Detection of lymphoid leukemia colony-forming cells in abelson virus infected mice: Differences in inbred strains

BALB/c or DBA/2 mice were infected with Abelson murine leukemia virus (A-MuLV), pseudotype Molony murine leukemia virus (M-MuLV). Infection of these mice with 10⁴ focus-forming units of A-MuLV (M-MuLV) induced overt leukemia, detectable grossly or microscopically in 90% of the mice at 20–38 days. However, these methods did not detect leukemia at 17 days or before. Bone marrow cells from A-MuLV-infected leukemic or preleukemic mice were placed in tissue culture in a soft agarose gel. Cells from leukemic or preleukemic BALB/c mice grew to form colonies of 10³ cells or more, composed of lymphoblasts, whereas marrow cells from normal uninfected mice did not. Cells from these colonies grew to form ascitic tumors after intraperitoneal inoculation into pristane-primed BALB/c recipient. Colony-forming leukemia cells could be detected in the marrow of A-MuLV-infected mice as early as 8 days after virus incoluation. The number of colony-forming leukemia cells increased as a function of time after virus inoculation. Colony-forming leukemia cells require other cells in order to replicate in tissue culture. Normal bone marrow cells, untreated or after treatment with mitomycin-C, provide this “helper” function. Only in the presence of untreated or mitomycin-C treated helper cells was the number of colonies approximately proportional to the number of leukemia cells plated. Marrow cells from leukemic BALB/c mice form more colonies than those from leukemic DBA/2 mice. The number of colonies formed per 10³ microscopically identifiable leukemia cells plated was determined to be 2–3 for leukemic BALB/c mice and 0.3 for DBA/2 mice. Cocultivation of leukemic DBA/2 marrow cells with mitomycin-C treated normal BALB/c cells did not increase the number of colonies formed by the DBA/2 leukemic cells. Thus, the decreased ability of DBA/2 leukemia cells to form colonies appears to be a property of the leukemia cell population.     

Murine acute leukemia cell line with megakaryocytic differentiation (MK-8057) induced by whole-body irradiation in C3H/He mice: cytological properties and kinetics of its leukemic stem cells

Five cases of murine leukemia with megakaryocytic differentiation were observed among the 417 cases of radiation-induced leukemias which developed in 30% of C3H/HeMs mice exposed at 8 to 10 weeks to 0.5 to 5 gy total body irradiation. Cells from individual leukemic colonies in the spleen of the irradiated mice, and cells from colonies in methylcellulose (MC) culture in vitro, derived from one of these leukemias, MK-8057, were injected into mice; both types of cells caused the deaths of the recipient mice by inducing the same type of leukemia. MK-8057 can be maintained in Dexter-type liquid culture with a feeder layer of irradiated bone marrow cells. There was a linear reciprocal relationship between the increasing number of MK-8057 cells injected versus the survival of the recipient mice. A reciprocal relationship also was seen between an increasing number of leukemic stem cells, corresponding to the number of MK-8057 cells, and the survival of mice injected with MK-8057. Giant nuclear megakaryocytes developed during the course of colony growth in the spleen as they did in the MC culture. Such megakaryocytes were acetylcholinesterase positive, whereas leukemic cells in the peripheral blood showed no sign of platelet production nor of a positive reaction to acetylcholinesterase. Cells maintained in culture were entirely positive in platelet glycoprotein IIb/IIIa when anti-human antibody was used. The larger cells in a splenic cell suspension derived from a moribund mouse were separated and enriched by velocity sedimentation using centrifugal elutriation (CE), and then subjected to flow cytometry using propidium iodide staining. Cells with up to 32N-DNA content were detected. After separating MK-8057 by counter-flow CE, the larger cell fraction (mode at 540 microns3) produced more leukemic colonies when injected into irradiated mice than did the small cell fraction (mode at 240 microns3). A higher percent of the larger cell fraction (61.9%) was killed by the addition of tritiated thymidine cytocide than in the smaller cell fraction (14.9%). Thus, the smaller cell fraction is considered to have more leukemic spleen colony-forming units (L-CFU-s) in the resting state.     

Similarities in long-term cultures of blood and bone marrow from patients with acute myelogenous leukemia

Dexter-type long-term cultures (LTC) were initiated with peripheral blood (PB) and/or bone marrow cells from 11 patients with acute myelogenous leukemia (AML), and 2 with myelodysplastic syndrome in blastic transformation. Marrow and PB cells from normal subjects served as controls. Assessment of nucleated cells and clonogenic progenitors in the adherent and nonadherent fractions of LTC revealed active hemopoiesis for greater than 5 wks in 4 of 8 cultures of AML blood, and 4 of 7 of AML marrow. The morphology and kinetics of nucleated cells and progenitors with putative normal (granulocyte-macrophage colony-forming units or CFU-gm), and abnormal (blast) phenotype in LTC from AML blood were similar to those from AML marrow, and adherent cells positive for collagen I and III and vimentin were found in both types of LTC. Growth of CFU-gm colonies ceased by wk 5-8 in AML cultures, significantly earlier than in LTC of normal marrow cells (survival of greater than 10 wks), which may indicate derivation of the CFU-gm from a transformed clone or deficiency of stromal function in the leukemic state. In most AML blood and AML marrow LTCs, growth of abnormal (blast) colonies continued until wk 4-6. This study demonstrates certain similarities of morphology and function between LTC of AML blood and AML marrow cells. LTC may provide a useful model for further analysis of circulating primitive hemopoietic progenitor cells in leukemic states.     

Proliferation and differentiation of Abelson virus-infected murine myeloid leukemia cell lines does not involve an autocrine mechanism

Culture in agar of cloned promonocytic leukemia cell lines derived from Abelson virus-infected mice produced colonies of both a compact and diffuse morphology. Diffuse colonies contained fewer cells capable of forming colonies when recultured in agar than did compact colonies. Serial subcloning of cells from diffuse, but not compact, colonies ultimately led to the complete loss of colony-forming cells, i.e., to clonal extinction. The production of both compact and diffuse agar colonies was independent of the cell density of either the static liquid culture from which cells were taken for culture in agar, or the number of cells per agar culture. Furthermore, bioassays of culture supernatants indicated the leukemia cells did not secrete hemopoietic growth factors active on normal hemopoietic cells, transforming growth factors active on adherent cell lines, or factors that influenced the growth of the leukemic cells themselves. Collectively, these data suggest neither growth-factor independent replication nor the spontaneous differentiation of Abelson virus-infected myeloid cells involves autocrine secretion of growth regulators.     

Multiple steps are required for the induction of tumors by Abelson murine leukemia virus.

Helper virus-free Abelson murine leukemia virus (A-MuLV) was used to induce monoclonal pre-B-cell tumors in mice. The clonality, patterns of immunoglobulin heavy-chain gene rearrangement, tumorigenicity, and v-abl oncogene expression in individual preleukemic and leukemic colonies were compared. Our results indicate that A-MuLV preleukemic cells with low or undetectable tumorigenic potential give rise to leukemic cells with high tumorigenic potential by a process of subclone selection. The levels of v-abl oncogene product in preleukemic and leukemic cell populations were not significantly different. These results suggest that an additional event(s) unrelated to the level of the v-abl protein product is required for A-MuLV-transformed cells to become fully malignant.     

Proliferation response of leukemic cells to CD70 ligation oscillates with recurrent remission and relapse in a low-grade lymphoma

Interactions of the TNF-related cell surface ligand CD70 with its receptor CD27 provide a costimulatory signal in B and T cell activation. Functional CD70-CD27 interactions could contribute to lymphoma and leukemia progression. This possibility was studied using DNA microarrays on a unique case of low-grade lymphoma/leukemia characterized by recurrent cycles of acute leukemic phase alternating with spontaneous remission. Upon induction of the acute phase expression of CD70 and CD27 in the leukemic cells increased 38- and 25-fold, respectively. Coexpression of membrane CD70 and CD27 on the leukemic (CD5+CD19+) cells was maximal 2-3 days following initiation of the attack. Soluble CD27 in the patient's serum was elevated during remission and further increased in the attack. Functional tests showed that neither anti-CD70 nor anti-CD27 Abs affect the rate of apoptosis. However, the anti-CD70 Ab specifically enhanced proliferation of the remission phase leukemic cells, whereas proliferation of the acute-phase counterparts that express higher level of membrane CD70 was unaffected. Hence, in this lymphoma/leukemia, membrane CD70 is presented on the leukemic cells in a responsive state during the remission and a nonresponsive state during the attack. Presumably, CD70 in its responsive state provides a costimulatory receptor for initiating the next acute phase while its nonresponsive state enables the remission.     

Treatment of a fatal transplantable erythroleukemia by procedures that lower endogenous erythropoietin

The in vitro growth of primary erythroleukemia cells has been examined in the presence and absence of the hormone erythropoietin (EPO). Although these leukemic cells had previously been considered to be hormone-independent, addition of EPO was found to be essential for maximum growth in culture. Erythroid colonies that grew in the presence of EPO were leukemogenic when returned to mice. Influence of EPO on the in vivo growth of leukemic cells was indicated by our findings that administration of the hormone caused a more severe leukemia and rapid death, and transfusion of red blood cells, which lowers endogenous EPO, led to decreased spleen size and increased survival of leukemic mice. We suggest from our results that hormone-associated therapy might be efficacious in the treatment of this and, perhaps, other leukemias.     

The role of humoral factors in the regression of leukemia in chickens as measured by in vitro colony formation

Leukemic myeloblasts induced by avian myeloblastosis virus in the chicken formed small compact (type II) colonies in semi-solid agar medium. Normal yolk sac cells from 12-day old embryos formed large diffuse (type I) colonies under the same conditions. Type I colony formation (but not type II) was strictly dependent upon the presence in the medium of a colony stimulating factor (CSF) present in fresh chicken serum or conditioned medium. Serum CSF levels were determined for normal, leukemic, and birds which had spontaneously regressed from myeloblastic leukemia. When type I colony formation was used as the assay, serum CSF levels of leukemic birds were found to be significantly lower than levels in either normal or regressed birds. When the same sera were tested for their ability to induce type II colonies, leukemic birds demonstrated a significantly higher CSF level than either normal or regressed sera. Regressed chickens had serum CSF levels similar to normal birds.     

Stability Analysis of a Model of Interaction Between the Immune System and Cancer Cells in Chronic Myelogenous Leukemia

We describe here a simple model for the interaction between leukemic cells and the autologous immune response in chronic phase chronic myelogenous leukemia (CML). This model is a simplified version of the model we proposed in Clapp et al. (Cancer Res 75:4053–4062, 2015). Our simplification is based on the observation that certain key characteristics of the dynamics of CML can be captured with a three-compartment model: two for the leukemic cells (stem cells and mature cells) and one for the immune response. We characterize the existence of steady states and their stability for generic forms of immunosuppressive effects of leukemic cells. We provide a complete co-dimension one bifurcation analysis. Our results show how clinical response to tyrosine kinase inhibitors treatment is compatible with the existence of a stable low disease, treatment-free steady state.     

Clonal analysis of the late stages of erythroleukemia induced by two distinct strains of Friend leukemia virus.

We observed striking differences between the tumorigenic colony-forming cells present in the spleens of mice late after infection with the anemia-inducing strain of Friend leukemia virus (strain FV-A) and those present after infection with the polycythemia-inducing strain (strain FV-P). Cells within primary colonies derived from FV-A- and FV-P-transformed cells (CFU-FV-A and CFU-FV-P, respectively) contained hemoglobin and spectrin, indicating that the CFU-FV-A and CFU-FV-P were transformed erythroid progenitor cells. The proportion of cells containing hemoglobin was relatively high (> 25%) in newly isolated cell lines derived from CFU-FV-P colonies, whereas cell lines derived from CFU-FV-A colonies had only low levels (0 to 2%) of hemoglobin-containing cells. A high proportion of the cell lines derived from CFU-FV-A colonies responded to pure erythropoietin and accumulated spectrin and hemoglobin, whereas the cell lines derived from CFU-FV-P colonies did not. A cytogenetic analysis indicated that primary CFU-FV-P colony cells were diploid, whereas chromosomal aberrations were observed in the immediate progeny of CFU-FV-A. The presence of unique chromosomal markers in the majority of the cells within individual colonies derived from CFU-FV-A suggested that these colonies originated from single cells. Finally, leukemic progenitor cells transformed by strain FV-A appeared to have an extensive capacity to self-renew (i.e., form secondary colonies in methylcellulose), whereas a significant proportion of the corresponding cells transformed by strain FV-P did not. In addition, the self-renewal capacity of both CFU-FV-A and CFU-FV-P increased as the disease progressed. From these observations, we propose a model for the multistage nature of Friend disease; this model involves clonal evolution and expansion from a differentiating population with limited proliferative capacity to a population with a high capacity for self-renewal and proliferation.     

Simultaneous occurrence of myelomonocytic leukemia and multiple myeloma: involvement of common leukemic progenitors and their developmental abnormality of "lineage infidelity".

We investigated the origin of leukemic progenitors in a case of the simultaneous occurrence of myelomonocytic leukemia and multiple myeloma (IgG-kappa). At presentation, myeloperoxidase and nonspecific esterase-positive myelomonocytic cells had proliferated up to 12.2 x 10(9)/liter in the peripheral blood. Bone marrow cell differentials revealed the coexistence of myelomonocytic cells (30%) and atypical plasmacytoid cells (26%). Myelomonocytic cells in peripheral blood expressed both myeloid antigens (CD11b, CD13, CD14, CD15, CD33) and T/B-lymphoid antigens (CD2, CD4, CD5, CD7, CD10, PCA-1). Bone marrow mononuclear cells (BMMC) could be divided into PCA-1 strongly positive and PCA-1 weakly positive populations, which were considered to represent myeloma cells and myelomonocytic cells, respectively; the former were CD2-positive (CD2+), CD14-, and CD15-, whereas the latter were CD2+, CD14+, and CD15+. Immunohistochemical analysis revealed that, in addition to plasmacytoid cells, a minority of myelomonocytic cells showed a positive reaction for IgG staining, and production of IgG was observed in the culture supernatant of CD14+ myelomonocytic cells in peripheral blood. Southern blot analysis revealed the presence of two identical rearrangement bands of immunoglobulin heavy chain gene in both BMMC containing myeloma cells and myelomonocytic cells and CD14+ myelomonocytic cells in peripheral blood. In a long-term methylcellulose assay, peripheral blood mononuclear cells produced large compact colonies consisting of macrophages and IgG+ plasmacytoid cells (M phi/P colonies), while BMMC produced a different type of colonies consisting of CD14+ myelomonoblasts, macrophages, and IgG+ plasma cells (Mb/M phi/P colonies) in addition to M phi/P colonies. Recloning experiments showed that primary Mb/M phi/P colonies gave rise to both secondary M phi/P and Mb/M phi/P colonies. These observations strongly suggest that common leukemic progenitors provide both myeloma and myelomonocytic leukemia cells, and the mechanism of "lineage infidelity" is probably involved in the development of their "bilineal" differentiation.     

Nature of leukemic stem cells in murine myelogenous leukemia

We investigated the nature of myelogenous leukemic stem cells in mice. L-8057, a megakaryoblastic leukemia cell line used in this study, produces in vivo and in vitro colonies. By means of typical chromosomal aberrations in L-8057, one can conveniently detect the origin of the cells in each colony derived from a leukemic stem cell. Direct evidence of whether cells from each colony had leukemogenicity in recipient mice was successfully obtained by the colony transplantation assay. Both leukemic colony-forming unit-spleen (L-CFU-s) and leukemic colony-forming unit-culture (L-CFU-c) in L-8057 may have belonged to the same differentiating stage in the stem cells because of their similar radiosensitivity, although some parts of the L-CFU of L-8057 seemed to have lost their capability to regenerate L-CFU-s when the cells were plated in dishes. This leukemic stem cell preserves high self-renewal ability in vitro after 10 passages. In addition, in vitro colony formation by this leukemic cell during the above course of serial passages did not require any additional exogenous stimulators. The same sort of trials have been made on other types of leukemias. Leukemic stem cells showed remarkable variety in their response to stimulating factors and in their self-renewal activity, which suggests that they may have consisted of heterogeneous populations.     

Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells

In individuals with chronic myeloid leukemia (CML) treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft contribute to leukemic relapse, but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Here we show that CD44 expression is increased on mouse stem-progenitor cells expressing BCR-ABL and that CD44 contributes functional E-selectin ligands. In a mouse retroviral transplantation model of CML, BCR-ABL1-transduced progenitors from CD44-mutant donors are defective in homing to recipient marrow, resulting in decreased engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engraft as efficiently as do wild-type HSCs. CD44 is dispensable for induction of acute B-lymphoblastic leukemia by BCR-ABL, indicating that CD44 is specifically required on leukemic cells that initiate CML. The requirement for donor CD44 is bypassed by direct intrafemoral injection of BCR-ABL1-transduced CD44-deficient stem cells or by coexpression of human CD44. Antibody to CD44 attenuates induction of CML-like leukemia in recipients. These results show that BCR-ABL-expressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML.     

Global dynamics of hematopoietic stem cells and differentiated cells in a chronic myeloid leukemia model

We consider a mathematical model describing evolution of normal and leukemic hematopoietic stem cells (HSC) and differentiated cells in bone marrow. We focus on chronic myeloid leukemia (CML), a cancer of blood cells resulting from a malignant transformation of hematopoietic stem cells. The dynamics are given by a system of ordinary differential equations for normal and leukemic cells. Homeostasis regulates the proliferation of normal HSC and leads the dynamics to an equilibrium. This mechanism is partially efficient for leukemic cells. We define homeostasis by a functional of either hematopoietic stem cells, differentiated cells or both cell lines. We determine the number of hematopoietic stem cells and differentiated cells at equilibrium. Conditions for regeneration of hematopoiesis and persistence of CML are obtained from the global asymptotic stability of equilibrium states. We prove that normal and leukemic cells can not coexist for a long time. Numerical simulations illustrate our analytical results. The study may be helpful in understanding the dynamics of normal and leukemic hematopoietic cells.     

Detection of minimal disease in acute leukemia using flow cytometry: studies in a rat model for human acute leukemia

The study was made using a rat model for human acute myelocytic leukemia (BNML), which shows striking similarities with human acute myelocytic leukemia (AML). A monoclonal antibody (MCA-Rm124), raised against BNML cells, allowed the recognition of the leukemic cell fraction. The discriminative capacity of the monoclonal antibody is based on differences in labeling intensities between normal and leukemic cells. After i.v. cellular transfer of leukemia, the growth of the leukemic cell population in the bone marrow, the liver, and the spleen was monitored using MCA-Rm124 and flow cytometry. For the bone marrow and the liver, a clonogenic assay for leukemic cells was used to quantify the cell content in these organs. A good correlation was found between the bioassay-derived data and the flow-cytometry-derived data. The doubling times of the leukemic cell population were not equal for the two organs studied, indicating that a number of different processes contribute to the net cell production per organ. Apart from their application in the detection of residual leukemia, monoclonal antibodies might be employed in the analysis of the growth kinetics of the "invisible" leukemic cell population.     

Acute nonlymphocytic leukemia (M2) with chromosome abnormality trisomy 4 developing eight years after radiation therapy for breast cancer

We report here the development, 8 years after radiation therapy for breast cancer, of acute nonlymphocytic leukemia (ANLL), type M2 of the FAB classification, in which trisomy 4 was detected as the only chromosomal abnormality. Simultaneous observation of cytologic and cytogenetic features of individual colonies derived from leukemic progenitor (L-CFU) and early progenitor (CFU mix) cultures in this patient revealed that all colonies examined had a normal karyotype, although the clone with trisomy 4 was predominant in the direct bone-marrow culture. These findings suggest that progenitor cells with trisomy 4 were less predominant in colony growth when stimulated by colony-stimulating factors (CSFs) than were stem cells with a normal karyotype.     

An evidence for adhesion-mediated acquisition of acute myeloid leukemic stem cell-like immaturities

For long-term survival in vitro and in vivo of acute myeloid leukemia cells, their adhesion to bone marrow stromal cells is indispensable. However, it is still unknown if these events are uniquely induced by the leukemic stem cells. Here we show that TF-1 human leukemia cells, once they have formed a cobblestone area by adhering to mouse bone marrow-derived MS-5 cells, can acquire some leukemic stem cell like properties in association with a change in the CD44 isoform-expression pattern and with an increase in a set of related microRNAs. These findings strongly suggest that at least some leukemia cells can acquire leukemic stem cell like properties in an adhesion-mediated stochastic fashion.     

Interaction between ZBP-89 and p53 mutants and its contribution to effects of HDACi on hepatocellular carcinoma

Normal hematopoiesis is suppressed during the development of leukemia. In the T-ALL leukemia mouse model described in our recent study (Hu X, et al. Blood 2009), the impacts of leukemic environment on normal hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) were distinct, in that normal HSCs were preserved in part because of increased mitotic quiescence of HSCs and resulting exhaustion of HPCs proliferation. Stem cell factor (SCF) secreted by leukemic cells in Nalm6 B-ALL model was previously suggested to force normal HSCs/HPCs out of their bone marrow niches and allow leukemic cells to occupy the niches (Colmone A, et al. Science 2008). Here we found that stem cell factor (SCF) expression in PB and BM of T-ALL model was increased, but SCF mRNA and protein levels in normal hematopoietic cells were higher than those in leukemia cells, which suggested that upregulated SCF was mainly contributed by non-leukemic cells in response to the leukemia development. To further elucidate the molecular mechanisms, microarray analysis was conducted on normal HSCs in this model and verified by real-time RT-PCR. The expression of Hes1 and its downstream target p21 were elevated in normal HSCs, whereas their expression showed no significant alteration in HPCs. Interestingly, although overexpression of Hes1 by retroviral infection inhibited the in vitro colony formation of normal hematopoietic cells, in vivo results demonstrated that normal Lin^- cells and HSPCs were better preserved when normal Lin^- cells with Hes1 overexpression were co-transplanted with T-ALL leukemia cells. Our results suggested that the differential expression of Hes1 between HSCs and HPCs resulted in the distinct responses of these cells to the leukemic condition, and that overexpression of Hes1 could enhance normal HSPCs in the leukemic environment.     

Active immunotherapy of friend virus-induced erythroleukemia and its spontaneous regression

Summary We have tested the effects of specific and nonspecific immunostimulation on the spontaneous regression and recurrence of erythroleukemia induced by a strain of the Friend murine leukemia virus complex, RFV. Subcutaneous inoculation of mice with UV-irradiated allogeneic leukemic spleen cells (LSC) protected against subsequent virus challenge. RFV-leukemic mice injected with LSC into subcutaneous BCG-primed sites had a significantly increased incidence of leukemia regression. When leukemic mice received BCG or LSC alone or normal allogeneic spleen cells (NSC) in place of LSC the incidence of regression was not different from that recorded in untreated controls. A single treatment of recently regressed mice with LSC given into BCG-primed sites prolonged the disease-free period, while LSC alone, BCG alone, or NSC had no such effect. Our data support an immunological basis for spontaneous regression of erythroleukemia and for maintenance of the regressed state. This system provides a model for testing the efficacy of immunotherapeutic protocols for maintenance of leukemia remission and tumor dormancy.