Fluorouracil selectively spares acute myeloid leukemia cells with long- term growth abilities in immunodeficient mice and in culture

A subset of leukemic cells is assumed to maintain long-term growth of acute myeloid leukemia (AML) in vivo. Characterization of these AML progenitor cells may further define growth properties of human leukemia. In vitro incubations with 5-fluorouracil (5-FU) have been used for enrichment of normal primitive hematopoietic stem cells. By analogy to normal hematopoiesis, it was hypothesized that primitive leukemic stem cells might be kinetically more inactive than colony- forming cells (colony-forming units-AML [CFU-AML]). To examine this hypothesis, conditions were established for incubation with 5-FU that eliminated all CFU-AML. These conditions selected a 5-FU-resistant AML fraction that was evaluated for its capacity for long-term growth by transplantation into mice with severe combined immunodeficiency (SCID) and long-term culture in the quantitative cobblestone area-forming cell (CAFC) assay. Transplantation of the 5-FU-resistant fraction of four cases of AML into SCID mice resulted in growth of AML. Whereas no CFU- AML survived, 31% to 82% of primitive (week-6) CAFC were recovered from the 5-FU-treated cells. Hematopoietic cells proliferating in the CAFC assay were shown to be leukemic by cytologic, cytogenetic, or molecular analysis. The reduction of AML growth as determined by outgrowth of AML in SCID mice was in the same order of magnitude as the primitive (week- 6) CAFC reduction. This indicates that both assays measure closely related cell populations and that the CAFC assay can be used to study long-term growth of AML. These results show a hierarchy of AML cells that includes 5-FU-resistant progenitors. These cells are characterized as primitive (week-6) CAFC and as leukemia-initiating cells in SCID mice.     

Megakaryocytopoiesis and granulopoiesis of W/Wv mice studied in long- term bone marrow cultures

Megakaryocytopoiesis and granulopoiesis of marrow cells from W/Wv mice were studied using a continuous liquid marrow culture system. Cells in the suspension phase were assayed weekly over a 16-week period for total nucleated cells, megakaryocytes, granulocytes, megakaryocytes and granulocyte-macrophage progenitor cells (CFU-Ms, CFU-GMs), and spleen colony-forming cells (CFU-Ss). Without hydrocortisone supplementation, proliferation of megakaryocytes, granulocytes, and their progenitor cells was significantly less in W/Wv cultures than in +/+ cultures. These cells became undetectable in both W/Wv and +/+ cultures at seven to 11 weeks in culture, after which only monocytes and macrophages proliferated in the cultures. Treatment of cultures with hydrocortisone improved megakaryocytopoiesis and granulopoiesis in both W/Wv and +/+ cultures. Following an initial lag phase of three to four weeks, proliferation of megakaryocytes, granulocytes, and their progenitor cells in W/Wv cultures equalled that observed in +/+ cultures and was sustained for 16 to 24 weeks. This improvement was associated with a sustained reduction in monocytes and macrophages. Despite improvements in megakaryocytopoiesis and granulopoiesis, production of macroscopic and microscopic spleen colonies by cells from W/Wv cultures remained severely reduced or absent. Studies of DNA synthesis rates of fresh marrow cells indicated that significantly fewer CFU-Ms and CFU-GMs were in cycle in W/Wv mice compared with +/+ mice. However, in hydrocortisone-treated W/Wv cultures, DNA synthesis rates of CFU-Ms and CFU-GMs increased markedly and equalled those observed for +/+ cultures. These results suggest that the improvements in megakaryocytopoiesis and granulopoiesis in hydrocortisone-treated liquid cultures is associated with a reduction in monocytes and macrophages and that progenitor cells of W/Wv mice have a proliferative defect that is correctable by hydrocortisone treatment in vitro.     

Preclinical assessment of purging with VP-16-213: key role for long- term marrow cultures

An evaluation of the effects of VP-16 on normal human marrow cells and representative lymphoma-leukemia cell lines was performed to assess this agent's applicability to ex vivo marrow purging. Tumoricidal dose curves were defined using malignant lymphoid (SK-DHL2 and Reh) and myeloid (HL-60) cells admixed with a 20-fold excess of irradiated marrow cells to simulate a borderline remission marrow. One-hour treatments yielded ID50 of less than 5 mumol/L of VP-16 for clonogenic units from each cell line; rare-to-zero clonogenic units survived exposure to 50 to 100 mumol/L. CFU-Mix, BFU-E, and CFU-GM were equal in their sensitivity to VP-16 (ID50s25 to 30 mumol/L). Marrows treated with 75 mumol/L were completely depleted of these colony-forming cells but produced CFU-GM in one-stage long-term marrow cultures (LTMCs). This dose had little adverse effect on the proliferative capacity of marrow stromal progenitors, as measured by CFU-F (ID50 271 mumol/L) and by the unperturbed development of adherent layers in LTMCs. Furthermore, these stromal layers were able to support hematopoiesis as well as controls in co-culture experiments with autologous marrow cells (two-stage LTMCs). In conclusion, doses of VP-16 that cleanse marrow of lymphoma-leukemia cells spare hematopoietic and stromal progenitors as demonstrated by LTMCs. These data favor the use of VP-16 in the clinical autotransplant setting.     

Clinical significance of long-term cultures of myeloid blood cells

The long-term maintenance of primitive hemopoietic precursor populations in cultures of human marrow was first described in 1981. This system, which was developed following previous work with murine marrow, appears to establish conditions that reproducibly allow the continuous turnover of a number of primitive progenitor cells, detected by their capacity upon transfer into semisolid assay cultures to generate limited numbers and types of mature blood cells. If not transferred, only those hemopoietic cells that are committed to the granulopoietic pathway are able to undergo terminal maturation. The demonstrated localization of the most primitive hemopoietic cells within the adherent fraction, primarily composed of nonhemopoietic mesenchymal elements expressing markers of fibroblasts, adipocytes, endothelial cells, and smooth-muscle cells has provided indirect evidence that interactions between these cells may be key to the survival and functional integrity of normal stem cells in this system. Such a concept has received additional support from recent studies on the cell cycle control of primitive hemopoietic cells located in and dependent on this adherent network of nonhemopoietic elements. Applications of this culture system to neoplastic populations of hemopoietic cells has revealed a number of intriguing differences in their behavior. Under conditions where maintenance of neoplastic hemopoiesis can be achieved, the most primitive progenitor classes remain continuously in cycle as they do in vivo. Thus the same inability to respond to signals that induce a noncycling state in their normal counterparts appears to be reproduced in the long-term culture system. For some populations, e.g., most CML marrows and many AML marrows, neoplastic hemopoiesis fails to become established. Although the reasons for this are not yet clear, this behavior is of interest, not only because it offers a sensitive method for detecting residual normal cells, but also as a practical approach to purging marrows of leukemic cells for autologous marrow transplantation.     

Long-term suspension cultures of human cord blood myeloid cells

A method is described that allows for the routine long-term (greater than 3 mo) growth of normal, immature human myeloid cells in liquid suspension culture. The techniques employ cell separation, utilization of special growth conditions (RPMI with hydrocortisone and vitamin D), and cord blood as the source of leukocytes. These cultures are composed predominantly of immature myeloid cells that have grown for over 3 mo in culture but eventually terminate as differentiated, mature granulocytes and monocyte-macrophages. Application of these techniques to other sources of fresh human leukocytes (bone marrow and adult peripheral blood) resulted in only short-term proliferation of myeloid cells. The techniques described can be used for the routine expansion of immature myeloid and monocytoid cell populations, particularly from newborns, and should be useful for systematic studies of normal myeloid- monocytoid cell growth and differentiation and providing normal control cells in studies employing human leukemic myeloid cells.     

Persistence of murine myeloid leukaemic cells in long-term bone marrow cultures

Patterns of growth in long-term bone marrow culture were compared for a number of murine myeloid leukaemias. A leukaemic pattern of haematopoiesis was maintained in culture for a period of at least 15 weeks for one of the lines. However in the other five murine myeloid leukaemic lines studies, the leukaemic cells appeared not to survive in culture and normal haematopoiesis was established. Transplantation of cells from these established cultures at weeks 7 or 11 into syngeneic recipients revealed that leukaemic cells were present. Thus leukaemic cells seem to persist in long-term bone marrow cultures even in morphologically normal cultures.     

Differentiation of myeloid dendritic cells into CD8alpha-positive dendritic cells in vivo

Bone marrow-derived dendritic cells (DC) represent a family of antigen-presenting cells (APC) with varying phenotypes. For example, in mice, CD8alpha(+) and CD8alpha(-) DC are thought to represent cells of lymphoid and myeloid origin, respectively. Langerhans cells (LC) of the epidermis are typical myeloid DC; they do not express CD8alpha, but they do express high levels of myeloid antigens such as CD11b and FcgammaR. By contrast, thymic DC, which derive from a lymphoid-related progenitor, express CD8alpha but only low levels of myeloid antigens. CD8alpha(+) DC are also found in the spleen and lymph nodes (LN), but the origin of these cells has not been determined. By activating and labeling CD8alpha(-) epidermal LC in vivo, it was found that these cells expressed CD8alpha on migration to the draining LN. Similarly, CD8alpha(-) LC generated in vitro from a CD8 wild-type mouse and injected into the skin of a CD8alphaKO mouse expressed CD8alpha when they reached the draining LN. The results also show that CD8alpha(+) LC are potent APC. After migration from skin, they localized in the T-cell areas of LN, secreted high levels of interleukin-12, interferon-gamma, and chemokine-attracting T cells, and they induced antigen-specific T-cell activation. These results demonstrate that myeloid DC in the periphery can express CD8alpha when they migrate to the draining LN. CD8alpha expression on these DC appears to reflect a state of activation, mobilization, or both, rather than lineage. (Blood. 2000;96:1865-1872)     

Kinetics of myeloid progenitor cells in human micro long-term bone marrow cultures

Hemopoiesis was analyzed in a miniaturized long-term culture of human bone marrow cells by quantifying the production of granulocyte-macrophage progenitor cells. As in the conventional long-term culture system, hemopoiesis was dependent on the presence of a marrow-derived adherent layer. Adipocytes proved to be essential for long-term proliferation. Optimal growth conditions were maintained by incubation in McCoy's medium supplemented with hydrocortisone, fetal calf serum, and horse serum. When calculated back to the volume of conventional cultures, the numbers and kinetics of nucleated cells and granulocyte-macrophage colony-forming cells were comparable in both culture systems. The microsystem is therefore suitable for performing multiple analyses on small samples of cells.     

Production of soluble CD34 by human myeloid cells

CD34, a glycophosphoprotein present in lymphohaematopoietic stem and progenitor cells, as well as in other cell types, exists in both transmembrane and intracytoplasmic forms. Transmembrane CD34 expression, which is high in the earliest haematopoietic precursors, decreases as cells mature. However, to our knowledge, there is no information on whether a decrease in transmembrane CD34 can also predict a release of the molecule from the cell membrane into the extracellular fluid. To investigate the above possibility, we studied conditions (incubation time, cell density and proliferative status) in human myeloid cells (lines KG-1a, KG-1 and cord blood-derived cells) that may cause a decrease in surface CD34 and the generation of a soluble form of the molecule. The latter, as demonstrated by Western blot analysis, adds more complexity to the proposed structural features and functional properties of CD34 in myeloid cells.     

Sequential expression of CD34 and CD33 antigens on myeloid colony-forming cells

The expression of CD33 and CD34 antigens on human bone marrow cells was examined by fluorescence-activated cell sorting and colony assays. A marked difference of antigen expression was observed on sorted progenitors of the granulocyte/macrophage lineage (CFU-GM) with respect to enrichment and maturation status. Single-color cell sorting revealed no difference of enrichment by anti-CD33 antibody between day-7 and d-14 progenitors, while anti-CD34 antibody preferentially enriched d-14 colony-forming units (CFU). By two-color cell sorting it could be shown that enrichment of d-14 CFU-GM occurred mostly in the CD34+CD33- and to a lesser extent in the double-positive fraction. In contrast, there was no difference in degree of enrichment between d-7 and d-14 CD34-CD33+ CFU-GM. From these data we conclude that, during early myelopoiesis, CD34 antigens are gradually lost while CD33 antigens are acquired.     

Partially differentiated ex vivo expanded cells accelerate hematologic recovery in myeloablated mice transplanted with highly enriched long- term repopulating stem cells

The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 10(3) enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1loLIN-Sca- 1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 +/- 19, 170 +/- 30, and 740 +/- 70 per 10(3) cells, respectively), and stem cells with competitive long-term repopulating potential (> or = 1 per 43 cells). Thy-1loSca-1+H-2Khl cells (TSHFU) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, > or = 1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 +/- 8, 0 and 15 +/- 4 per 10(3) cells, respectively). Recipients of 10(3) TLS cells transiently recovered leukocytes to > or = 2,000/microL in 12 days, but sustained engraftment required 25 days. Platelets recovered to > or = 200,000/microL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 10(3) TSHFU cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 10(3) TLS or TSHFU cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-,83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSHFU cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSHFU cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 10(3) TSHFU cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 10(3) TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSHFU stem cells depleted of progenitors.     

Blood-derived macrophage layers in the presence of hydrocortisone support myeloid progenitors in long-term cultures of CD34+ cord blood and bone marrow cells

 Monocytes/macrophages secrete various cytokines that induce proliferation of colony-forming unit granulocyte-macrophage (CFU-GM) in short-term assays. To determine whether macrophages also support proliferation of more primitive progenitors, i.e., cells that give rise to colony forming cells in a 5-week long-term culture (LTC), we established plastic-adherent macrophage layers from human peripheral blood (PB) and filgrastim (G-CSF)-mobilized progenitor cell collections in the presence of hydrocortisone, and compared these layers with bone marrow (BM) stroma regarding their suitability to support proliferation and differentiation of CD34⁺ BM and cord blood (CB) cells in 5-week LTCs. CD34⁺ cells were seeded onto irradiated macrophage and BM stromal layers, as well as without any preformed layer. After 5 weeks, colony formation (CFU-GM, BFU-E/CFU-E) and cell expansion were determined. CD34⁺ cells from BM and CB yielded more CFU-GM and total nucleated cells at 5 weeks in the presence of both types of adherent layer compared with cultures without a layer (p<0.05). For CD34⁺ BM cells, macrophage layers were superior to BM stroma in enhancing CFU-GM and CFU-E/BFU-E output (p<0.05). In contrast, BM stroma was favorable compared with macrophages concerning nucleated cell expansion from CD34⁺ CB cells (p=0.027). The macrophage nature of PB-derived adherent cells was confirmed immunocytochemically by positive staining for CD68, Ki-M1p, CD31, CD54, inconstant staining for CD14, and negative staining for CD1a, CD3, CD15, CD34, and CD62E. Cytochemical reactions were positive for α-naphthyl acetate esterase and negative for peroxidase and periodic acid-Schiff, consistent with the immunophenotype. In conclusion, the results show that blood-derived macrophages support CFU-GM generation from CD34⁺ CB and BM progenitors for 5 weeks in vitro. Differential effects on proliferation and maturation of BM versus CB progenitors are discussed. Received: February 16, 1999 / Accepted: June 21, 1999     

Interleukin-9 stimulates the proliferation of human myeloid leukemic cells

Human interleukin-9 (IL-9) stimulates the proliferation of primitive hematopoietic erythroid and pluripotent progenitor cells, as well as the growth of selected colony-stimulating factor (CSF)-dependent myeloid cell lines. To further address the role of IL-9 in the development of acute leukemia, we evaluated the proliferative response of three leukemic cell lines and 32 primary samples from acute myeloblastic leukemia (AML) patients to recombinant human (rh)-IL-9 alone and combined with rh-IL-3, granulocyte-macrophage CSF (GM-CSF), and stem cell factor ([SCF] c-kit ligand). The colony-forming ability of HL60, K562, and KG1 cells and fresh AML cell populations upon IL-9 stimulation was assessed by a clonogenic assay in methylcellulose, whereas the cell-cycle characteristics of leukemic samples were determined by the acridine-orange flow cytometric technique and the bromodeoxyuridine (BRDU) incorporation assay. In addition, the terminal deoxynucleotidyl transferase assay (TDTA) and standard analysis of DNA cleavage by gel electrophoresis were used to evaluate induction of prevention of apoptosis by IL-9. Il-9, as a single cytokine, at various concentrations stimulated the colony formation of the three myeloid cell lines under serum-containing and serum-free conditions, and this effect was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL- 9 resulted in an increase of blast colony formation in all the cases studied (mean +/- SEM: 19 +/- 10 colony-forming unit-leukemic [CFU- L]/10(5) cells plated in control cultures v 107 +/- 32 in IL-9- supplemented dishes, P < .02). IL-9 stimulated 36.8% of CFU-L induced by phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM), and it was the most effective CSF for promoting leukemic cell growth among those tested in this study (i.e., SCF, IL-3, and GM-CSF). The proliferative activity of IL-9 was also observed when T-cell-depleted AML specimens were incubated with increasing concentrations of the cytokine. Addition of SCF to IL-9 had an additive or synergistic effect of the two cytokines in five of eight AML cases tested for CFU-L growth (187 +/- 79 colonies v 107 +/- 32 CFU-L, P = .05). Positive interaction was also observed when IL-9 was combined with IL-3 and GM-CSF. Studies of cell-cycle distribution of AML samples demonstrated that IL-9 alone significantly augmented the number of leukemic cells in S-phase in the majority of cases evaluated. IL-9 and SCF in combination resulted in a remarkable decrease of the G0 cell fraction (38.2% +/- 24% v 58.6% +/- 22% of control cultures, P < .05) and induced an increase of G1- and S- phase cells. Conversely, neither IL-9 alone nor the combination of IL-9 and SCF had any effect on induction or prevention of apoptosis of leukemic cells. In summary, our results indicate that IL-9 may play a role in the development of AML by stimulating leukemic cells to enter the S-phase rather than preventing cell death. Moreover, IL-9 acts synergistically with SCF for recruiting quiescent leukemic cells in cell cycle.     

Acute myeloid leukemia stem cells and CD33-targeted immunotherapy

Although the identification of cancer stem cells as therapeutic targets is now actively being pursued in many human malignancies, the leukemic stem cells in acute myeloid leukemia (AML) are a paradigm of such a strategy. Heterogeneity of these cells was suggested by clonal analyses indicating the existence of both leukemias resulting from transformed multipotent CD33(-) stem cells as well others arising from, or predominantly involving, committed CD33(+) myeloid precursors. The latter leukemias, which may be associated with an intrinsically better prognosis, offer a particularly attractive target for stem cell-directed therapies. Targeting the CD33 differentiation antigen with gemtuzumab ozogamicin was the first attempt of such an approach. Emerging clinical data indicate that gemtuzumab ozogamicin is efficacious not only for acute promyelocytic leukemia but, in combination with conventional chemotherapy, also for other favorable- and intermediate-risk AMLs, providing the first proof-of-principle evidence for the validity of this strategy. Herein, we review studies on the nature of stem cells in AML, discuss clinical data on the effectiveness of CD33-directed therapy, and consider the mechanistic basis for success and failure in various AML subsets.     

Increased serum levels of interleukin-15 in rheumatoid arthritis with long- term disease

OBJECTIVE: To study the serum levels of IL-15 in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), seronegative spondyloarthropathies (SSd) and healthy donors. METHODS: The IL-15 serum levels were measured by ELISA in sera from 50 RA patients, 30 patients with SLE, 30 patients with SSd and 30 healthy donors. In RA patients, several clinical and demographic parameters were also obtained at the time of sample collection. IL-15 levels were compared in different RA subpopulations (positive or negative rheumatoid factor [RF], long term or recent onset disease, high or low disease activity). In addition, the possible association with other demographic and clinical parameters (gender, age, disease duration, etc) was also analysed. RESULTS: RA patients had significantly higher serum levels of IL-15 (102.4 +/- 150 pg/ml; p = 0.0001) than SLE patients (9.8 +/- 15.3 pg/ml), SSd patients (7.9 +/- 14.6 pg/ml) and healthy donors' (5.2 +/- 11.6 pg/ml). RA patients with a disease evolution less than 2 years showed lower IL-15 levels (33.7 +/- 62.2 pg/ml) than those with long-term disease (152.4 +/- 64.6 pg/ml; p = 0.004). In addition, a significant correlation between IL15 in serum and the number of disease-modifying antirheumatic drugs (DMARDs) prescribed was detected in RA patients (r = 0.42; p = 0.002). No association between IL-15 levels and age, gender, RF or disease activity was observed in this group. CONCLUSION: IL-15 is elevated in RA patients, specially in those with long term disease, compared to other rheumatic disorders. This finding supports that IL-15 is involved in the perpetuation of RA synovitis.