Adhesion patterns of cell interactions revealed by reflection contrast microscopy

Reflection contrast in combination with phase contrast microscopy was utilized for the study of adhesion patterns of locomotive L5222 rat leukemia cells. It was found that for cells moving in a spherical shape on the glass surface, adhesions were very faint. This inconspicuous pattern, however, became very distinct, as soon as the cells changed to a flattened configuration. Such a change took place when leukemia cells came into contact with other spread cells and started to move under these cells. Reflection contrast further showed that in the pathway of the locomoting L5222 cells the adhesions of the overlying spread cells were momentarily detached from the substrate. It is concluded that the combination of reflection contrast and phase contrast represents a good tool for gaining new information on the interaction of motility and formation of adhesions.     

Ultrastructural evidence for contacts between migrating L5222 rat leukemia cells and extracellular matrix components of the rat mesentery

The nature of interactions between cells migrating through tissues and their structural surroundings are largely unknown. We have therefore examined the ultrastructural relationship between L5222 rat leukemia cells, moving through the loose connective tissue of the mesentery, and components of the extracellular matrix (ECM). Ultrathin tissue sections, fixed in the presence of ruthenium hexammine trichloride (RHT), revealed the following: Constitutents of fibrillar and nonfibrillar elements of the ECM are in contact with the plasma membrane of L5222 cells. Linear nonfibrillar ECM elements contact the plasma membrane at point-like sites, often associated with root-like structures present within the submembraneous microfilament mesh. Aggregates of ECM material are connected to patch-like cell membrane sites, associated with a condensed, plate-like part of the microfilament mesh. Point-like and patch-like contacts are more numerous at the anterior part of polarized migrating L5222 cells than on the posterior end. In round resting leukemia cells they are evenly distributed around the cell periphery. We suggest that the ECM-cell membrane contacts represent tissue adhesion sites. We therefore hypothesize that in migrating cells a coordinate interaction occurs between the contact sites and the continuous microfilament meshwork which results in a simultaneous backward movement of ECM-membrane contacts on the cell body and in a net forward movement of the whole cell. Since Dembo et al. (1981) present a similar mechanism for in vitro locomotion of granulocytes, we assume that blood cell locomotion in vivo and in vitro depends on similar molecular mechanisms: force generation by the cell, transmembraneous linkage between cytoskeletal and ECM elements, and membrane fluidity. The major difference in blood cell locomotion through a three-dimensional tissue or on a plane substratum would then be given by the distribution of contact sites, occurring around the cell periphery or limited to the ventral cell surface, respectively.     

Motility of L 5222 rat leukemia cells in the flattened state. Evidence against emperipolesis.

Emperipolesis is the term for the assumed penetration of living cells into other living cells. As reported earlier, L 5222 rat leukemia cells, migrating in vitro, change from a spherical to a spread configuration when they meet flat cells, and continue to move in this shape within the contours of the target cells. Whether or not this close cellular association corresponded to emperipolesis could not be determined with phase and interference contrast cinemicrography alone. In combination with transmission electron microscopy, it could be demonstrated that the compartment, in which the spread leukemia cells move, is not the cytoplasm of the target cells, but the narrow space created by the target cells and the underlying glass surface. Thus, emperipolesis could be ruled out for L 5222 leukemia cells. On this basis the reported observations on emperipolesis are reviewed, and a critical attitude regarding the occurrence of emperiopolesis in general is advocated.     

Fibrillar bodies in leukemic cells revealed by polarization microscopy.

An optical polarizing microscope with a good coefficient of extinction permits the visualization of the cytoplasmic fibrillar body in living preparations and smears of leukemic cells (human leukemias and the L 5222 experimental leukemia). These inclusions are not visible by phase contrast microscopy nor in fixed and stained smears. The detection in living cells of fibrillar bodies makes it possible to study directly the conditions for their formation and their reaction to the effect of certain drugs.     

Different modes of mesenteric infiltration displayed by two rat leukemias. A study by scanning and transmission electron microscopy and by microcinematography

Infiltration of the mesentery after intraperitoneal implantation of two transplantable rat leukemias, the undifferentiated L5222 and the myeloid BNML, was studied by means of scanning and transmission electron microscopy, and microcinematography. In animals implanted with L5222 cells, contraction of the mesenteric mesothelium is a conspicuous feature. It occurs within the first 24h after implantation and influences decisively the course of infiltration. In contrast, The presence of BNML cells leads to mesothelial contraction only in the terminal stage and, therefore, exerts no direct effect on infiltration. In addition, the two leukemias differ with regard to their cellular motility. Whereas L5222 cells locomote within the mesentery, only stationary movements are recorded with BNML cells. Based on the different interactions with the mesothelium and cell motilities, two distinct modes of infiltrating the mesentery could be ascertained for the two rat leukemias.     

Augmentation of host antitumor immunity by low doses of cyclophosphamide and mafosfamide in two animal tumor models

Summary By cloning in vitro we have obtained two sublines of the L5222 rat leukemia, one with high (L5222-S) and the other with low (L5222-R) in vivo sensitivities to non-toxic doses of mafosfamide, a stabilized derivative of 4-hydroxy-cyclophosphamide. This sensitivity in vivo was not related to the cytotoxic activity of the drug in vitro. Treatment of rats bearing the L5222-S and of mice transplanted with the MOPC-315 plasmocytoma with low doses of mafosfamide or cyclophosphamide resulted in a high percentage of surviving animals, which were resistant to a subsequent tumor challenge. Viable leukemic cells were needed to establish antitumor immunity, since it was not possible to induce resistance by injection of mitomycin-C-treated, non-viable L5222 cells. The adoptive transfer of spleen cells from animals immune against the L5222-S and the MOPC-315 resulted in resistance of the syngeneic recipients against a rechallenge with tumor cells, provided that the animals were treated with an immunosuppressive dose (100 mg/kg) of cyclophosphamide prior to the spleen cell implantation. In nude mice treatment of the L5222 with low doses of mafosfamide also resulted in surviving animals, however resistance to a second tumor challenge occurred only sporadically.The data presented confirm that therapy with cyclophosphamide or mafosfamide enhances host antitumor immunity but, contrary to previous reports, it could be demonstrated that successful tumor rejection was independent of T cells.Supported by the Federal Ministry of Research and Technology (BMFT), Bonn-Bad Godesberg, FRG     

Comparative morphology of cells located on glass and in the loose connective tissue: a study by scanning electron microscopy

Most of the studies dealing with cellular shape, surface configuration, and motility are carried out in vitro on plane substrata. During the past years, the direct transfer of results obtained under these conditions to the cellular behavior displayed in the living organism, has been increasingly challenged. For this reason we have investigated the above mentioned functions of different cell classes localized on glass and in the loose connective tissue. The cells utilized were: fibroblasts and macrophages from normal rat and rabbit mesenteries, V2 rabbit carcinoma cells and L5222 rat leukemia cells. The combination of time-lapse cinematography and scanning electron microscopy revealed that motility and surface features are the same, irrespective of the immediate surrounding. Cellular shape and attachment, on the other hand, are dependent on the substrate. Fibroblasts, macrophages and cells of epithelial origin, including carcinoma cells, flatten on glass, but have a rounded configuration in the tissue. The flat leading lamellae displayed during locomotion on glass, are not evident in cells migrating through tissues. What regards attachment devices, extensively studied on glass, their formation and position within a tissue are, at present, a matter of speculation. Although it can be assumed that a similar process is operable in vivo and in vitro, clarification rests upon the use of ultrahistochemical techniques.     

Intermediate-sized filaments in leukemia cells

Electron microscopic studies on human acute leukemias have shown that leukemic populations contain spherical and polarized cells in various proportions. As recorded by time-lapse cinematography, the two cell configurations represent different functional states: resting cells are completely spherical, locomotive cells are polarized with a conspicuous extension posteriorly. In 9 out of 12 cases of acute myeloid leukemia the two cell configurations were found to coincide with a different pattern of intermediate-sized filaments (ISF). Most spherical myeloblasts possessed large bundles of ISF (a minority had small bundles), whereas polarized myeloblasts showed small groups or single filaments. A similar correlation between cell shape and arrangement of ISF was observed in a transplantable undifferentiated rat leukemia. Two concepts can be distinguished with regard to the role of fibrillar structures in leukemic myeloblasts: thick bundles of ISF either represent a pathological state or have a functional significance. A tentative interpretation of our own results provides some arguments in favor of a disaggregation-reaggregation cycle of thick ISF bundles, whereas a pathological ("end stage") nature of these structures appears less likely.     

Formation and function of Vibrio parahaemolyticus lateral flagella.

Formation of the lateral flagella (L-flagella) of Vibrio parahaemolyticus was studied immunologically, using specific antiserum against L-flagella. On solid medium, L-flagella were formed at both high (37 degrees C) and low (25 degrees C) temperatures, although at high temperatures they became dissociated from the cells and decomposed in the medium. L-flagella were not formed in liquid or soft-agar medium. Formation of L-flagella was decreased by lowering the pH of the medium and repressed by transferring the cells from solid medium to liquid medium. Mutants possessing L-flagella but not a polar monotrichous flagellum (M-flagellum) swarmed on solid medium, whereas mutants were grown on solid medium and then transfered to liquid medium, the cells oscillated until they lost L-flagella. It is postulated that L-flagella are locomotive organelles on solid medium and in some cases also in liquid medium, whereas M-flagella are locomotive organelles only in liquid medium.     

Identification of hepatic niche harboring human acute lymphoblastic leukemic cells via the SDF-1/CXCR4 axis

In acute lymphoblastic leukemia (ALL) patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc (null) mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.     

Complete mimicry: a case of alveolar rhabdomyosarcoma masquerading as acute leukemia

Background

A small number of rhabdomyosarcoma (RMS) cases involve the bone marrow. A leukemic presentation of RMS has been reported in a few case series, although almost all cases of leukemic RMS are not completely mimicking leukemia. We encountered a case with RMS cell infiltration of the bone marrow that resembled floating hematological cells.

Case presentation

We encountered a rare case of a 15-year-old boy with a 2-week history of left femoral pain. Upon admission, he was afebrile with no other symptoms. No apparent cause of femoral pain was detected on an initial examination. Laboratory findings revealed normal white blood cell (WBC) count and hemoglobin concentration, with a platelet count of 10.3 × 10⁴/μL. WBCs included 2.0% metamyelocytes, 4.5% myelocytes, and 0.5% blasts. Lactate dehydrogenase concentration was 1299 U/L, creatine kinase was 437 U/L, and C-reactive protein was 1.25 mg/dL. Bone marrow aspiration demonstrated hypercellular marrow (nucleated cell count 1.84 × 10⁴/μL) and 89.0% of blast-like cells of all nucleated cells. The proliferating cells were negative for myeloperoxidase and esterase, and strongly positive for CD56. Positron emission tomography exhibited extensive accumulation of ¹⁸F–fludeoxyglucose with a SUVmax of 7.09. Magnetic resonance imaging revealed T1-low intensity, gadolinium-enhanced, diffuse, and irregular lesions on his pelvis and bilateral femurs. These laboratory and imaging findings suggested hematological malignancy with diffuse bone involvement, suggestive of acute leukemia. However, the pathological diagnosis of bone marrow and basal penile muscle biopsy was alveolar RMS. Karyotype analysis of bone marrow cells revealed the characteristic translocation of t(2;13)(q35;q14). The final diagnosis was alveolar RMS with massive involvement of the bone marrow and the primary site in the perineal muscles. The tumor cells both of the primary site and bone marrow were positive for myogenin.

Conclusions

A literature review found a misdiagnosed case of completely mimicking leukemic RMS as natural-killer (NK)-cell leukemia. Such a misdiagnosis can have critical consequences. We experienced a rare case of alveolar RMS with symmetrical diffuse bone marrow involvement completely masquerading as acute leukemia. The results of a surface marker study showing that the tumor cells had a near NK-cell phenotype were misleading.

     

Action of temperature-sensitive mutants of myeloproliferative sarcoma virus suggests that fibroblast-transforming and hematopoietic transforming viral properties are related

The myeloproliferative sarcoma virus is molecularly related to the Moloney sarcoma virus (Pragnell et al., J. Virol. 38:952-957, 1981), but causes both fibroblast transformation in vitro and leukemic changes--including spleen focus formation--in adult mice. The fibroblast transforming properties of myeloproliferative sarcoma virus were used to select viral temperature-sensitive mutants at 39.5 degrees C, the nonpermissive temperature. These mutants are temperature sensitive in the maintenance of the transformed state. This was also shown by cytoskeletal changes of the infected cells at permissive and nonpermissive temperatures. Viruses released from cells maintained at both the permissive and nonpermissive temperature are temperature sensitive in fibroblast transformation functions. All temperature-sensitive mutants show only a low reversion rate to wild-type transforming function. The myeloproliferative sarcoma virus temperature-sensitive mutants are inefficient in causing leukemic transformation (spleen enlargement, focus formation) in mice at the normal temperature. A method to maintain a low body temperature (33 to 34 degrees C) in mice is described. One temperature-sensitive mutant was checked at low body temperature and did not induce leukemia. These data thus indicate that the same or related viral functions are responsible for hematopoietic and fibroblast transformation.     

Cytology of leukemic lymphadenopathy

OBJECTIVE: To describe the cytomorphologic features of leukemic cells in lymph node aspiration material. STUDY DESIGN: We studied lymph node fine needle aspiration (FNA) smears of 36 leukemic patients. In 23 cases the lymphadenopathy was noticed simultaneously with marrow leukemia, and in the other 13 cases the lymphadenopathy was noticed during a relapse. Special stains, such as periodic acid Schiff, Sudan Black-B, Oil Red-O and nonspecific esterase, were used in special cases. RESULTS: Thirty-three cases were diagnosed as lymphoma, 1 as extramedullary hematopoiesis and 2 as leukemic involvement. CONCLUSION: Leukemic lymphadenopathy can be misdiagnosed as lymphoma on FNA smears. The clinical findings, previous history, hematologic studies and immunocytochemical studies are essential to the differentiation of leukemic smears from lymphoma. However, in some cases the leukemic infiltration can be diagnosed with certainty, provided that the smears show the characteristic findings, such as Auer rods and neoplastic promyelocytes with azurophilic granules.     

Immunocytochemical diagnosis of acute leukemia with pleural involvement

Cytologic examination of the pleural effusion from a patient with acute leukemia, leukocytosis and bleeding revealed the presence of many leukemic cells, "lymphocytes" and erythrocytes. The significance of these cellular changes was investigated by simultaneous study of blood and effusion leukocytes by morphologic, cytochemical and immunochemical methods. Both the leukemic blasts and the "lymphocytes" in the effusion and the blood were found to be neoplastic and contained antigens characteristic of both myeloid cells (OKM-1) and lymphoblasts (C-ALLA, common acute lymphoblastic leukemia antigen). These results, when analyzed in the context of the clinical findings, were indicative of acute leukemia with pleural involvement. Such a clinically oriented approach may further enhance the potential of cytodiagnosis in patients with serous effusions.     

Matrix metalloproteinase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia.     

Bilateral tympanic cavity infiltration with effusion in a patient with acute myeloblastic leukemia

A case of acute myeloblastic leukemia with bilateral tympanic cavity infiltration with effusion in addition to right facial and bilateral auditory nerve involvement is described. Tympanotomy demonstrated the presence of leukemic cells in the effusion of bilateral tympanic cavities. While repeated examinations of the cerebrospinal fluid using the cytocentrifuge failed to show any leukemic cells, it was suspected that the bilateral tympanic cavity infiltration was associated with meningeal involvement.     

Lack of mast cell reactivity during leukemic infiltration of the rat mesentery under syngeneic and allogeneic conditions. A study by transmission electron microscopy (TEM)

The ultrastructural morphology of mast cells localized in rat mesenteries was studied after intraperitoneal implantation of L5222 rat leukemia cells in syngeneic and allogenic hosts. It became evident that the mast cells in the syngeneic (BDIX rat) as well as the allogeneic system (BN rat) showed nor morphological alterations. Degranulation was never observed. This is in contrast to the behavior of macrophages which displayed a strong phagocytotic activity in allogeneic hosts. Thus, it seems that mast cells, under the present experimental conditions, remained inactive during a phase of intense tumor rejection.     

Small oligomers of immunoglobulin E (IgE) cause large-scale clustering of IgE receptors on the surface of rat basophilic leukemia cells

We examined the distribution of small oligomers of IgE bound to rat basophilic leukemia cells using fluorescence microscopy. The oligomers were seen to cluster into visible patches on the cell surface at 4 degrees C; at higher temperatures internalization also was observed. In contrast, cells labeled with IgE monomers remained predominantly ring-stained. Evidence is provided that the observed clustering of IgE oligomers is a cell-induced phenomenon, and the possible significance of this clustering is discussed in the context of the oligomer-triggered degranulation of rat basophilic leukemia cells.     

Communication between white cells and the abnormalities of this in leukemia.

The production of white blood cells is mediated by cellular communication. Proliferation and differentiation of granulocytic and monocytic progenitor and immature cells have been studied in detail and are regulated by stimulatory and inhibitory molecules produced and released from the progeny of the progenitor cells. The resultant interactions between cells and cell-derived molecules suggest operable positive and negative feed-back mechanisms during normal hematopoiesis, and what one sees in the end is the net result of these interactions. The complexities of cellular regulation are partially unravelled by physical separation of the different populations and biochemical analysis of the regulatory molecules. Subpopulations of granulocyte-monocyte progenitors (CFU-c) exist and evidence suggests that they vary in responsiveness to different molecules. Stimulatory molecules are themselves chemically and physically heterogenous and, until recently, believed to have similar biological actions, but this concept must be re-evaluated. Different molecules may activate different subsets of progenitor cells, and there is now a role for substances which enhance the stimulatory interactions. Many studies on normal and leukemic cell responsiveness to stimulation must be re-examined in light of this recent information. Several inhibitory substances operate during normal hematopoiesis. Mature granulocytes, progeny of CFU-c, appear to elaborate at least two inhibitory activities. One activity influences immature, recognizable granulocytes and the other indirectly reduces progenitor cell proliferation by decreasing the production and release of molecules which stimulate CFU-c. In addition, mononuclear phagocytes produce and release E-type prostaglandins in direct response to elevated levels of the stimulatory molecules and E-type prostaglandins counteract increased stimulatory levels by decreasing the sensitivity of CFU-c to stimulators. Much of our present level of sophistication derives from in vitro experimentation and it is apparent that we are only beginning to understand these inter-relationships and their relevance to the in vivo situation. However, these in vitro studies have shed light on the interactions occurring during leukemia. Leukemic cells retain the capacity to respond to normal regulators and must therefore be considered dependent rather than autonomous neoplasms. Abnormalities do exist in leukemic cell interactions: the progenitor cells themselves may be defective and leukemic cells may respond to molecules which normal cells do not. The degree of sensitivity to stimulators and inhibitors will have to be carefully investigated to determine if and what differences may exist between normal and leukemic cells. Normal mature granulocyte derived inhibitory activity is quantitatively deficient in leukemic cells but another inhibitory activity which appears to be specifically present in cells from patients with leukemia and some cases of myelodysplasia is present...