Stem Cells, Vol. 19, No. 1, 1-11,
January 2001
© 2001 AlphaMed Press
New Strategies in the Treatment of Acute Myelogenous Leukemia (AML): In Vitro Culture of AML Cells—The Present Use in Experimental Studies and the Possible Importance for Future Therapeutic Approaches
Øystein Bruseruda,
Bjørn Tore Gjertsena,
Brynjar Fossa,
Tien-Sheng Huangb
a Division for Hematology, Department of Medicine, Haukeland University Hospital, Bergen, Norway;
b Institute of Molecular Biology, The University of Bergen, Bergen, Norway
Key Words. Acute myelogenous leukemia • Experimental models • In vitro culture • Chemotherapy • Apoptosis
Øystein Bruserud, M.D., Medical Department, Haukeland University Hospital, N-5021 Bergen, Norway. Telephone: 47-55-29-80-60; Fax: 47-55-97-29-50; e-mail: oystein.bruserud{at}haukeland.no
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ABSTRACT
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In vitro studies of cultured native acute myelogenous leukemia (AML) blasts and cell lines have contributed significantly to our present knowledge about the pathogenesis of AML. In the present article we review different techniques for preparation and in vitro culture of AML blasts. Well-characterized serum-free in vitro conditions can now be used in experimental studies of AML, and this makes comparisons between different studies easier. We also describe assays for characterization of AML progenitor subsets (i.e., suspension cultures, colony assays, long-term in vitro culture, xenotransplantation in immunocompromised mice), and we discuss the possible use of AML cell lines as experimental models in AML. Furthermore, clinical studies suggest that the in vitro growth characteristics of AML blasts assayed by short-term culture of the total native populations can be used as a predictor of prognosis after intensive chemotherapy. These in vitro assays may therefore be used for more accurate identification of prognostic parameters and thereby form a basis for the development of simplified laboratory techniques suitable for routine evaluation of patients undergoing risk-adapted therapy. However, it will be equally important to further evaluate the clinical relevance of assays for primitive AML progenitors, and to develop simplified methods that can be used to characterize these progenitor subsets in the routine clinical evaluation.
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INTRODUCTION
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Studies of in vitro cultured acute myelogenous leukemia (AML) cell lines and native blasts have been important for the characterization of proliferation, differentiation, and apoptosis in leukemic hematopoiesis, and for our understanding of chemotherapy effects in AML [1-6]. One would also expect experimental studies to become important in the future characterization of genetic abnormalities as pathogenetic factors in AML [3, 4], and based on the in vitro results several new therapeutic strategies have already been suggested [1, 5, 6]. Furthermore, in vitro growth characteristics of native AML blasts may be useful as a prognostic parameter in AML [7-12]. An understanding of these AML models is therefore essential for the interpretation of experimental data and for the understanding of possibilities and limitations of the new therapeutic approaches.
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WHAT KIND OF AML CELL POPULATIONS SHOULD BE STUDIED?
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Bone Marrow Versus Peripheral Blood-Derived Native AML Blasts
The bone marrow of AML patients includes by definition at least 20% or 30% leukemia blasts [1, 13], whereas only a subset of the patients has high blast counts in peripheral blood (Table 1
). A high blast count in the blood seems to be an adverse prognostic factor [14-16], but it is not known whether this prognostic impact reflects a more advanced disease status (simply a quantitative difference) or a qualitative difference between the malignant cells. Thus, if AML blasts are derived from peripheral blood, the experimental results may be representative only for this particular subset of patients. Furthermore, even AML cells derived from marrow and blood in the same patients may have different phenotypes [17]. Taken together these observations emphasize the importance of standardized cell sampling from the same in vivo compartment in comparative AML studies.
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Table 1. Preparation of enriched AML blast populations and culture of human AML cells; the importance of separation procedures and culture conditions for interpretation of experimental results
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Gradient Separation and Positive/Negative Selection for Preparation of Native AML Blasts
Gradient separation (specific density 1.077 g/ml) is widely used for preparation of AML cells [7-12], and this method results in populations of >95% purity when samples are derived from selected patients with a high number of AML blasts relative to other low density cells [18]. Additional positive and/or negative selection is commonly used when gradient-separated blasts contain a relatively high percentage of contaminating cells. Enriched AML blast population can then be prepared by depletion of lymphocytes and monocytes, for example, by using CD2- (T lymphocyte) and CD19-specific (B lymphocyte) immunomagnetic negative selection in combination with removal of adherent cells [18]. Various methods for positive selection may also become useful for preparation of AML cell subsets, for example, CD34+ AML cells [19, 20]. However, extensive ex vivo manipulation, including cell separation procedures, can induce gene expression and thereby alter the release of soluble mediators as well as the expression of membrane molecules by native AML blasts [21-24].
The Platelet Contamination of Native AML Blast Populations
Gradient-separated AML blasts are contaminated with platelets, but in our experience the platelet numbers are usually too low to have any major influence on the functional characteristics of native AML blasts. First, we determined the number of platelets in gradient-separated mononuclear cells (>95% AML blasts after separation; peripheral blood samples with blood leukocyte counts >30 x 109/l and >80% AML blasts) derived from 18 patients, and the AML blast:platelet ratio was always 
1.0 even for patients with normal platelet counts. At this ratio, normal platelets do not alter AML blast proliferation and cytokine secretion [25, 26]. Second, although activated platelets can adhere to native AML blasts [27], detectable platelet adhesion is not observed after incubation of gradient-separated AML populations under in vitro conditions that would be expected to activate the platelets (Foss et al., unpublished data). Third, we have also examined the release of platelet-derived growth factor (PDGF) isoform AB and vascular endothelial growth factor (VEGF) by gradient-separated AML populations. Although both these cytokines can be released by activated platelets, PDGF and VEGF levels showed no significant correlation, and only a minority of patients showed detectable levels of PDGF (22 of 51 patients) or VEGF (16 of 40 patients) (Foss, unpublished data). This observation indicates that both these mediators are released independently by AML blasts without detectable contribution from a low number of contaminating platelets. Taken together, the observations suggest that the platelet contamination of gradient-separated AML blasts is of minor functional importance.
AML Cell Lines: An Alternative to Native AML Blasts?
Human AML cell lines are often used as in vitro models for AML disease (Table 1
). Several cell lines have been extensively characterized and are regarded as typical for different AML subtypes [28, 29]. However, AML cell lines often show extensive chromosomal abnormalities, and this is a fundamental difference from native AML blasts that usually show normal karyotypes or only a limited number of abnormalities [4, 30]. Thus, results from studies of cell lines have to be interpreted with great care and need to be verified by studies of native AML blasts.
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IN VITRO CULTURE OF NATIVE AML BLASTS
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The Culture Medium
Both native AML blasts and AML cell lines can be cultured in medium with inactivated fetal calf or human serum [7-12, 31-33], and the serum then represents a nonstandardized parameter with unidentified mediators that may have effects on AML blasts [34]. For example, cytarabine cannot induce differentiation of the AML cell line ML-1 when cultured in serum-free medium, but the drug induces differentiation of ML-1 cells cultured in serum-containing medium by rendering the cells responsive to differentiation-inducing cytokines in the serum [34].
Salem et al. [35] concluded that bovine serum albumin (15 mg/ml), cholesterol (7.8 µg/ml), transferrin (7.7 x 10–6 M), and insulin (1 µg/ml) were important for proliferation of native AML blasts under serum-free conditions. The relative importance of each factor differs between patients [35], and the experiences of Blair et al. [19] and Bruserud et al. [32] suggest that the presence of all four factors is not essential for most patients. First, although both cholesterol and fatty acids seem to be important for regulation of proliferation and apoptosis of human AML cells [35-37], serum-free Iscove's modified Dulbecco's medium (IMDM) supplemented with only bovine serum albumin (2%), transferrin (200 µg/ml), and insulin (10 µg/ml) can be used for in vitro culture of native AML blasts [19]. Second, a medium developed for culture of normal stem cells and contaning IMDM supplemented with bovine serum albumin (20 ng/ml), low-density lipoprotein (0.05 mg/ml), human iron-saturated holotransferrin (0.2 mg/ml), and 2-mercaptoethanol 5 x 10–5 M (no insulin) can be used for culture of native AML blasts [32]. Several standardized serum-free media that are commercially available, can also be used for in vitro culture of native AML blasts [32, 33]. However, even serum-free culture conditions can modulate the functional characteristics of native AML blasts, particularly the constitutive cytokine secretion and the ability to function as accessory cells during T cell activation [32, 33].
The Effects of Endotoxin on In Vitro Cultured AML Blasts
Endotoxin can induce cytokine secretion by normal monocytes, and it can also alter the functional characteristics of native AML blasts [38]. It is therefore important to culture AML blasts in media with a minimal endotoxin level for two reasons: A) to minimize the influence of a minor population of contaminating, endotoxin-activated normal monocytes, and B) to avoid induction of functional alterations in AML cells.
Addition of Antibiotics to the Medium
Antibiotics are usually added to culture media, and an aminoglycoside (e.g., streptomycin, gentamicin) alone or in combination with penicillin is often used. An effect of penicillin on in vitro-cultured AML blasts has not been described. On the other hand, the aminoglycoside neomycine has a PDGF-isoform- and receptor-specific antagonistic effect, whereas gentamicin and streptomycin have weaker effects [39]. PDGF receptors can also be expressed by native AML blasts (Foss, manuscript in preparation), and for these patients aminoglycosides may interfere with the functional characteristics of AML cells. In fact, we found that neomycin (3.3 mM) had a minor effect on the constitutive cytokine secretion (interleukin 1ß [IL-1ß], IL-6, tumor necrosis factor 
[TNF-]) by native AML blasts for a subset of PDGF-receptor positive patients (Foss et al., unpublished data). However, one would expect other aminoglycosides to have no or only minimal effects on AML blast functions.
Growth Factor-Dependent Proliferation of Native AML Blasts
For a subset of patients the AML blasts show autocrine (spontaneous) in vitro proliferation when cultured in medium alone, whereas for other patients the leukemia cells can only proliferate in the presence of exogenous growth factors [7-12, 18, 31-33]. These growth factors may, in addition, induce AML blast differentiation [1]. Several cytokines can function as growth factors for AML blasts as exemplified by the results summarized in Table 2. It can be seen that the optimal growth factor for expansion of AML cells differs between patients/FAB subclasses. AML-M2 cells show generally higher proliferative responses than AML-M0/M1 and AML-M4/M5 cells. For AML-M2 cells the highest number of responders was observed in the presence of IL-1ß, stem cell factor (SCF) and G-CSF, whereas the highest number of responders for AML-M0 and AML-M4/5 cells was detected with GM-CSF. IL-9 is another cytokine that also functions as a growth factor for a majority of AML patients [40]. However, when standardized in vitro conditions have to be used throughout an experimental study, we have often used the combination IL-3 + SCF + GM-CSF that initiates a strong proliferative response for a majority of patients [33, 41]. Combinations that include additional factors (e.g., erythropoietin/IL-6/G-CSF) have also been used [19, 42].
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Table 2. Cytokine-dependent proliferation of native AML blasts, a summary of the results for 50 consecutively examined AML patients1
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Apoptotic Cell Death During In Vitro Culture
An effective treatment of AML seems to depend on induction of apoptosis, and resistance against chemotherapy can often be explained by altered regulation of apoptosis [43]. Apoptosis is detected for an increasing fraction of cells during in vitro culture of native AML blasts [18], and the proliferative in vitro response of the leukemic cells is then caused by another subset of cells [7-12]. The number of apoptotic cells is determined by several factors, for example, a decreased fraction is observed in AML populations that show autocrine proliferation, in the presence of exogenous growth factors, and when an optimal culture medium is used [33, 42, 44-45]. The occurrence of spontaneous in vitro apoptosis may thus serve as an experimental model of cell death regulation in AML.
Since the first definition of apoptosis in 1972 [46], a wide array of methods have been developed for detection of apoptosis (Table 3) [47]. Apoptosis was initially a morphological diagnosis [46], and transmission electron microscopy still appears to be the gold standard [47]. Our suggestion is that experimental studies of apoptosis should include initial examination by two or three independent methods, and thereafter the most convenient assay should be chosen for further studies. The results always have to be interpreted with great care because diverging apoptotic phenotypes are frequently detected both by morphological and biochemical examination, and the apoptotic phenotype may also differ between cell types. For example, in AML cells, different types of apoptosis result in differences in cell surface and nuclear morphology, as well as differences in the levels of internucleasomal fragmentation and TUNEL positivity [48].
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Table 3. Methods used for detection and analysis of apoptotic cell death (for detailed discussion and additional references [47])
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Simple morphological examination of the nucleus (e.g., bisbenzamide or May-Grünewald-Giemsa staining) is suitable for the initial examination of apoptosis in experimental studies. If there is doubt about the apoptotic morphology, for example, by atypical or lacking nuclear condensation or fragmentation, annexin V binding may be the method of choice. Flow cytometric analysis of annexin V can also be combined with immunostaining to allow examination of apoptosis in smaller subsets in mixed populations, for example, gradient-separated AML populations with relatively high contamination of other low-density cells [47]. However, there are certain cell types that do not expose enough phosphatidylserine on the cell surface during apoptosis to be labeled with annexin V [49].
The initial studies of nuclear morphology or annexin labeling can be supplemented with characterization of molecular fragmentation or studies of selected intracellular pathways that are involved in the regulation of apoptosis, for example, assays detecting mitochondrial alterations, caspase activation, or cleavage of caspase substrates like polyadenosine 5'-diphosphate-ribose polymerase. These techniques have in common that they often describe population (and not single cell) characteristics, and mixed cell populations are thus difficult to characterize. With the growing knowledge of signaling pathways directing apoptosis [50], techniques involving detection of the caspase activation or detection of caspase substrates may be used in immunoblotting or immunostaining protocols. Again, careful interpretation of the results is necessary because not all cases of apoptosis include caspase activity, and certain cells may even lack caspase-3 [51]. These observations thus support our suggestion that an established method based on nuclear morphology in AML cells should be chosen as an initial screening assay before further characterization of the molecular mechanisms.
Gene Induction During In Vitro Culture
As described above, extensive cell separation procedures seem to induce gene expression in AML blasts. Several studies have also demonstrated that gene expression can be induced or modulated by in vitro culture conditions, either by the culture medium itself or by agents added to the medium (e.g., by the presence of agents interacting with histone acetylation [52]). First, altered gene expression possibly contributes to the increased expression of certain membrane molecules after in vitro culture [23, 24]. Second, the ability of native AML blasts to respond to differentiation induction depends on the culture conditions [34], and experimental evidence suggests that altered gene expression is involved in medium-induced modulation of blast differentiation [53]. Third, alterations in iron metabolism of cultured cells will also result in complex network events acting at the transcriptional and translation levels to change the expression of proteins involved in transport/uptake/utilization/storage of iron [54]. The importance of iron-saturated transferrin for optimal in vitro proliferation of native AML blasts indicates that similar alterations may occur in cultured AML cells.
The occurence of spontaneous in vitro apoptosis in native AML blasts is modulated by serum-free culture conditions [33]. Other studies have described that induction of spontaneous apoptosis in AML cells cultured under serum-free conditions can be suppressed by inhibition of c-jun expression [55]. Taken together, these observations thus suggest that altered gene expression is involved in the regulation of spontaneous in vitro AML blast apoptosis and possibly also in the suppression of apoptosis when using optimal serum-free conditions.
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THE HIERARCHICAL ORGANIZATION OF THE AML CLONE
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Experimental Studies
Experimental data suggest that AML populations have a hierarchical organization from relatively rare primitive progenitors down to more mature cells that dominate in native AML blast populations derived from blood or bone marrow [56] (Table 4). The cells at different steps in this hierarchy differ in their expression of cell surface molecules and their self-renewal (proliferative) capacity.
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Table 4. Experimental models for the study of AML cell proliferation; a summary of studies indicating a hierarchy of the human AML clone
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Short-term suspension cultures of native AML populations have been used for characterization of both proliferative responses (seven days of culture) and constitutive cytokine secretion (two days of culture) by AML cells [18]. An increasing fraction of apoptotic cells is then detected during culture [18], and assays based on 3H-thymidine incorporation of the total AML population after seven days of culture seem to reflect enrichment of more primitive colony-forming (clonogenic) progenitors [57]. This 3H-thymidine incorporation assay seems to be more sensitive than the colony-formation assay for detection of cytokine-induced proliferation [58], and proliferative characteristics reflected in this assay have also been used as a determinant of prognosis in AML [11].
Autocrine or spontaneous AML blast proliferation can be assayed by the ability to form colonies during culture in a semisolid medium (e.g., containing methylcellulose) without exogenous growth factors [7-11]. Although colony-forming (clonogenic) cells constitute a minority of the total AML cell population [7-11], their frequency is increased when cells are cultured at high densities [57]. This observation suggests that spontaneous colony formation is determined both by the growth characteristics of the clonogenic subset, and by the ability of nonproliferating neighboring AML cells to release soluble mediators that facilitate the proliferation of clonogenic cells. This dependency of clonogenic cells on local cytokine release by nonproliferating blasts is reduced when colony formation is assayed in the presence of exogenous growth factors [57], but even then the colony-forming cells constitute a minority of the total AML cell population. The frequency of clonogenic cells shows a wide variation and depends both on the culture conditions and on differences between patients. As an example, in the study of Sutherland et al. [59] the colony-forming AML cells that could respond to exogenous growth factors had a frequency between 3 and 2,600 per 105 cells and were usually derived from the CD34– subset of native AML cells.
The most primitive AML progenitors can be characterized by their self-renewal capacity during long-term in vitro culture (Table 4): A) the identification of suspension-culture initiating cells (SCIC) is based on the ability of these progenitors to give rise to new colony-forming cells even after several weeks of cytokine-dependent proliferation in suspension culture [19, 42, 59], and B) the cobblestone area forming cells are characterized by their ability to form cobblestone colonies after six weeks of culture on a stromal layer [59, 60]. These long-term cultured AML cells are regarded as more primitive and are detected at a lower frequency than the colony-forming cells [59]. The primitive progenitors also differ from more mature AML cells by their relative resistance to fluorouracil [60]. In contrast to colony-forming cells that usually are derived from CD34– AML cells, for most patients the SCIC are derived from the minor population of CD34+/CD38–CD71– AML cells [19, 59]. However, CD34– SCIC have been detected for a minority of patients [19, 42, 61].
AML cells with the ability to engraft in immunocompromized severe combined immunodeficient (SCID) mice are also regarded as primitive progenitors (Table 4) [61]. The transplanted AML cells that initiate the leukemic disease in mice, fulfill two important criteria of leukemic stem cells: A) the cells can proliferate, differentiate and thereby recapitulate the pathology of the original human disease, and B) the cells are able to self-renew and thereby reestablished the disease after transplantation [60-62]. Most cells capable of engraftment are included in the minor subset of CD34+CD71– AML cells, but engraftment of CD34– cells has also been observed [19, 42, 59]. Thus, SCID repopulating cells and AML progenitors capable of self-renewal during long-term in vitro culture seem to be derived from the same AML subsets and may be identical or overlapping populations.
Are Experimental Studies of the Total AML Blast Population Still Relevant?
Are experimental models based on short-term culture of the whole native AML cell population still relevant, or should future studies be based on the complex models requiring long-term culture or xenotransplantation of selected progenitors? Because the fundamental aim of all AML studies is to characterize the disease and thereby improve treatment and increase long-term disease-free survival, the answer to this question should be based on both clinical and experimental data. First, all AML cells in the native population are characterized by identical genetic abnormalities that are regarded as fundamental in the pathogenesis of AML, and one would therefore expect that the characteristics of the total cell population reflect important disease characteristics. The clinical relevance of cell population characteristics is also demonstrated by two observations: A) detection of autocrine proliferation in assays based on characterization of the whole native AML population in short-term culture can be used as a prognostic parameter in AML [7-12], and B) autocrine proliferation is also associated with decreased in vitro apoptosis [63-65], and expression levels of apoptosis-regulatory genes in the whole AML population seem to be a predictor of survival/AML relapse after intensive chemotherapy [66-68]. Furthermore, a recent study described that the two most important prognostic factors (i.e., predictors of later leukemia relapse) in AML were cytogenetics (a population characteristic) and the achievement of complete hematological remission after the first induction course [30]. Remission induction mainly reflects the chemosensitivity of the whole native AML population and not only residual progenitors. Taken together these observations thus support the hypothesis that cell population characteristics reflect important disease characteristics. Second, only assays based on native AML populations and short-term culture are suitable for evaluation of large patient populations, and a further characterization and development of such assays is therefore important for the characterization of AML heterogeneity and the development of risk-adapted therapy. Third, cultures including the total AML population are important for the functional characterization of the natural AML microenvironment (e.g., cytokine network, release of other soluble mediators, cell adhesion effects), and these interactions are also important for the proliferation of more primitive AML progenitors [57]. At last, the possible future use of ex vivo modulated AML blasts in T cell targeting immunotherapy will probably be based on the use of the whole native AML cell population [69, 70]. We therefore conclude that the experimental models utilizing short-term culture of the total native AML populations are still important and relevant in the study of AML, and they are even the experimental models with the best documented clinical relevance [7-12].
Long-term culture and xenotransplant assays of AML cells have only been used for evaluation of relatively small patient populations. Patient heterogeneity has also been observed with all these assays and especially with the SCID-mouse model where the degree of engraftment shows a wide variation [19, 42, 59, 71]. A recent study demonstrated that high peripheral blood blast counts at the time of diagnosis had a much stronger impact on engraftment than cytogenetics and response to induction chemotherapy [71], whereas cytogenetics and therapy response are more important than the blast count as predictors of relapse after chemotherapy [30]. The long-term culture/xenograft models may thus reflect disease characteristics with less clinical impact. The observation of a phenotype shift after xenografting [71] also supports our conclusion that the clinical relevance of the SCID/long-term culture models needs further evaluation.
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POSSIBLE CLINICAL USE OF IN VITRO CULTURE IN AML
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Prognostic Evaluation in Risk-Adapted Therapy
Several studies have described an association between the ability of autocrine AML blast proliferation (3H-thymidine incorporation or colony formation) and disease-free survival after intensive chemotherapy [7-12], but this association could not be detected in other studies [72, 73]. This discrepancy may be due to differences between in vitro assays or therapeutic regimens. The autocrine proliferation is caused by constitutive growth factor secretion [7], is associated with decreased spontaneous in vitro apoptosis [63, 64], and may therefore reflect prognostically important differences in the regulation of apoptotic AML cell death. Functional assays of autocrine proliferation/apoptosis/constitutive cytokine secretion may thus become useful for clinical evaluation and a more accurate identification of prognostic factors in future risk-adapted therapy.
Immunotherapy
Native AML blasts can be induced to differentiate into a dendritic cell phenotype with increased ability to induce specific antileukemic T cell reactivity [69, 70]. If one can ensure that such differentiated populations do not include leukemic progenitors that give rise to later AML relapse, these cells may be used in future immunotherapy for induction of antileukemic reactivity by autologous T cells. One possible approach could then be to use nonproliferating 
-irradiated AML blasts that have maintained several functional characteristics, including: A) constitutive secretion of the immunomodulatory cytokines IL-1ß, IL-6, and TNF-, and B) the ability to function as accessory cells during mitogenic T cell activation [74, 75]. These characteristics are maintained after 50 Gy irradiation, but are decreased at higher doses (75-150 Gy) [74].
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CONCLUDING REMARKS
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Native AML blast populations can now be cultured in vitro under well-characterized serum-free conditions, and the use of such standardized conditions in future experimental investigations is important to allow comparison between different studies (e.g., in vitro modulated AML cells used for immunotherapy). Furthermore, clinical studies suggest a prognostic impact of AML blast growth characteristics assayed by short-term culture of native AML populations. These assays may therefore be used for more accurate identification of prognostically important cell characteristics and thereby form a basis for the development of simplified laboratory techniques that are suitable for routine evaluation of AML patients undergoing risk-adapted therapy. However, it will also be very important to further evaluate the clinical relevance of primitive AML progenitor assays and to develop simplified methods that can be used for characterization of these progenitors as a part of large clinical studies.
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ACKNOWLEDGMENTS
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The study was supported by The Norwegian Cancer Society and the Meltzer Foundation. The advice of Professor Peter Ernst is gratefully acknowledged.
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Received October 3, 2000;
accepted for publication October 3, 2000.
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Abstract
Introduction
