Antibody-producing human-human hybridomas. III. Derivation and characterization of two antibodies with specificity for human myeloid cells

Peripheral blood mononuclear cells from a patient with acute myeloid leukemia (AML) and spleen cells from a patient with chronic myeloid leukemia (CML) were fused with HAT-sensitive human B lymphoma cells (RH-L4) in attempts to generate human monoclonal antibodies (Mab) against antigens with high specificity for myeloid leukemia cells. Forty-seven of 246 hybridomas secreted Ig that bound to AML cell surface constituents, as determined by FACS analysis of viable cells that were FITC-stained with the human Mab as the first-step reagent and FITC-conjugated rabbit anti-human Ig as second-step. Two of the 47 human Mab (one from each patient and designated AML-19 and CML-20, respectively) bound to both autologous and allogeneic myeloid leukemia cells. No significant binding was observed to cell surface constituents on human bone marrow cells, granulocytes, lymphocytes, erythrocytes, thymocytes, monocytes, lymphoblastic leukemia cells, fibroblasts, malignant B and T lymphocytic cell lines, and murine bone marrow cells. Both human Mab were IgG and were cytotoxic to myeloid leukemia cells in the presence of complement. About 70% of peripheral blood cell samples from 46 AML patients contained AML-19- and CML-20-positive cells, but the reactivity pattern had no correlation to the morphologic FAB classification of the samples. The promyelocytic HL60 cell line and the K562 cell line reacted with the two antibodies. Dot blot analysis of binding of AML-19 and CML-20 to cellular extracts immobilized on nitrocellulose paper showed that both human Mab in this assay also reacted with normal bone marrow cells. This was supported by microscopic immunofluorescence because both human Mab stained intracytoplasmatic structures in normal bone marrow cells, but both intracytoplasmatic and cell surface components stained in myeloid leukemia cells. Moreover, immunoblotting demonstrated that both human Mab in leukemia cells reacted with two cellular proteins with Mr approximately 14,500 and 18,000, and in normal bone marrow cells with a molecule with Mr approximately 20,000. Immunoprecipitation of cell membrane molecules with both the AML-19 and CML-20 antibody precipitated from leukemic cells only the molecule with Mr approximately 18,000 and no components from normal bone marrow cells. It is concluded that myeloid leukemogenesis may result in generation of cell surface expression of either new or abnormally processed molecules that are immunogenic in the autochthonous host. These molecules may also be useful as markers in diagnosis of myeloid leukemia.     

Normal hematopoiesis and hematologic malignancies: Role of canonical Wnt signaling pathway and stromal microenvironment

Wnts are a family of evolutionary-conserved secreted signaling molecules critically involved in a variety of developmental processes and in cell fate determination. A growing body of evidence suggests that Wnt signaling plays a crucial role in the influence of bone marrow stromal microenvironment on the balance between hematopoietic stem cell self-renewal and differentiation. Emerging clinical and experimental evidence also indicates Wnt signaling involvement in the disruption of the latter balance in hematologic malignancies, where the stromal microenvironment favors the homing of cancer cells to the bone marrow, as well as leukemia stem cell development and chemoresistance. In the present review, we summarize and discuss the role of the canonical Wnt signaling pathway in normal hematopoiesis and hematologic malignancies, with regard to recent findings on the stromal microenvironment involvement in these process and diseases.     

Monosomy 7 in donor cell-derived leukemia after bone marrow transplantation for severe aplastic anemia: Report of a new case and review of the literature

Monosomy 7 arises as a recurrent chromosome aberration in donor cell leukemia after hematopoietic stem cell transplantation. We report a new case of donor cell leukemia with monosomy 7 following HLA-identical allogenic bone marrow transplantation for severe aplastic anemia (SAA). The male patient received a bone marrow graft from his sister, and monosomy 7 was detected only in the XX donor cells, 34 months after transplantation. The patient’s bone marrow microenvironment may have played a role in the leukemic transformation of the donor hematopoietic cells.     

Pathophysiological Scenario of Hematopoietic Disorders: A Comparative Study of Aplastic Anemia,Myelodysplastic Syndrome and Leukemia in Experimental Animals

The conversion of physiology to pathophysiology in hematological disorders viz: aplastic anemia, myelodysplastic syndrome (MDS) and leukemia in murine models was the subject of study in the present programme. Peripheral blood hemogram, spleno-somatic index, bone marrow smear study, cytochemical staining of marrow, cell release kinetics study during marrow explants culture, hematopoietic niche assessment, chromosomal aberration study, plasma membrane stability study of marrow cells, lysosomal membrane and mitochondrial membrane stability study and innate immune parameters were performed in the aplastic anemia, leukemia and MDS mouse model. In bone marrow aplasia, peripheral blood pancytopenia, marrow hypocellularity, decreased marrow cellular viability, deterioration of bone marrow hematopoiesis as well as hematopoietic microenvironment and extramedullary hematopoiesis were noticed. In addition, disruption of mitochondrial and lysosomal membrane integrity along with reduction of innate immune parameters were found in the hematopoietic suppressed condition. Surprisingly, no noticeable chromosomal aberration was found in the aplastic condition. Ineffective marrow hematopoiesis together with the disruption of hematopoietic microenvironment was observed in MDS. Also, extramedullary hematopoiesis, increased marrow cellular death, chromosomal aberration and loss of innate immunity were the common events. During leukemia, the number of functionally and structurally immature cells in the peripheral blood and bone marrow was increased together with malignant conversion of hematopoietic cells in the presence of malignancy supportive stromal microenvironment. Chromosomal aberration, decrease of cell mediated immunity with least mitochondrial apoptotic damage were also found in leukemic condition as well.     

How do normal and leukemic white blood cells egress from the bone marrow? Morphological facts and biochemical riddles

Under normal circumstances only mature granulocytes and monocytes cross the bone marrow sinus wall, a trilaminar structure consisting of endothelial cells, a discontinuous basal membrane and an adventitial cell layer in order to get access to the blood circulation. In leukemia, however, immature white blood cells are able to traverse the barrier and to appear in the blood stream. Very little is known about the regulatory processes which govern the egress of white blood cells in healthy individuals and their malignant counterparts in patients with leukemia. The results of the few studies performed to address this question in animal and human leukemias all agree that the extent to which adventitial cells (fibroblasts) cover the endothelium in bone marrow is drastically reduced. This implies altered interactions between the leukemic and adventitial cells and their extracellular matrix. We propose here a model to explain the egress of normal cells and their leukemic counterparts. It is based upon our own experimental data and the general at present limited knowledge of the subject. It is hoped that this model will stimulate further research into this important aspect of leukemogenesis.     

Functional ramifications for the loss of P-selectin expression on hematopoietic and leukemic stem cells

Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between blood cells and their microenvironment to preserve the homeostatic balance of long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and differentiated cells. Adhesion molecules like P-selectin (encoded by the Selp gene) are essential to hematopoiesis, and their dysregulation has been linked to leukemogenesis. Like HSCs, leukemic stem cells (LSCs) depend upon their microenvironments for survival and propagation. P-selectin plays a crucial role in Philadelphia chromosome-positive (Ph(+)) chronic myeloid leukemia (CML). In this paper, we show that cells deficient in P-selectin expression can repopulate the marrow more efficiently than wild type controls. This results from an increase in HSC self-renewal rather than alternative possibilities like increased homing velocity or cell cycle defects. We also show that P-selectin expression on LT-HSCs, but not ST-HSCs and MPPs, increases with aging. In the absence of P-selectin expression, mice at 6 months of age possess increased levels of short-term HSCs and multipotent progenitors. By 11 months of age, there is a shift towards increased levels of long-term HSCs. Recipients of BCR-ABL-transduced bone marrow cells from P-selectin-deficient donors develop a more aggressive CML, with increased percentages of LSCs and progenitors. Taken together, our data reveal that P-selectin expression on HSCs and LSCs has important functional ramifications for both hematopoiesis and leukemogenesis, which is most likely attributable to an intrinsic effect on stem cell self-renewal.     

Bone marrow microenvironment: The guardian of leukemia stem cells

Bone marrow microenvironment(BMM) is the main sanctuary of leukemic stem cells(LSCs) and protects these cells against conventional therapies. However, it may open up an opportunity to target LSCs by breaking the close connection between LSCs and the BMM. The elimination of LSCs is of high importance, since they follow cancer stem cell theory as a part of this population. Based on cancer stem cell theory, a cell with stem cell-like features stands at the apex of the hierarchy and produces a heterogeneous population and governs the disease.Secretion of cytokines, chemokines, and extracellular vesicles, whether through autocrine or paracrine mechanisms by activation of downstream signaling pathways in LSCs, favors their persistence and makes the BMM less hospitable for normal stem cells. While all details about the interactions of the BMM and LSCs remain to be elucidated, some clinical trials have been designed to limit these reciprocal interactions to cure leukemia more effectively. In this review, we focus on chronic myeloid leukemia and acute myeloid leukemia LSCs and their milieu in the bone marrow, how to segregate them from the normal compartment, and finally the possible ways to eliminate these cells.     

Development of a bone marrow culture for maintenance and growth of normal human B cell precursors.

The absence of long term bone marrow cultures for studying the growth and differentiation of human B cell precursors (BCP) has placed restrictions on the ability to analyze the early stages of human B cell ontogeny. We now describe a bone marrow-derived adherent cell microenvironment that maintains human BCP for several weeks in vitro. The adherent cells are maintained in a serum-free tissue culture medium, and consist of a predominant population of CD10+ fibroblast-like cells and a minor population of CD10+/nonspecific esterase+ macrophages. Adherent cell cultures seeded with fresh or cryopreserved fetal bone marrow, or purified CD10+/surface IgM- cells, provide a supportive microenvironment for lymphoid cells with a predominant phenotype of CD10+/CD19+/HLA-DR+/surface IgM-. Supplementation of the adherent cell cultures with human IL-7 induces active growth of BCP during the first 14 to 21 days of culture. However, the expansion of these cells does not continue past 21 days, and the cultures undergo a steady decline in BCP. Analysis of adherent cell conditioned medium revealed the presence of an unidentified soluble factor (or factors) that acts in concert with IL-7 to promote the growth of CD10+/surface IgM- cells. This culture system will be useful in elucidating the patterns of gene expression and growth factor requirements that characterize normal human B cell ontogeny, and perturbations of normal B cell ontogeny that lead to immunodeficiency and leukemia.     

Pronounced hypoxia in models of murine and human leukemia: high efficacy of hypoxia-activated prodrug PR-104

Recent studies indicate that interactions between leukemia cells and the bone marrow (BM) microenvironment promote leukemia cell survival and confer resistance to anti-leukemic drugs. There is evidence that BM microenvironment contains hypoxic areas that confer survival advantage to hematopoietic cells. In the present study we investigated whether hypoxia in leukemic BM contributes to the protective role of the BM microenvironment. We observed a marked expansion of hypoxic BM areas in immunodeficient mice engrafted with acute lymphoblastic leukemia (ALL) cells. Consistent with this finding, we found that hypoxia promotes chemoresistance in various ALL derived cell lines. These findings suggest to employ hypoxia-activated prodrugs to eliminate leukemia cells within hypoxic niches. Using several xenograft models, we demonstrated that administration of the hypoxia-activated dinitrobenzamide mustard, PR-104 prolonged survival and decreased leukemia burden of immune-deficient mice injected with primary acute lymphoblastic leukemia cells. Together, these findings strongly suggest that targeting hypoxia in leukemic BM is feasible and may significantly improve leukemia therapy.     

Molecular Signatures of Quiescent,Mobilized and Leukemia-Initiating Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) are rare, multipotent cells capable of generating all specialized cells of the blood system. Appropriate regulation of HSC quiescence is thought to be crucial to maintain their lifelong function; however, the molecular pathways controlling stem cell quiescence remain poorly characterized. Likewise, the molecular events driving leukemogenesis remain elusive. In this study, we compare the gene expression profiles of steady-state bone marrow HSC to non-self-renewing multipotent progenitors; to HSC treated with mobilizing drugs that expand the HSC pool and induce egress from the marrow; and to leukemic HSC in a mouse model of chronic myelogenous leukemia. By intersecting the resulting lists of differentially regulated genes we identify a subset of molecules that are downregulated in all three circumstances, and thus may be particularly important for the maintenance and function of normal, quiescent HSC. These results identify potential key regulators of HSC and give insights into the clinically important processes of HSC mobilization for transplantation and leukemic development from cancer stem cells.     

CD28 expressed on malignant plasma cells induces a prosurvival and immunosuppressive microenvironment

Interactions between the malignant plasma cells of multiple myeloma and stromal cells within the bone marrow microenvironment are essential for myeloma cell survival, mirroring the same dependence of normal bone marrow-resident long-lived plasma cells on specific marrow niches. These interactions directly transduce prosurvival signals to the myeloma cells and also induce niche production of supportive soluble factors. However, despite their central importance, the specific molecular and cellular components involved remain poorly characterized. We now report that the prototypic T cell costimulatory receptor CD28 is overexpressed on myeloma cells during disease progression and in the poor-prognosis subgroups and plays a previously unrecognized role as a two-way molecular bridge to support myeloid stromal cells in the microenvironment. Engagement by CD28 to its ligand CD80/CD86 on stromal dendritic cell directly transduces a prosurvival signal to myeloma cell, protecting it against chemotherapy and growth factor withdrawal-induced death. Simultaneously, CD28-mediated ligation of CD80/CD86 induces the stromal dendritic cell to produce the prosurvival cytokine IL-6 (involving novel cross-talk with the Notch pathway) and the immunosuppressive enzyme IDO. These findings identify CD28 and CD80/CD86 as important molecular components of the interaction between myeloma cells and the bone marrow microenvironment, point to similar interaction for normal plasma cells, and suggest novel therapeutic strategies to target malignant and pathogenic (e.g., in allergy and autoimmunity) plasma cells.     

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.     

Remission of acute myeloid leukemia: studies of in vitro colony formation by bone marrow cells

Human bone marrow contains cells which form leukocyte colonies in semisolid culture media. Each leukocyte colony arises from a single colony-forming cell which is thought to be a unipotential stem cell, and which is subject to regulation in vitro by colony-stimulating factor. In acute myelogenous leukemia variable abnormalities in colony formation by marrow cells occur. Usually colony formation either fails to occur or the colonies that are formed are small and contain fewer than 50 cells. Similar abnormalities have been described in bone marrow dysfunction preceding overt leukemia. Usually remission of leukemia is accompanied by improved cloning by marrow cells. In this study three patients are reported in whom remission was associated with impaired cloning, and one of these patients has remained in continuous remission for a further 18 months. These observations suggest that remission status is not necessarily associated with repopulation of the bone marrow by normal hematopoietic cells.     

The transmural migration and release of blood cells in acute myelogenous leukemia.

The transmural passage of malignant blood cells from the extravascular parenchyma into sinusoidal lumen has been studied in the bone marrow of rats with myelogenous leukemia. The Shay myelogenous leukemia was chosen as a model system because an increased bone marrow cellularity is, in this leukemia, usually accompanied by an increase in circulating myeloid cells. By means of light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) it was found that the sinusoidal endothelial lining of the bone marrow remains intact and continuous even in advanced stages of the disease. SEM shows that the malignant myeloblast-like cell enters the sinusoidal lumen by means of a temporary migration pore, which appears only during the transmural passage of the cell. Certain nondegenerative changes in the sinusoidal blood vessels are associated with the myelogenous leukemia. The normal radial alignment of sinusoids about the central sinusoid is changed into a tortuous pattern, and intraluminal cytoplasmic bridges which impede the blood flow are formed by the endothelial cells.     

Increased mobilization of c-kit+ Sca-1+ Lin- (KSL) cells and colony-forming units in spleen (CFU-S) following de novo formation of a stem cell niche depends on dynamic, but not stable, membranous ossification

Stem cells are thought to inhabit in a unique microenvironment, known as "niche," in which they undergo asymmetric cell divisions that results in reproducing both stem cells and progenies to maintain various tissues throughout life. The cells of osteoblastic lineage have been identified as a key participant in regulating the number of hematopoietic stem cells (HSCs). HSCs receive their regulatory messages from the microenvironment in the bone marrow. This would account for a reason why the localization of hematopoiesis is usually restricted in the bone marrow. To clarify the above possibility we employed a cell implantation-based strategy with a unique osteoblast cell line (KUSA-A1) derived from a C3H/He mouse. The implantation of KUSA-A 1 cells resulted in the generation of ectopic bones in the subcutaneous tissues of the athymic BALB/c nu/nu mice. Subsequently the mice obtained a greater amount of the bone marrow than normal mice, and they showed an increased number of HSCs. These results indicate that the newly generated osteoblasts-derived ectopic bones are responsible for the increase in the number of the HSC population. Furthermore, the increased number of HSCs directly correlates with both the magnitude of dynamic osteogenic process and the size of the newly generated bone or "niche."     

Radiobiological properties of human hematopoietic and stromal marrow cells

Effective hematopoiesis requires the presence of normal hematopoietic progenitors and a supporting microenvironment. Impairment of one of these marrow compartments will result in marrow failure. Total body irradiation (TBI) followed by bone marrow transplantation (BMT) is becoming an established modality in the treatment of malignant hematopoietic disorders. The objectives of irradiation are to ablate host marrow and immunocompetent cells as well as to eradicate neoplastic cells. Although leukemic cells are thought to have the same radiobiological characteristics as their normal counterparts, it has been proposed recently that some leukemic cells may possess a substantial capacity to repair sublethal radiation damage. Thus, radiation administered at different dose rates or fractions might differ in its ability to ablate malignant cells and consequently affect the relapse rate in the post-transplant period. Different modes of irradiation can also affect the proliferative capacity and the hematopoietic supportive function of the marrow microenvironment. Bone marrow ablation must be accomplished with the least possible damage to other tissues. Impairment of the proliferative capacity of the marrow microenvironment or its hematopoietic supportive function can result in graft failure in the post-transplant period. In this review, we discuss the radiobiological characteristics of normal hematopoietic, leukemic and stromal cells and their relevance to bone marrow transplantation.     

K562 chronic myeloid leukemia cells modify osteogenic differentiation and gene expression of bone marrow stromal cells

Bone marrow (BM) microenvironment plays an important role in normal and malignant hematopoiesis. As a consequence of interaction with the leukemic cells, the stromal cells of the bone marrow become deregulated in their normal function and gene expression. In our study, we found that mesenchymal stem cells (MSC) from BM of chronic myeloid leukemia (CML) patients have defective osteogenic differentiation and on interaction with K562 CML cells, the normal MSC showed reduced osteogenic differentiation. On interaction with K562 cells or its secreted factors, MSC acquired phenotypic abnormalities and secreted high levels of IL6 through NFκB activation. The MSC derived secreted factors provided a survival advantage to CML cells from imatinib induced apoptosis. Thus, a therapy targeting stromal cells in addition to leukemia cells might be more effective in eliminating CML cells.     

Disruption of Annexin II /p11 Interaction Suppresses Leukemia Cell Binding,Homing and Engraftment,and Sensitizes the Leukemia Cells to Chemotherapy

The bone marrow microenvironment plays an important role in acute lymphoblastic leukemia (ALL) cell proliferation, maintenance, and resistance to chemotherapy. Annexin II (ANX2) is abundantly expressed on bone marrow cells and complexes with p11 to form ANX2/p11-hetero-tetramer (ANX2T). We present evidence that p11 is upregulated in refractory ALL cell lines and patient samples. A small molecule inhibitor that disrupts ANX2/p11 interaction (ANX2T inhibitor), an anti-ANX2 antibody, and knockdown of p11, abrogated ALL cell adhesion to osteoblasts, indicating that ANX2/p11 interaction facilitates binding and retention of ALL cells in the bone marrow. Furthermore, ANX2T inhibitor increased the sensitivity of primary ALL cells co-cultured with osteoblasts to dexamethasone and vincristine induced cell death. Finally, in an orthotopic leukemia xenograft mouse model, the number of ALL cells homing to the bone marrow was reduced by 40–50% in mice injected with anti-ANX2 antibody, anti-p11 antibody or ANX2T inhibitor compared to respective controls. In a long-term engraftment assay, the percentage of ALL cells in mouse blood, bone marrow and spleen was reduced in mice treated with agents that disrupt ANX2/p11 interaction. These data show that disruption of ANX2/p11 interaction results in reduced ALL cell adhesion to osteoblasts, increased ALL cell sensitization to chemotherapy, and suppression of ALL cell homing and engraftment.