ESb Cells Research Articles

Determination of cell-free interleukin 2 receptor level in the serum of normal animals and of animals bearing IL-2 receptor positive tumours with high or low metastatic capacity.

Serum levels of cell-free interleukin-2 receptors were elevated above normal in mice bearing the IL-2R positive T-cell lymphoma Eb or its highly metastatic variant ESb. Although ESb cells expressed less IL-2R molecules than Eb cells on their cell surface, serum receptor levels were raised more quickly in ESb than in Eb tumour bearing animals. Elevated IL-2R serum levels were a sensitive tumour marker in animals bearing the aggressive variant ESb but not in animals bearing the low metastatic line Eb. Peritoneal ascites tumour-bearing animals had higher serum IL-2R levels than corresponding animals with subcutaneously growing tumours. Thus, serum IL-2R levels in tumour-bearing animals were dependent on the tumour line and influenced by the site and mode of tumour growth.

Prolongation of survival of mice bearing the Eb and ESb lymphoma by treatment with interferon inducers alone or in combination with Corynebacterium parvum.

The objective of this study was to evaluate if pretreatment with Corynebacterium parvum (C. parvum) augments the effects of interferon (IFN) inducers on survival of DBA/2 mice transplanted with two syngeneic lymphoma variants, the low metastatic Eb and the high metastatic ESb tumor. The involvement of IFN in the treatment effects was investigated. As inducers of IFN-alpha/beta Newcastle disease virus (NDV), polyinosinic-polycytidylic acid (polyI:polyC), and 10-carboxymethyl-9-acridanone (CMA) were injected i.p. at the site of tumor transplantation. The Eb tumor was found to be sensitive to the antiproliferative action of IFN-alpha/beta in vitro. In vivo single injections of each of the inducers retarded growth of the Eb tumor. In C. parvum-pretreated mice the effects of the inducers on survival were markedly increased. There was a correlation between prolonged survival and local IFN levels in response to polyI:polyC or CMA but not upon NDV. Injections of each of the inducers increased cytotoxicity of peritoneal exudate cells against the Eb tumor cells in vitro especially when mice were pretreated with C. parvum. Although other mechanisms cannot be excluded IFN-mediated activation of host defence and also direct antiproliferative effects of endogenously produced IFN seem to be involved in the antitumor effects by these IFN inducers in the Eb model. In the ESb tumor model irrespective of additional pretreatment with C. parvum survival was only slightly prolonged by the treatments and endogenous IFN induction did not result in any real benefit for the animals. When compared with Eb cells the ESb cells were less sensitive to the antiproliferative action of IFN-alpha/beta in vitro and less sensitive to in vitro cytotoxicity by the host cells. Although other mechanisms may additionally be active in vivo the different susceptibility of the Eb and ESb tumor cells to the direct and indirect actions of IFN seems to contribute to the different responsiveness of these tumor cell lines to the treatments with IFN inducers.

Immunoresistant metastatic tumor variants can re-express their tumor antigen after treatment with DNA methylation-inhibiting agents.

Immuno-escaping variants which arise during metastasis of ESb lymphoma cells in syngeneic DBA/2 mice have been shown to exhibit selective resistance to lysis by ESb-specific cytotoxic T-lymphocytes (CTL). The immuno-resistant variants present no changes in the expression of H-2Kd molecules which appear to be the restricting elements for ESb-specific CTL. We now show that treatment of clonal immuno-resistant ESb variant cells with MNNG or 5'azacytidine can restore the sensitivity to tumor-specific CTL lysis in a high percentage of cloned progenitor cells. The acquisition of susceptibility to lysis by these clones is most likely due to re-expression of ESb-type tumor antigens because such cells regain the capacity to compete with original 51Cr-labelled ESb cells for lysis by ESb-specific CTL, and regain the capacity to induce ESb-specific CTL in vivo. Our data suggest that the immuno-resistant variants are not cellular mutants but rather gene regulatory variants. This could explain: their high frequency of occurrence during metastasis; the relative stability of the variant phenotype; and the reversibility observed after the use of DNA-demethylating and gene-activating drugs like 5'-azacytidine or MNNG.

Analysis of protective and cytotoxic immune responses in vivo against metabolically inactivated and untreated cells of a mutagenized tumor line (requirements for tumor immunogenicity)

The immunogenicity of a mutagenized subline (ESb-D) of the weakly immunogenic T-cell lymphoma L 5178 Y ESb has been characterized. The injection of 10 6 ESb-D cells ip did not establish lethal tumors in untreated DBA/2 mice but established tumors in sublethally irradiated mice. Injection of ESb-D cells into otherwise untreated DBA/2 mice established also a state of protective immunity against the subsequent injection of otherwise lethal doses of ESb tumor cells. Protection was only obtained after injection of intact but not UV-irradiated or mitomycin-C-treated ESb-D cells. A direct T-cell-mediated cytotoxic activity was also demonstrable in the spleen cells of DBA/2 mice after injection of ESb-D cells but not ESb cells. The cytotoxic activity was variant specific for ESb-D target cells, and it was induced only with intact but not UV-irradiated or mitomycin C-treated ESb-D cells. This suggested that the induction of protective and cytotoxic immunity may require the persistence of the antigen or unusually high antigen doses. The in vivo priming for a secondary in vitro cytotoxic response, in contrast, was achieved with intact and also with mitomycin C-treated ESb-D cells but again not with UV-irradiated ESb-D cells. This indicated that the metabolic activity was a minimal requirement for the in vivo immunogenicity of the ESb-D tumor line. The secondary cytotoxic activity was demonstrable on ESb-D and ESb target cells and could be restimulated in vitro about equally well with ESb-D and ESb cells. But the in vivo priming was again only obtained with ESb-D cells and not with ESb cells. These experiments thus demonstrated that the requirements for immunogenicity are more stringent in vivo than in vitro, and more stringent for the induction of direct cytotoxic and protective immunity in vivo than for the in vivo priming for secondary in vitro responses.

Release of L-alanine by tumor cells.

Culture supernatants from the weakly immunogenic T cell lymphoma L5178Y ESb were found to contain substantial amounts of alanine and lactate at a ratio of about 1:10. Supernatants from cells of the highly immunogenic mutant line ESb-D also contained lactate but only minute amounts of alanine. Moreover, ESb cells converted 14C-labeled glucose or pyruvate into labeled alanine and lactate at a ratio of about 1:10, whereas ESb-D cells yielded only labeled lactate and no detectable alanine. The injection of L-alanine in combination with L-lactate into mice strongly suppressed the capacity of their spleen cells to generate cytotoxic responses. The injection of L-alanine also suppressed the immunogenicity of ESb-D cells, as demonstrated by the generation of cytotoxic activity in vivo and by the in vivo immunization (priming) for secondary cytotoxic responses against ESb-D cells in vitro. Taken together, these experiments suggest the possibility i) that the ESb cells prevent the induction of cytotoxic responses by releasing immunosuppressive alanine, and ii) that the immunogenic mutant ESb-D may have gained immunogenicity by losing this immunosuppressive property.

Involvement of both heparanase and plasminogen activator in lymphoma cell‐mediated degradation of heparan sulfate in the subendothelial extracellular matrix

The effect of plasminogen on the ability of highly metastatic ESb mouse lymphoma cells to degrade heparan sulfate (HS) in the subendothelial extracellular matrix (ECM) was studied. A metabolically sulfate-labeled ECM was incubated with the lymphoma cells, and labeled degradation products were analyzed by gel filtration on Sepharose 6B. Heparanase-mediated release of low-Mr (0.5 less than Kav less than 0.85) HS cleavage products was stimulated fourfold in the presence of plasminogen. Incubation of plasminogen alone with the ECM resulted in its conversion into plasmin, which released high-Mr (Kav less than 0.33) labeled proteoglycans from the ECM. Heating the ECM (80 degrees C, 1 hr) abolished its ability to convert plasminogen into plasmin, yet plasminogen stimulated, through its activation by the ESb plasminogen activator, heparanase-mediated release of low-Mr HS fragments. Heparin inhibited both the basal and plasminogen-stimulated degradation of HS side chains but not the total amount of labeled material released from the ECM. In contrast, aprotinin inhibited the plasminogen-stimulated release of high- as well as low-Mr material. In the absence of plasminogen, degradation of heated ECM by ESb cells was completely inhibited by aprotinin, but there was only a partial inhibition of the degradation of native ECM and no effect on the degradation of soluble HS proteoglycan. These results demonstrate that proteolytic activity and heparanase participate synergistically in the sequential degradation of ECM HS and that the ESb proteolytic activity is crucial for this degradation when the ECM-associated protease is inactivated. Plasminogen may serve as a source for the proteolytic activity that produces a more accessible substrate to the heparanase.

Characterization of cellular and extracellular plasmamembrane vesicles from a low metastatic lymphoma (Eb) and its high metastatic variant (ESb): inhibitory capacity in cell-cell interaction systems

Spontaneously shed extracellular plasma membrane vesicles (ECM) of a highly metastatic murine tumor line (ESb) were compared (i) with plasma membrane vesicles (PM) of the same cells prepared by the nitrogen cavitation method and (ii) with ECM and PM preparations of the related low metastatic tumor line Eb. From a previous biochemical analysis it was concluded that the exfoliation of ECM vesicles, which is very pronounced in metastatic ESb cells, is not a random process. This conclusion is further corroborated by the present functional analysis. Compared to ESb PM, ESb-derived ECM were selectively enriched for Fc receptors and depleted in glycoproteins with affinity for hepatocytes. Tumor-derived ECM carried the same tumor antigen as the corresponding tumor line and showed in comparison to PM material an increased inhibitor capacity in a T-cell-mediated tumor-specific cytotoxicity test.

Expression of Ia antigens in a murine T-lymphoma variant

ESb, a cellular high metastatic variant derived from the murine T-cell lymphoma L5178Y (Eb), was found to synthesize la antigens. Ia-specific antibodies reacted with the ESb cells and precipitated Ia-like molecules from them. Two-dimensional gel electrophoretic analysis of immunoprecipitates of metabolically labeled ESb cells indicated that the la molecules on ESb were indistinguishable from those on murine B-cells. No la antigens were detectable on the parental tumor line Eb. Treatment with recombinant interferon-γ (IFN-γ) caused enhancement of class I histocompatibility antigen expression on Eb and ESb tumor lines. In ESb cells the expression of la and of Ia-associated invariant chain (Ii) was also increased upon IFN-γ treatment. No induction of either la and li antigens was observed upon IFN-γ treatment of the Eb line. These studies demonstrate a substantial difference between the Eb and ESb tumor lines with respect to: (i) constitutive expression of class II major histocompatibility antigens, and (ii) response to IFN-γ treatment.

Characterization of cellular and extracellular plasma membrane vesicles from a non-metastasizing lymphoma (Eb) and its metastasizing variant (ESb)

Plasma membrane fragments from two variants of a murine lymphoma, Eb and ESb, with different metastatic capacity were investigated. Plasma membranes were isolated from tumor cells recovered from the peritoneal cavity. They differed in their lipid composition, indicating a more fluid state of the plasma membranes derived from the highly metastatic tumor line ESb. Extracellular membrane vesicles could be isolated from the ascites of the tumor-bearing mice. The shedding capacity of ESb cells was much higher than that of Eb cells. The extracellular membranes by chemical analysis and the measurement of marker enzymes proved to be derived from the plasma membranes. However, they differed from the plasma membranes from which they were derived in several aspects: (i) the lipid to protein ratio was diminished; (ii) the activities of some plasma membrane-associated enzymes were lower while other were identical in plasma membranes and extracellular membranes; (iii) the content of saturated fatty acids in phopholipids was enhanced in extracellular membranes. These effects were more pronounced in the highly metastasizing tumor line ESb. It is thus concluded that shedding of extracellular membranes is not a random process. The biochemical differences found in the plasma membranes and the extracellular membranes of the two tumor lines are discussed with respect to the different metastatic capacity of the tumors.

Suggestive evidence that the highly metastatic variant ESb of the T-cell lymphoma Eb is derived from spontaneous fusion with a host macrophage.

Two lines of evidence are reported which suggest that the highly metastatic variant ESb of the T-cell lymphoma Eb is derived from spontaneous fusion with a host macrophage. Firstly, ESb cells are shown to express the macrophage differentiation antigen Mac-1 which was not found on Eb cells or on any other tumor cells tested except the macrophage tumor line Pu5. Secondly, the progression from low to high metastatic capacity could be reproduced in vitro following hybridization of thioguanine-resistant Eb cells (EbTGR) with syngeneic bone-marrow-derived macrophages. Two HAT medium-selected hybrid tumor lines (Eb-F1 and Eb-F2) could be established. They were found to express cell surface markers of both parental lines: T lymphoid differentiation antigens from T-lymphoma and macrophage antigens (Mac-1, class II MHC antigens) from the normal cell fusion partner. The antigens were identified on the hybrids and subclones thereof by means of monoclonal antibodies and 3 different detection assays: cytofluorography, complement-dependent cytotoxicity and immunoprecipitation followed by gel electrophoresis. Animals inoculated s.c. with the parental line EbTGR developed local tumors but not metastases and survived for more than 40 days. In contrast, animals inoculated similarly with Eb-F1 or Eb-F2 cells quickly developed metastases in visceral organs and died as early as 10-14 days following inoculation. In many but not all respects, the in vitro-derived T-lymphoma-macrophage hybrids resembled the spontaneous in vivo-derived variant ESb. These findings, together with the presence of Mac-1 antigen on ESb cells, suggest (1) that ESb variant cells may be derived from spontaneous fusion with a host cell, most likely a macrophage and (2) that somatic cell fusion may be an important mechanism of genetic rearrangements leading to metastatic variants. The new highly metastatic tumor lines which were developed under well-defined in vitro conditions, and their subclones, may become very useful tools for studying the contribution of specific genetic traits and of membrane-related structures to various steps of the metastatic process.

Acquisition of high metastatic capacity after in vitro fusion of a nonmetastatic tumor line with a bone marrow-derived macrophage.

A low metastatic, thioguanine-resistant murine T lymphoma line (EbTGR) was hybridized in vitro, with the help of polyethylene glycol, with syngeneic bone marrow-derived macrophages. Two HAT-resistant hybrid lines (Eb-F1 and Eb-F2) were obtained from independent fusion cultures. A cytogenetic analysis revealed that most of the macrophage chromosomes except No. 12 had segregated or become rearranged 60 d after fusion, a time at which the cell lines had become stabilized in culture. Syngeneic mice inoculated subcutaneously with the tumor macrophage hybrid lines developed, very quickly, visceral metastases and died after less than 2 wk, while those inoculated with the parental line lived for greater than 6 wk and developed only localized, large primary tumors. The metastatic hybridomas expressed a similar tumor antigen as a spontaneous, in vivo derived, high metastatic variant (ESb) of the same tumor. This suggests that ESb cells might have arisen from a spontaneous fusion with a host macrophage.

New plasma membrane proteins expressed by a high metastatic variant of a chemically induced tumor

The composition of plasma membrane proteins of related low and high metastatic tumor cells were compared in order to elucidate possible genetic changes which may occur during progression of tumor cells towards metastatic capacity. For this purpose a procedure was worked out which allows the purification of mouse plasma membrane (PM) proteins from biosynthetically labeled cells. PM isolated from the two related T lymphoma lines Eb and ESb, which differ greatly in metastatic capacity, as well as from Con A-activated T cell blasts displayed similar degrees of purification as shown by marker enzyme analysis. When [ 35S]-methionine-labeled proteins of such PM preparations from cloned Eb and ESb cells were compared by 2-D gel electrophoresis it was found that maps of the highly metastatic variant ESb cells permitted detection of about 50 new protein spots which were not found on the 2-D gel maps of the parental Eb cells. Approximately 80% of these new spots were also found on maps derived from syngeneic splenic Con A blasts. This observation indicated that most of the newly expressed membrane proteins of ESb cells were possible differentiation or cell activation-related and not tumor cell-specific.

Interactions Between Lymphoid Tumor Cells and Isolated Liver Endothelial Cells

Interactions were studied between highly metastatic murine MB6A lymphosarcoma cells and rat liver endothelial cells that had been isolated by collagenase perfusion and purified by unit gravity sedimentation. Experiments were performed on the day of isolation. MB6A cells were observed to adhere to the endothelial cells. Addition of rat serum had a striking effect: The endothelial cells spread over the MB6A cell surface, engulfing the tumor cells. The factor involved was nondialyzable and also was present in rat plasma. Similar interactions were seen with highly metastatic ESb and MDAY-D2 lymphoma cells and with nonmetastatic Eb cells. Low-metastatic GRSL 34 leukemia and TA3/Ha ascites mammary carcinoma cells did not adhere to the endothelial cells. With this in vitro model the molecular mechanisms of adhesion to liver endothelium were studied. As a first step, univalent antibodies against MB6A cells were found to inhibit adhesion, indicating involvement of specific cell surface molecules.

Metastatic potential severely altered by changes in tumor cell adhesiveness and cell-surface sialylation.

A plastic adherent variant line (ESb-M) of a highly invasive and metastatic murine T cell lymphoma (ESb) was found to have lost its metastatic potential while still being tumorigenic in normal syngeneic hosts. The variant retained most of its ESb-derived antigenic and biochemical characteristics but differed at binding sites for certain lectins with specificity for terminal N-acetylgalactosamine residues. Whereas such sites were masked by sialic acid on metastatic ESb cells, they became unmasked on the adherent variant line. Metastatic revertants of ESb-M cells did not express the respective lectin receptor sites because these were again masked by sialic acid. It is suggested that the masking of specific lectin receptors sites on the tumor cell surface is of crucial importance for metastatis. If freely exposed, these sites may change adherence characteristics of the cells possibly not only in vitro (to plastic) but also in vivo.

Cytogenetic changes during tumor progression towards invasion, metastasis and immune escape in the Eb/ESb model system.

Related tumor lines which represent different stages in their progression towards metastatic capacity were investigated and compared at the chromosomal level. The parental low-metastatic tumor line (L5178Y/Eb) was derived from a long-term transplanted, chemically induced T-cell lymphoma of the DBA/2 mouse. The cytogenetic analysis included this Eb line, a spontaneous high metastatic variant thereof which expressed a distinct tumor-associated transplantation antigen (ESb TATA+), and an immunoresistant TATA-negative variant of the latter (ESb TATA-). All three cell lines were characterized by a near-diploid chromosome count and by some common chromosomal markers derived from Nos. 6, 13 and 16 Large-scale chromosomal rearrangments resulted in the formation of eight marker chromosomes in Eb cells, 16 in ESb TATA+ cells and 18 in ESb TATA- cells. Tumor progression in this system showed a tendency to monosomies, which could bring the corresponding genes to a hemizygous state and possibly to a release from repression. Chromosome 15 was trisomic in Eb cells, monosomic in ESb TATA+ cells and hardly detectable in ESb TATA- cells. The Ig heavy-chain gene-carrying region of both chromosomes No. 12 was found in translocation with chromosomes Nos. 5, 13 and 14 (Eb cells) and with Nos. 1 and 17 (ESb cells). ESb TATA- cells differed from ESb TATA+ cells at four different chromosomes (Nos. 5, 8, 14 and 15).

Different expression of Lyt differentiation antigens and cell surface glycoproteins by a murine T lymphoma line and its highly metastatic variant.

Cloned lines of the methylcholanthrene-induced DBA/2 T lymphoma Eb and its highly metastatic variant line ESb were analyzed for differences in the expression of serologically detectable cell surface differentiation markers. Flow cytofluorographic analysis of cells stained with fluorescein isothiocyanate-conjugated monoclonal rate anti-mouse Thy-1, Lyt-1, Lyt-2 and complement-dependent cytotoxicity with mouse alloantisera against Lyt-3.2 and Ly-6.2 revealed, for the parental low metastasizing line, Eb, a phenotype of Thy-1+, Lyt-1-, Lyt-2+, Lyt-3+, Ly-6+, whereas the highly metastasizing variant line typed as Thy-1-, Lyt-1+, Lyt-2-, Lyt-3-, Ly-6-. Analysis of galactose oxidase/NaB3H4-labeled glycoproteins from Eb and ESb clones by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed further phenotypic differences. Selective binding of radiolabeled glycoproteins to Helix pomatia or Vicia villosa-Sepharose, respectively, allowed the identification of T130 to be expressed on Eb cells and T145 to be expressed on some ESb clones. The latter antigen is expressed on murine cytotoxic T lymphocytes. Immune precipitation analysis revealed that Eb and ESb bear different molecular forms of the T200 antigen. Comparisons of iodinated surface proteins derived from tumor cells either treated or untreated with tunicamycin indicated that many of the differences in membrane proteins between Eb and ESb cells could be attributed to differences in glycosylation. Our results, derived from a defined tumor system of lymphoid origin, show that the progression from a low to a high malignant tumor line can be associated with changes in the expression of various defined cell surface differentiation antigens. The question of a possible relationship between tumor progression and cell differentiation or dedifferentiation is discussed.

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. X. Immunoselection of tumor variants differing in tumor antigen expression and metastatic capacity.

We previously described morphological, functional and antigenic differences between a chemically-induced DBA/2 lymphoma, Eb, and a spontaneous variant, ESb, which arose in 1968 and had highly increased metastatic capacity. Now we present evidence that the two cell lines, in spite of the differences observed, are still related. (1) Shifts from Eb to ESb can be reproduced after 11 years. They occur during routine i.p. transplantation, especially when high cell numbers are passaged. (2) ESb variant cells can be recovered from Eb tumor populations after immunoselection in vivo with specific anti-Eb cytolytic T lymphocytes (CTL). (3) Parental-type Eb cells can be isolated from ESb tumor populations after immunoselection in vitro with specific anti-ESb CTL Ten ESb cells mixed with 10(6) Eb cells and inoculated s.c. caused a shift in the mortality curve suggesting that a possible contamination of the Eb population with pre-existing ESb variant cells was less than 1 in 100,000.