Antibodies to CD44 enhance adhesion of normal CD34+ cells and acute myeloblastic but not lymphoblastic leukaemia cells to bone marrow stroma

The role of CD44 in the adhesion of haemopoietic cells to bone marrow stromal layers has not been clearly defined in humans, although its importance in the murine system has been well documented. We have demonstrated that the CD44 antibody, NIH44-1, enhances the adhesion of haemopoietic cells to bone marrow stroma. Normal human CD34⁺ haemopoietic progenitors and blasts from patients with acute myeloblastic, but not lymphoblastic, leukaemia responded to NIH44-1. All CD44 antibodies tested which bound the same epitope as NIH44-1 also augmented haemopoietic cell adhesion to bone marrow adherent layers; however, antibodies which bound to other CD44 epitopes showed mixed responses. Augmented adhesion was independent of cell metabolism, suggesting that antibody binding resulted in direct activation of the CD44 molecule. However, hyaluronic acid was not the ligand for induced adhesion, nor could we show a role for other CD44 ligands including fibronectin, laminin, collagen or chondroitin sulphate proteoglycan. Similarly, none of the 22 CD44 antibodies tested inhibited the stimulatory effect of the NIH44-1. Expression of CD44 was not sufficient to determine NIH44-1 responsiveness since cell lines and leukaemic cells which failed to respond to NIH44-1 expressed high levels of CD44. Neither CD44 isoforms nor glycosylation patterns could be identified as predictive of response. CD44 antibodies enhanced binding of normal and leukaemic haemopoietic progenitors to bone marrow fibroblasts via an unidentified stromal ligand.     

Serum-free liquid marrow culture in patients with acute lymphoblastic leukaemia: a potential application to purge marrow for autologous transplantation

We have previously established a serum-free (SF) culture medium, which supports normal haemopoietic progenitor cell growth for at least 4 weeks as does conventional serum dependent (SD) medium. In the present study, we investigated the efficacy of such a defined SF liquid medium which sustained in vitro residual normal haemopoietic proliferation of marrow derived from ALL patients and which was detrimental for the leukaemic population. Evidence for a potential selective effect of SF culture was obtained by a leukaemic progenitor cell assay (ALL-CFU) and the detection of the bcr/abl translocation by polymerase chain reaction (PCR). In 13 experiments including 12 patients, morphological blast cells and ALL-CFU were dramatically reduced within 3 weeks of incubation in both SF and SD cultures. Likewise, in 5/5 experiments in SD and 2/5 experiments in SF conditions, leukaemic cells expressing the bcr/abl fusion gene disappeared within 3-4 weeks. In contrast, the absolute numbers of supernatant cells harvested weekly from SF and SD cultures were similar. No difference in CFU-GM production was detected for the two culture systems. Erythropoiesis in SF medium exhibited a slower decline than that found in SD. These results indicate that liquid marrow culture may selectively deplete leukaemic lymphoblastic cells and enable repopulation by residual normal haemopoietic cells. This technique may be useful to purge leukaemic cells for clinical autologous bone marrow transplantation in patients with ALL.     

Leukaemic transformation of donor cells in a patient receiving a second allogeneic bone marrow transplant for severe aplastic anaemia

Allogeneic blood or bone marrow transplantation is a successful treatment for leukaemia and severe aplastic anaemia (SAA). Graft rejection following transplantation for leukaemia is a rare event but leukaemic relapse may occur at varying rates, depending upon the stage of leukaemia at which the transplant was undertaken and the type of leukaemia. Relapse is generally assumed to occur in residual host cells, which are refractory to, or escape from the myeloablative conditioning therapy. Rare cases have been described, however, in which the leukaemia recurs in cells of donor origin. Lack of a successful outcome of blood or bone marrow transplantation for severe aplastic anaemia (SAA), however, is due to late graft rejection or graft-versus-host disease. Leukaemia in cells of donor origin has rarely been reported in patients following allogeneic bone marrow transplantation for SAA. This report describes leukaemic transformation in donor cells following a second allogeneic BMT for severe aplastic anaemia. PCR of short tandem repeats in bone marrow aspirates and in colonies derived from BFUE and CFU-GM indicated the donor origin of leukaemia. Donor leukaemia is a rare event following transplantation for severe aplastic anaemia but may represent the persistence or perturbation of a stromal defect in these patients inducing leukaemic change in donor haemopoietic stem cells.     

Differential action of diffusible molecules in long-term marrow culture on proliferation of leukaemic and normal haemopoietic cells

Summary. The capacity of diffusible molecules in the fluid phase of long-term human bone marrow culture (LTMC) to exert preferential adverse effects on leukaemic relative to normal haemopoietic cells has been investigated. Responses of isolated cell populations were assessed in diffusion chamber inserts which permitted contact with fluid phase molecules but not with the adherent stromal cell layer of the LTMC system. Growth of AML cells in diffusion chambers was inhibited during co-culture with LTMC of autologous leukaemic bone marrow, and the same effect was produced during co-culture with normal LTMC. No inhibitory action was exerted on growth of normal haemopoietic precursors under the same conditions. Comparable responses were observed with human leukaemic cell lines and patient leukaemic cells, and studies on cell lines demonstrated inhibition of growth was induced by molecules generated in LTMC which caused accumulation in G1 phase of leukaemic cells of both myeloid and lymphoid lineage. The inhibitory effect was not reproduced by TGFβ, IFNγ, IFNα, TNFα, LIF, SCF or II-6, and was not impaired by inhibitors of nitric oxide or PGE production in the LTMC. These observations suggest the action of diffusible molecules of undefined constitution contributes to the preferential loss of leukaemic cells in LTMC.     

Differences in the distribution of CD34 epitopes on normal haemopoietic progenitor cells and leukaemic blast cells

The CD34 molecule expressed on haemopoietic progenitor cells contains a large number of epitopes whose expression may be related to the maturation or function of the cells. Monoclonal antibodies specific for different epitopes have been reported to detect different numbers of CD34⁺ leukaemic blast cells. We wanted to confirm this observation and study whether parallel findings could be observed for normal haemopoietic progenitor cells. The cells were immunophenotyped by flow cytometry with a series of monoclonal antibodies reactive with different CD34 epitopes. Class III epitopes (resistant to enzymatic cleavage with neuraminidase, chymopapain and a glycoprotease from Pasteurella haemolytica) showed a broader distribution on normal haemopoietic progenitor cells and leukaemic blast cells than class I epitopes (sensitive to cleavage with all three enzymes) and class II epitopes (sensitive to degradation with glycoprotease and chymopapain, and resistant to neuraminidase). The subpopulation of normal progenitor cells which exclusively expressed class III epitopes had flow cytometric characteristics compatible with mature myeloid progenitor cells, whereas class I, II and III epitopes were equally expressed on cells enriched for immature subsets. No discordant epitope expression could be observed for the more immature leukaemias (AML-M0/1) and a higher percentage of the more mature leukaemic blast cells (AML-M3 and AML-M4/5) expressed class III epitopes compared to the percentage expressing class I and II epitopes. These data indicate that CD34 class III epitopes are more broadly distributed on normal haemopoietic progenitor cells and leukaemic blast cells than class I and II epitopes, and that class I and II epitopes may be down-regulated prior to class III epitopes during normal haemopoietic progenitor cell differentiation. These findings should be considered when selecting CD34 mabs for quantification and positive selection of haemopoietic progenitor cells for research and clinical purposes.     

A model system illustrating the isolation and enrichment of a rare population of tumour cells in bone marrow

A model system employing a modified nylon matrix is described for the separation of rare cells titrated into either a leukaemic cell line or normal bone marrow. A 75- to 125-fold enrichment and recovery of the rare cell population was achieved, starting from an initial level of 0.014 to 0.2% of the total population. The rare cell population was identified by pre-labelling with Hoechst 33342, which intercalates into the DNA, and renders cells highly fluorescent. Separation and recovery of cells was totally dependent on the use of a panel of monoclonal antibodies binding to the labelled population. The nylon matrix, precoated with an anti-mouse immunoglobulin, traps the cells coated with monoclonal antibodies, and these can be released simply by gentle manipulation of the matrix. The matrix employed has been shown to not specifically trap committed bone marrow progenitors as determined by CFU-GM, BFU-E and CFU-GEMM assays. The use of this technique should simplify the isolation of rare tumour cells metastasizing to bone marrow.     

Reduction of heat-induced haemotoxicity in a hyperthermic purging protocol of murine acute myeloid leukaemic stem cells by AcSDKP

The tetrapeptide AcSDKP (Goralatide) is a cytokine with known inhibitory effects on cell proliferation. Many purging agents used in autologous bone marrow transplantation protocols, including hyperthermia, preferentially kill cycling cells. A pretreatment with Goralatide offers a possibility to reduce the haemotoxicity in many purging settings. The impact of Goralatide on the hyperthermic purging protocol was investigated in normal and myeloid leukaemic (SA8) murine cells. The median survival time after transplantation (i.e. leukaemia incidences) was used as an in vivo parameter to determine the effects on leukaemic cells. The hyperthermic effect on normal and leukaemic cells was also investigated in vitro using the cobblestone area-forming cell (CAFC) assay. A heat treatment of 90 min at 43°C resulted in a 4-log depletion of leukaemic stem cells. For normal progenitor cells (CFU-GM) a 2-log cell kill was shown. The reduction in proliferative activity of the CFU-GM after an 8 h incubation with 10^?9 m Goralatide resulted in a decrease in the heat sensitivity of the progenitor subset to approximately a 1-log cell kill. The leukaemic precursor cells seem insensitive to Goralatide inhibition, implicating an increase in the therapeutic window of the hyperthermic purging protocol. Finally, simulated remission bone marrow (5% leukaemic blasts) was incubated with Goralatide followed by a heat treatment of 90 min at 43°C. Lethally irradiated (10 Gy) mice transplanted with heat-treated remission bone marrow (10⁶ normal bone marrow cells versus 5 × 10⁴ leukaemic cells) died of aplasia while Goralatide-pretreated remission bone marrow could rescue the irradiated mice without revealing leukaemic engraftment. These findings confirmed the enhanced protection against hyperthermia of the normal haemopoietic subsets by Goralatide and thus increased the success of the hyperthermic purging protocol.     

Persistence of an activating N-RAS oncogene mutation in clonogenic progenitor cells from an acute myeloid leukaemia patient in remission

Summary A patient with acute myeloid leukaemia (AML) with an activating N- RAS oncogene mutation was studied in a haemopoietic clonogenic progenitor cell assay. Individual colonies and clusters were analysed by polymerase chain reaction and oligonucleotide hybridization for the original mutation. The mutation was detected in a majority of leukaemic clusters, but also in almost half of the differentiated colonies. After chemotherapy the patient entered clinical remission. However, the mutation could still be detected in the bone marrow. Only differentiated colonies and no leukaemic clusters were grown from the remission bone marrow, but the original mutation was still detectable in almost half of the colonies.     

Suppressor T cell chronic lymphocytic leukaemia associated with red cell hypoplasia

A case of T cell chronic lymphocytic leukaemia (CLL) with red cell hypoplasia and neutropenia is reported. WBC was 10.0 X 10(9)/l with 78% being T lymphocytes. These T lymphocytes were positive for Fc gamma receptor and had OKT8 and Leu 2a antigens on the cell surfaces. They suppressed both erythroid and granulocyte-macrophage colony formation in normal bone marrow cultures and suppressed immunoglobulin production by normal B lymphocytes in vitro. Though the myeloid cells were preserved in the bone marrow and the values of serum immunoglobulins were within normal limits, it can be estimated from these results that red cell hypoplasia and neutropenia were partially due to the suppression of haemopoietic precursor cells by leukaemic T lymphocytes.     

Diverse effects of anti-CD44 antibodies on the stromal cell-mediated support of normal but not leukaemic (CML) haemopoiesis in vitro

We have identified three non-cross-reacting anti-human CD44 monoclonal antibodies that have significant positive or negative (or no) effects on normal human haemopoiesis in the long-term culture (LTC) system. These effects manifested as increases or decreases in the number of LTC-initiating cells (LTC-IC), and the number of colony-forming cells (CFC) recovered from cultures in which either unseparated or highly purified CD34⁺CD38⁻ normal marrow cells were placed on pre-established normal marrow feeder layers in the presence or absence of each antibody. The effects seen were rapid and sustained, and dependent on the presence of a preformed feeder layer. Interestingly, the same anti-CD44 antibodies had no effect on the maintenance of leukaemic (Ph⁺) progenitors (from patients with chronic myeloid leukaemia) when these cells were cultured on preformed feeder layers established from normal marrow. CD44 appears to be part of a mechanism by which stromal elements can regulate primitive normal haemopoietic cells but not their leukaemic (Ph⁺) counterparts.     

Changing bone marrow micro-environment during development of acute myeloid leukaemia in rats

The Brown Norwegian rat transplanted with promyelocytic leukaemic cells (BNML) has been used as a model for human acute myeloid leukaemia. We have previously shown that both the blood supply to the bone marrow and the metabolic rate decrease in relation to the leukaemic development in these rats. Here we have investigated how the development and progression of this leukaemia affect oxygenation, pH and proliferation of normal and leukaemic cells in vivo . Bone marrow pH was measured by a needle electrode. Nitroimidazol-theophylline (NITP) was used to identify hypoxic cells, and we applied bromodeoxyuridine (BrdUrd) to identify DNA replicating cells.   The leukaemia progressed slowly until day 27 after which a rapid deterioration could be observed leading to severe changes over the following 5 d. In whole blood there was evidence of progressing metabolic acidosis. In bone marrow the fraction of leukaemic cells increased to > 90% and the pH dropped to about 6.5. The fraction of NITP⁺ cells increased to > 80% in bone marrow and to about 40% in blood. The fraction of BrdUrd⁺ cells was unchanged in blood, but decreased in bone marrow both for normal cells (from about 20% to 5%), and for leukaemic cells (from about 45% to 25%), evidently as a result of the severely changed micro- environment. In this study we have demonstrated in vivo the development of an acidic and hypoxic bone marrow hampering normal haemopoiesis during leukaemic growth. Our data support the notion of BNML as a valuable tool for studying leukaemogenesis.     

Selective secretion of chemoattractants for haemopoietic progenitor cells by bone marrow endothelial cells: a possible role in homing of haemopoietic progenitor cells to bone marrow

To elucidate the mechanisms by which haemopoietic progenitor cells lodge in the bone marrow, we examined the secretion of chemoattractants for haemopoietic progenitor cells by bone marrow and lung endothelial cells. The bone marrow endothelial cells, but not lung endothelial cells, secreted chemoattractants for the haemopoietic progenitor cell line, FDCP-2, and normal haemopoietic progenitor cells. Checkerboard analysis demonstrated that the conditioned medium of the bone marrow endothelial cells had chemotactic activity and random motility-stimulating activity. The bone marrow endothelial cells expressed stromal-cell-derived factor-1 (SDF-1) mRNA and produced SDF-1 protein, whereas the lung endothelial cells did not. Adhesion of FDCP-2 cells to the bone marrow endothelial cells was partially inhibited by anti-SDF-1 antibody. These findings suggest that the chemoattractants for haemopoietic progenitor cells including SDF-1 and random motility-stimulating factor(s) selectively secreted by the bone marrow endothelial cells may contribute to the homing of haemopoietic progenitor cells to bone marrow.     

Haemopoiesis in long-term bone marrow cultures. A review.

Bone marrow-derived adherent cell layers, containing endothelial cells, fat cells and macrophages will support prolonged haemopoiesis in vitro. Evidence suggests that the adherent layer is acting as an in vitro haemopoietic inductive environment for stem cell proliferation and differentiation into the variety of committed progenitor cells of the granulocytic, erythroid, megakaryocytic and lymphoid lineages. Using this system we have analysed the factors controlling proliferation of stem cells (CFU-S), differentiation and maturation of granulocyte/macrophage precursor cells (CFU-C), leukaemic transformation by chemicals and viruses, the role of environment versus stem cells in the aetiology of haemopoietic aplasias and the possible usefulness of long-term cultures for bone marrow transplantation.     

Isolation and culture of endothelial cells from human bone marrow

Summary : Adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelium are potentially important in the homing of these cells back to re-establish haemopoiesis following stem cell transplantation. A simple method for the isolation and culture of human bone marrow endothelial cells is described using bone marrow aspirates obtained from patients undergoing bone marrow harvests for autologous or syngeneic bone marrow transplantation. The method is based on the selective binding of the lectin Ulex europaeus agglutinin-1 (UEA-1) to endothelial cells. Magnetic Dynabeads coupled with UEA-1 were incubated with single cell suspensions of bone marrow following red cell lysis, and bound cells were isolated with a magnet. The isolated cells demonstrated positive immunofluorescence staining for von Willebrand factor. Cells were plated onto tissue culture flasks coated with extracellular matrix derived from human umbilical vein endothelial cells in an endothelial serum-free medium together with 5% fetal calf serum for 24h. Cells were then cultured in endothelial serum-free growth medium supplemented with 5% fetal calf serum, endothelial cell growth supplemented with 5% fetal calf serum, endothlial cell growth supplement and heparin. After 2–4 weeks in culture, two morphologically different cell populations can be identified. One has a polygonal spindle-shaped morphology with a rapid growth rat, the other a rounded morphology and a slow growth rate. Both population have a vasiculated cytoplasm. Positive immunostaining of the cells was demonstrated with a number of endothelial cell markers including von Willebrand factor, and antibodies to ICAM-1, VCAM-1, E-wlectin, CD31 and BMA120. Weibel-Palade bodies were observed by electron microscopy.
Culture of these cells will allow detailed in vitro studies of adhesion mechanisms in the homing of haemopoietic progenitor cells.     

Expression of integrins in human bone marrow

Expression of integrins, a superfamily of glycoprotein alpha/beta heterodimers which integrate the cytoskeleton with the extracellular matrix and/or mediate cell-cell adhesive interactions, was examined on normal and leukaemic bone marrow cells by immunohistochemistry and immunotransmission electron microscopy (immuno-TEM). Among the beta 1/VLA molecules studied, VLA-2 and 6 were expressed on megakaryocytes and platelets, while VLA-4 was present on 40% of haemopoietic cells, including monocytes, erythroblasts and immature cells; this molecule was typically localized at sites of intercellular contact, as seen by immuno-TEM, suggesting it may be involved in interactions among haemopoietic cells during differentiation. In human long-term bone marrow cultures (LTBMC), VLA-1 and 3 were present respectively on 35% and 40% of the adherent cells which included fibroblasts and endothelial cells, as shown by double-labelling experiments; VLA-2 was expressed only on a subpopulation of fibroblasts. beta 2/LeuCAM molecules were absent from platelets, megakaryocytes and HLA-DR+/myeloperoxidase- early myeloid precursors, and appeared progressively during maturation in both lymphoid and myeloid cells. Expression of beta 3/cytoadhesin molecules was restricted to megakaryocytes and platelets and, in the adherent layer of LTBMC, to endothelial cells. The regulated expression and specific localization of integrins in the bone marrow suggest that these molecules may have a role in normal haemopoiesis.     

Contrasting patterns of neoplastic cell behaviour in long-term culture of bone marrow from patients with acute leukaemia and myelodysplastic disorders

Alterations in neoplastic cell behaviour responsible for increased production of terminally-differentiated granulocytes during long-term culture of bone marrow in different categories of acute leukaemia and myelodysplasia have been investigated. An increase in neutrophils associated with transition to a morphological picture identical to normal control cultures occurred in 15 of 25 studies on acute leukaemia in contrast to one of six studies on myelodysplastic disorders. An abnormal neoplastic karyotype was employed as a marker for monitoring the course of the neoplastic cell population in 11 studies in which there was progression towards a normal pattern of differentiation. An increase in differentiation was shown by this means to represent increased maturation of cells of the neoplastic process in one study on a myelodysplastic disorder, demonstrating domination of proliferative activity in culture by all of the myelodysplastic disorders examined. Transition towards normal differentiation in nine studies on acute leukaemia, however, correlated with partial or complete replacement of the acute leukaemic cells by normal haemopoietic series in de novo acute leukaemia, and by Ph positive cells in blast crisis of CML. Conversion to morphologically and cytogenetically normal cell populations in five studies on de novo acute leukaemia occurred in four cases which failed to respond to remission-induction therapy, suggesting the selective toxic effect capable of purging acute leukaemic cells from bone marrow operated by a mechanism which lacked cross-resistance to currently-employed cytotoxic agents.     

Platelet-derived growth factor stimulates growth of highly enriched multipotent haemopoietic progenitors

Platelet-derived growth factor (PDGF) has been shown to stimulate growth of normal and malignant fibroblasts, glial cells and smooth muscle cells. A growth promoting effect on human haemopoietic precursors has also been described, but the interpretation of this haemopoietic proliferative response to PDGF has been hampered by the lack of purity of the target population. In this study we show that PDGF promotes growth of early bone marrow haemopoietic progenitors depleted of either monocytes or T lymphocytes which are known to influence haemopoiesis. Moreover, the action of PDGF is even increased on a highly enriched BI-3C5 early bone marrow population. BI-3C5 is a novel monoclonal antibody which recognizes an antigen present on all multilineage colony-forming cells (CFU-mix) (Tindle et al. 1985). BI-3C5 positively and negatively sorted fractions were obtained by fluorescence activated cell sorting (FACS) and PDGF was found to stimulate growth of CFU-mix in the BI-3C5-positive fraction (consisting of only 4-6% of the marrow population), the effect being more marked than that on unsorted bone marrow. The results suggest that the product of the cellular proto-oncogene c-sis (the putative structural gene for the beta chain of PDGF) may play a regulatory role in the in vivo proliferation of multipotent haemopoietic progenitors.     

Haemopoietic progenitor cell lines generated by the myeloproliferative leukaemia virus: a model system to analyse murine and human lineage-affiliated genes

Multipotential progenitor and stem cells occur with a low frequency in haemopoietic tissue. As a result, it is often difficult to obtain sufficient numbers of cells to undertake many of the assays that would be informative about the molecular events involved in the regulation of lineage-affiliated genes within these multipotent cells. To circumvent this problem, we have used the myeloproliferative leukaemia virus (MPLV) to generate a phenotypically diverse array of haemopoietic progenitors from adult mouse bone marrow and embryonic blood. These cells could be expanded to perform a variety of analyses that would not previously have been possible using analogous primary cells. The validity of these assays was supported by the observation that the phenotype of several MPLV-infected lines was very similar to previously described primary haemopoietic progenitor cells. By using mice transgenic for the human alpha and beta globin gene clusters, we have shown that human genes may also be investigated. In addition, this strategy has a wide potential applicability including the rescue of haemopoietic progenitors from mouse embryos lacking genes critical for their survival as well as the study of any haemopoietic gene for which an appropriate transgenic mouse is available.     

Competition between recipient and donor cells after bone marrow transplantation for chronic myeloid leukaemia

The cytogenetic and clinical course of three patients allografted for Ph positive chronic myeloid leukaemia are reported. All patients had a peculiar pattern of relapse. Two out of three patients had donor marrow graft pretreated with monoclonal antibody for graft versus host prevention. The cytogenetic relapse was invariably associated with major morphological changes in the marrow indicating that these were also haematological relapses. However, no changes in the peripheral blood count were observed. When relapse occurred in these patients, Ph positive marrow metaphases and host red blood cells ranged from 75% to 100% of the total cell population: thereafter they spontaneously reverted to complete chimaerism. Therefore the presence of leukaemic cells even in considerable amount was not sufficient, per se, to prevail over normal marrow. In addition these observations indicate that relapse was not associated with elimination of the graft: while haemopoiesis was entirely of recipient origin the donor normal stem cells were present and vital although functionally silent. These data suggest that, although TBI remains the more effective tool for eradicating the majority of leukaemic cells, haemopoietic competition between host and donor marrow may have a major impact on leukaemic relapse.