in Fig. 1. As shown in Fig. 1a and b, live BMCs were collected and SSC low /CD45 dim cells were obtained. The BMCs were stained with various combinations of monoclonal antibodies for 30 min such as isotype 1, isotype 2, and sample (Fig. 1c). The CD133 positive cells in the R1, R2, R3-gated cells were measured using flow cytometry, and the results were expressed as percentage changes from the isotype 2 (Fig. 1d). A total of 40 AML patients were examined for the expression of the target antigens, CD45 dim CD34 + CD38 − CD133 + on the surface of BMCs. These cells were present in high numbers in the bone marrow samples isolated from patients with AML, but not in those of healthy controls (Fig. 2). These results indicated that CD45 dim CD34 + CD38 − CD133 + cells in bone marrow are potential AML stem cells.
Elevated IL-1β, IL-6, IL-17 and IL-23 cytokine production of plasma in patients with AML Recently, Th17 related cytokines such as IL-1β, IL-6, IL-17, IL-21, IL-22, and IL-23 play crucial roles in the pathogenesis of many diseases, including inflammatory diseases, autoimmune diseases, and cancers [24]. They have been shown related to Th17 cells. Especially, elevated frequencies of these cytokines in patients with AML have been associated with prognosis [25]. Therefore, we examined the levels of IL-1β, IL-6, IL-17 and IL-23 in the bone marrow plasma samples, which were matched to BMCs in AML patients. Plasma samples from the AML patients exhibited higher levels of IL-1β, IL-6, IL-17, and IL-23 than those from healthy controls (Fig. 3).
The CD45 dim CD34 + CD38 − CD133 + cells are prominently detected in the bone marrow of patients with AML and CML As shown in Fig. 4, the CD45 dim CD34 + CD38 − CD133 + cells were examined by four-color flow cytometry in diverse hematological malignancies including AML (n = 40), CML (n = 6), DLBCL (n = 19), MM (n = 10), MDS, (n = 5), HL (n = 4), ALL (n = 3), and CLL (n = 2). These cells are significantly detected in the bone marrow of patients with AML and CML, but not in those with DLBCL, MM, MDS, ALL, CLL, and HL. These results indicated that CD45 dim CD34 + CD38 − CD133 + cells in bone marrow are potential of AML stem cells. In addition, these cells might be used for the detection of AML stem cells.

Discussion
The hypothesis that cancer stem cells including LSCs are responsible for the initiation, relapse, and drug resistance of cancers has caused a great deal of excitement in this area of research. The importance of cancer stem cells has been demonstrated in a variety of tumors [6,[25][26][27][28][29]. Especially, LSCs have unlimited capacity of self-renewal and are responsible for the maintenance of leukemia. Because selective eradication of LSCs could lead to considerable therapeutic benefits, there has been an interest in the identification and characterization of the LSC population that controls their development [30,31]. Therefore, studies related to prognostically relevant and potentially reliable molecular targets are needed.
AML is a hematopoietic disease that is characterized by clonal growth and the accumulation of myelopoietic progenitor cells [31]. It is a devastating disease that is mostly incurable [4]. Moreover, the treatment for AML involves intense cytotoxic treatment as approximately 70% of the patients with AML are refractory to initial therapy or undergo relapse [2]. This is at least partially driven by the chemo-resistant nature of the LSCs that maintain the disease. Therefore, novel anti-LSC therapies could decrease the number of relapses and improve survival.
In the present study, CD34 + AML was found in 75% of patients with AML and 6 patients had CD34 − AML. The clinical implications of CD45 dim CD34 + CD38 − CD133 + cells might be different in CD34 − AML. The CD34 − AML had lower proportion of CD45 dim CD34 + CD38 − CD133 + cells. Even though we could not evaluate prognostic impact of CD45 dim CD34 + CD38 − CD133 + cells in CD34 − AML due to small number of patients, some portion of CD45 dim CD34 + CD38 − CD133 + cells in CD34 − AML might contain normal hematopoietic stem cells as well as LSCs.
CD133 has been reported to be a cancer stem cell marker in solid tumors [14,[32][33][34]. Several studies have shown that CD133 positive cells have the capacity for selfrenewal, differentiation, high proliferation, and forming tumors in xenografts [33,34]. Although the precise function of CD133 remains unknown, it is associated with aggressive cancers and poor prognosis. CD133 is known to be required for tumor growth and survival [14,29,32].  5 High proportion of the CD45 dim CD34 + CD38 − CD133 + cells predicts poor survival in AML patients. a Higher CD45 dim CD34 + CD38 − CD133 + cell proportion was significantly associated with worse OS (P < 0.001). b Poor EFS was significantly associated with higher proportion of CD45 dim CD34 + CD38 − CD133 + cells (P = 0.002) However, in hematological malignancies including AML, the clinical implications of CD133 expression are not well known. Interestingly, CD45 dim CD34 + CD38 − CD133 + cells are present in more numbers in the bone marrow of patients with AML, but not in healthy controls (Fig. 2). In a further study, only CD133 expression in AML need to be investigated if CD133 marker positivity regardless of CD34 + CD38 − might be a significant marker for discriminating LSC and a prognostic biomarker. Moreover, the asynchronism of CD133 + expression should be also evaluated in CD34 − AML in the future. In other lymphoid hematologic malignancies such as lymphoma, MM, ALL, and CLL than AML, there could be some differences according to the percentages of both malignant cells and CD34 + CD38 − compartments within bone marrows because of niche competition between two cell populations.
We also found increased production of IL-1β, IL-6, IL-17 and IL-23 in the bone marrow microenvironment of AML patients at the time of diagnosis (Fig. 3). These findings suggest that IL-1β, IL-6, IL-17 and IL-23 may be associated with leukemogenesis or pathophysiology of AML. Carey et al. also reported that IL-1 and IL-1β might be associated with AML cell growth [35]. IL-3 plays a key role within the network of cytokines involved in the regulation of hematopoiesis and leukemic blast formation. However, IL-3 has no prognostic significance [20]. As expected, the plasma samples from the AML patients at diagnosis exhibited higher levels of Th17 related cytokines, including IL-1β, IL-6, IL-17 and IL-23, than those from healthy controls (Fig. 3). To be honest with you, we expected these cytokines to have some degree of association with LSCs, but it was difficult to find the correlation in the experimental results. More specifically, the prognostic impact of IL-17 in AML is not clear, although higher serum IL-17 levels have been reported to be an adverse prognostic factor of AML in a univariate analysis of IL-17 by Han et al. [25]. In our results, however, IL-17 did not seem to have an adverse impact on prognosis of AML, because IL-17 was inversely correlated with the percentage of CD45 dim CD34 + CD38 − CD133 + LSCs which was shown to be a significant negative prognostic marker, considering together clinical factors. There is little data regarding IL-23 levels in AML, although IL-23 levels have been reported to be associated with AML leukemogenesis and disease susceptibility in a previous study [36]. Based on our findings, it may be more advantageous to investigate T helper type 17 (Th17) cell or cell level associations than to monitor cytokines expressed in plasma to understand the association between LSC and Th17.
We applied the gate of CD45 dim population using the same criteria. Also, our results showed that the individual differences were large for CD45 dim population ( Supplementary  Fig. 1). In addition, the CD45 dim CD34 + CD38 − CD133 + cells were prominently detected in the bone marrow of patients with AML and CML, but not in those with DLBCL, MM, MDS, ALL, CLL, and HL (Fig. 4). Moreover, the prognostic significance of LSCs has been reported in previous studies [1,17]. Tervinjin et al. showed that higher CD34 + CD45 − LAP + cell proportions were related to poor survival [1]. However, our study demonstrated that higher levels of the CD45 dim CD34 + CD38 − CD133 + cells predict poor OS and EFS in AML (Fig. 5). These results also indicate that the CD45 dim CD34 + CD38 − CD133 + cell compartment in the bone marrow could help discriminate between LSCs and normal hematopoietic stem cells, and can serve as a strong prognostic marker. Therefore, targeting CD45 dim CD34 + CD38 − CD133 + cells could serve as a novel therapeutic strategy in AML. Future studies will focus on the elimination of the CD45 dim CD34 + CD38 − CD133 + cells in patients with AML. Also, it needs to make sure that CD45 dim CD34 + CD38 − CD133 + cells actually work as LSCs in the future. And it is necessary to assess whether the CD45 dim CD34 + CD38 − CD133 + cells have the same characteristics as the stem cells. Therefore, our results indicate that CD45 dim CD34 + CD38 − CD133 + cells have the potential of leukemic stem cells in acute myeloid leukemia.