ESb Lymphoma Research Articles

Cytoplasmic domain mutants of beta1 integrin, expressed in beta 1-knockout lymphoma cells, have distinct effects on adhesion, invasion and metastasis.

Structural requirements for beta 1 integrin cytoplasmic domain functions in adhesion, migration and signaling have been studied mainly for fibroblasts in vitro. The relevance for beta 1-dependent in vivo migration of lymphoid cells has not been assessed. To study this, we transfected beta 1 mutants into beta 1-deficient double knockout (DKO) ESb lymphoma cells, and tested the capacity of the cells to metastasize to liver and spleen. This was compared to alpha 4 beta 1-dependent invasion into cell monolayers in vitro and Mn2+-induced adhesion to fibronectin. Deletion of the five C-terminal residues or mutation of both threonines T788 and T789 to alanines blocked invasion and metastasis and greatly reduced adhesion, in line with known in vitro effects. However, mutations of the NPXY motif tyrosines had unexpected consequences. A Y783F mutation had no effect at all, but a Y783,795F double mutation strongly reduced Mn2+-induced adhesion, whereas it had limited effects on invasion and metastasis. Furthermore, cells expressing a beta 1 beta 2 chimeric subunit, which contains phenylalanines in the NPXY/F motifs, adhered poorly but invasion and metastasis was fully restored to the same levels as for cells expressing wild-type beta 1. We conclude that part of the functions of the beta 1 cytoplasmic domain that are required for adhesion are not essential for beta 1-dependent invasion and metastasis.

Tumor stimulator cell modification by infection with Newcastle Disease Virus: analysis of effects and mechanism in MLTC-CML cultures.

Effects of tumor stimulator cell modification by infection with Newcastle Disease Virus (NDV) are described as analysed in vitro in mixed lymphocyte tumor cell cultures (MLTC). Direct antitumor effects were seen with human melanoma or colon-carcinoma cells in a dose- and time-dependent manner when using live but not UV inactivated virus. When T cell stimulation was measured by [3H]-thymidine uptake, NDV infected tumor stimulator cells did not show an augmentation but rather an inhibitory effect in comparison to non-infected stimulator cells. Virus infected tumor stimulator cells were, however, capable of augmenting the induction of tumor specific cytotoxic T cells in MLTC-CML assays when using murine ESb lymphoma immune cells and syngeneic NDV modified ESb cells as stimulators. A CML stimulatory effect was also shown for NDV modified third party cells and thereof derived conditioned medium. These effects are most likely explained by interferon- which is induced in tumor cells by NDV infection and by interferon-á which is induced in responder cells when stimulated with NDV infected stimulator cells.

Role of cytokines in GVL (ESb lymphoma) and GVHD after adoptive transfer of allogeneic T lymphocytes in mice.

ESb lymphoma cells injected i.v. into DBA/2 (H-2d) mice multiply rapidly in the liver and kill all mice in a few days. Adoptive transfer of allogeneic C57B1/6 (H-2b) tumor-immune or normal splenic lymphocytes to sublethally irradiated DBA/2 mice induced a marked antitumor state, graft-versus-leukemia (GVL), increasing the mean survival time 2-3-fold, but also induced an acute and lethal graft-versus host disease (GVHD). We have undertaken experiments to try to dissociate GVL from GVHD. Transfer of immune spleen cells induced a greater GVL than transfer of normal spleen cells with an equivalent to GVHD. Three to five million immune or normal CD8+ T lymphocytes were sufficient to induce both GVL and GVHD. Individual DBA/2 mice were labeled and followed. In mice undergoing GVHD, the spleens were repopulated by donor (H-2b) lymphocytes, and tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were present in the sera of 26 of 27 and 18 of 20 mice, respectively, together with increased amounts of TNF-alpha and IL-6 mRNA in their spleens. This was in contrast to DBA/2 mice receiving allogeneic cells but not developing GVHD. Both interferon-alpha/beta (IFN-alpha/beta) and IL-12, which had proven very effective in association with adoptive transfer of syngeneic immune T lymphocytes in inhibiting ESb metastases, enhanced GVHD when administered with allogeneic immune or normal spleen cells, and >90% of mice died. Intensive IL-2 treatment inhibited GVHD while maintaining GVL.

Superiority of the ear pinna over a subcutaneous tumour inoculation site for induction of a Th1-type cytokine response.

This study examines whether a correlation may be found between Th1- or Th2-type cytokine responses and resistance or susceptibility to tumour growth. Cytokine profiles were investigated in a well-defined mouse tumour model in which the injection site and the genetic background determine the phenotype of either tumour resistance or tumour susceptibility. DBA/2-derived ESb lymphoma variant cells with high metastatic capacity were inoculated into syngeneic mice either s.c., where they grow and metastasize, or into the ear pinna (i.e.), where they do not grow because of induction of protective immunity. Alternatively, the tumour cells were injected s.c. or i.e. into allogeneic B10.D2 mice, which are resistant to the tumour although they are identical at the MHC locus. Between 1 and 10 days after tumour cell injection the spleen-derived mRNA was tested for cytokine gene expression or the spleen cells were analysed by FACScan for T cell activation. The strongest cytokine response was observed in i.e. inoculated B10.D2 mice. This was characterized by an early (days 2-3) peak of interferon gamma (INF-gamma), interleukin-2 (IL-2), IL-2 receptor alpha (IL-2Ralpha) and IL-4. The cytokine mRNA response of i.e. inoculated DBA/2 mice was quite similar except that no IFN-gamma could be detected. In s.c. inoculated B10.D2 mice, the IL-2, IL-2Ralpha and IFN-gamma responses were weaker than after i.e. injection while the IL-4 response was comparable. The most striking difference between these cytokine profiles from tumour-resistant mice and those of s.c. inoculated tumour-susceptible DBA/ 2 mice was a delay in the latter in the IL-2, IL-2Ralpha and IFN-gamma responses and the observation that the IL-4 response was not down-regulated. The persisting IL-4 response could down-regulate a Th1-type response and thereby explain tumour susceptibility as a consequence of host conditioning.

Effective introduction of T cell costimulatory molecules into virus modified tumor cell vaccines by modification with bispecific antibodies

This report describes the generation of bispecific antibodies which bind with one arm to virus modified tumor cell vaccines and introduce with the other arm anti-murine CD28 T cell costimulatory molecules. This is an effective alternative to somatic gene therapy strategies using genes coding for ligands of CD28 such as CD80 (B7-1) or CD86 (B7-2). While these B7 molecules interact not only with CD28 but also with CLTA-4, thereby generating a negative signal, agonistic anti CD28 antibodies only bind to CD28 and therefore deliver only positive costimulatory signals. The new bispecific antibody (bsAb) HN x CD28 allows the introduction of anti-CD28 antibodies into the tumor cell vaccine ATV-NDV, an autologous tumor cell vaccine already modified by infection with Newcastle Disease Virus (NDV). The bsAb HN x CD28 attaches with its anti-HN binding site to the NDV derived hemagglutinin-neuraminidase (HN) molecule which serves as a common foreign anchoring molecule in the vaccine. NDV infected tumor cells which were further modified with HN x CD28 on their cell surface (bs-vaccine), showed increased T cell stimulatory capacity in vitro. This was revealed by augmented proliferation as well as augmented CTL activity. When syngeneic mice were injected with aggressive murine ESb lymphoma cells which were infected with NDV and further modified with the bsAb HN x CD28, delayed tumor development and prolonged survival was observed in comparison to respective controls.

Intra-pinna induction of specific antitumor immune T cell functions

Upon inoculation of highly metastatic ESb lymphoma cells into the ear pinna of syngeneic mice a potent specific antitumor immune response is induced which prevents the outgrowth of the tumor cells at this particular site. When the tumor cell inoculated pinna was resected at different times after antigen application, systemic protective antitumor immunity, tested by s.c. tumor challenge 7 days later, could still be induced. This was true even for a situation in which the pinna was resected as early as two hours after tumor cell inoculation. Protective antitumor immunity was found to be even augmented when the pinna was removed three days after tumor cell inoculation as compared to the non-resected situation. Pinna resection had no effect on the induction of a tumor specific cytotoxic T lymphocyte response while resection after 48 h had an augmenting effect on the delayed type hypersensitivity (DTH) response. An explanation for some of these findings was found when lacZ-labeled lymphoma cells were used for single cell detection in tissue sections. As early as 15 min after intra-pinna inoculation disseminated single tumor cells were detected in the local draining lymph node as well as in the spleen. Whether these tumor cells can prime T cells for protective immunity directly or only after processing and presentation by specialized host cells remains to be elucidated.

Muscle cells produce a low molecular weight factor with anti-cancer activity

The present study describes a new low molecular weight factor released by muscle cells, which inhibits proliferation of tumor cells in vitro and in vivo, is highly specific towards tumor cells, and has no observable effect on normal cells' proliferation. What first prompted us to investigate this factor was the observation that tumor metastases are extremely rare in striated muscles. Co-culturing of striated muscle cells with malignant cells led to marked morphological alterations in the latter, in contrast to the same cells when incubated without muscle cells. A conditioned medium of striated muscle cells was prepared and its effect tested on a variety of cells. This conditioned medium (CM) inhibited proliferation of tumor cell lines of murine (B16 melanoma, Madison 109 lung carcinoma, MCA-105 sarcoma, ESB lymphoma), or of human origin (HTB-38 adenocarcinoma, T47D breast carcinoma, CX1 colon carcinoma). The proliferation of normal cells (bone marrow cells, fetal liver erythroid cells) was not affected by the CM. Flow cytometric analysis of B16 melanoma cells following incubation with the CM revealed that 63% +/- 12 of the cells were in the G0/G1 phase of the cell cycle, compared to 47.8% +/- 8 of cells incubated with a medium (not conditioned) only. The activity of the CM and of certain fractions thereof was also demonstrated in vivo: they prevented tumor growth in mice inoculated intraperitoneally with MCA-105 sarcoma cells. Partial purification of the CM revealed that the active component was a non-proteinaceous compound with a molecular weight of about 500 D. The results clearly suggest that muscle cells produce a low molecular weight factor which can selectively inhibit the proliferation of tumor cells in vitro and in vivo. This factor is neither species nor tumor specific.

Correlation between the sensitivity or resistance to IL-2 and the response to cyclophosphamide of 4 tumors transplantable in the same murine host.

We have studied the anti-tumor response to cyclophosphamide (CTX) in DBA/2 mice transplanted s.c. with 4 tumors exhibiting different responses to IL-2: ESb lymphoma and Friend leukemia cells (non-responsive or poorly responsive, respectively), p11-R-Eb and Eb lymphoma cells (both highly responsive to IL-2). CTX injections on days 7, 14 and 21 resulted in a significant anti-tumor response in mice transplanted s.c. with Friend leukemia cells or ESb cells, whereas no anti-tumor effect was observed in mice injected with Eb or p11-R-Eb cells. All 4 tumor cell lines were equally sensitive to the cytotoxic effects of mafosfamide, an in vitro active analogue of CTX. To define the host mechanisms responsible for the lack of an anti-tumor effect of CTX in mice transplanted with IL-2-responsive tumors, we studied several aspects of the spontaneous or IL-2-induced anti-tumor response in mice transplanted with p11-R-Eb cells. Injection of monoclonal antibodies (MAbs) to IFN-gamma completely abolished the anti-tumor effects of IL-2. Using a Winn assay, clear-cut anti-tumor activity was found in spleen cells from mice transplanted with the IL-2-responsive tumors. This activity was abolished by CTX, which also abrogated the anti-tumor response to IL-2 in mice injected with p11-R-Eb cells. Our results indicate an inverse correlation between sensitivity to IL-2 and response to CTX and emphasize the importance of initial host-tumor interaction in determining the type of response to IL-2 or CTX.

Adhesion of lymphoma cells to fibronectin: differential use of alpha 4 beta 1 and alpha 5 beta 1 integrins and stimulation by the 9EG7 mAb against the murine beta 1 integrin subunit.

Murine ESb and MDAY-D2 lymphoma cells are highly metastatic, in particular to the liver, and are highly invasive in hepatocyte cultures. This may involve adhesion to hepatocyte surface-associated fibronectin (Kemperman et al., 1994, Cell Adh. and Communic. 2:45). Both ESb and MDAY-D2 cells express the fibronectin receptor alpha 4 beta 1, and MDAY-D2 cells in addition also alpha 5 beta 1. Yet, adhesion of ESb cells to fibronectin was low, and MDAY-D2 cells did not adhere at all, but adhesion of both cells was stimulated by phorbol myristate acetate (PMA) and Mn2+. In ESb cells, this adhesion was mediated by alpha 4 beta 1. In MDAY-D2 cells, however, only alpha 5 beta 1 was involved, despite alpha 4 beta 1 levels similar to ESb cells. The alpha 4 beta 1 integrin was functional since it mediated adhesion of MDAY-D2 cells to VCAM-1. An alpha 5 beta 1-negative variant of MDAY-D2 adhered to fibronectin and this was mediated by alpha 4 beta 1. These results indicate that alpha 4 beta 1 function in these cells is suppressed in the presence of alpha 5 beta 1. Adhesion of ESb cells to hepatocytes was inhibited by anti-alpha 4 antibody, but only by 30%, and fibronectin adhesion was found to have no role in the interaction of MDAY-D2 cells with hepatocytes. This suggests that alpha 4 beta 1 and alpha 5 beta 1 function is not activated during this interaction. The 9EG7 antibody against mouse beta 1 integrin was described to inhibit beta 1 integrins (Lenter et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 9051). In contrast, we observed that 9EG7 stimulated beta 1-integrin function: Adhesion of ESb and MDAY-D2 cells not only to fibronectin, but also to laminin was induced or enhanced.

Successful immunotherapy of the highly metastatic murine ESb lymphoma with sensitized CD8+ T cells and IFN‐α/β

Daily IFN-alpha/beta therapy was totally ineffective in inhibiting the development of visceral metastases in DBA/2 mice injected i.v. with the ESb lymphoma regardless of the number of tumor cells injected. The finding that IFN-alpha/beta therapy increased the survival time of ESb-immunized mice rechallenged with ESb cells suggested that cooperation between the immune system and IFN-alpha/beta was important. Adoptive transfer of Esb-immune spleen cells (but not normal cells) together with IFN-alpha/beta treatment did inhibit the development of ESb metastases in immunocompetent DBA/2 mice. Either treatment alone was ineffective. The anti-metastatic effect was specific for the ESb lymphoma as spleen cells from ESb-immunized mice together with IFN-alpha/beta treatment did not inhibit the development of metastases in mice challenged with IFN-alpha/beta-resistant 3C18 FLC. Depletion of CD8+ T cells (but not CD4+ T cells or B lymphocytes) prior to transfer eliminated the protective effect of ESb-immune splenocytes in IFN-alpha/beta-treated mice. As few as 1 x 10(6) ESb-immune spleen cells highly enriched for CD8+ cells increased the survival time of IFN-alpha/beta-treated ESb-challenged DBA/2 mice. The combined therapy of ESb-specific immune cells and IFN-alpha/beta resulted in long-term immunity to this tumor.

Inactivation of erythrocytic, lymphocytic and myelocytic leukemic cells by photoexcitation of endogenous porphyrins

The photodynamic sensitization of leukemic cells (erythrocytic, myelocytic and lymphocytic) via light activation of endogenous porphyrins is described. Human myelocytic—erythrocytic K562 cells and murine Friend erythroleukemia (FELC) and T-cell lymphoma Eb—Esb cells were stimulated to synthesize and accumulate porphyrins. K562 cells accumulated high amounts of protoporphyrin by stimulation with 5-aminolevulinic acid (ALA) plus sodium butyrate or hemin. For Friend and Eb—Esb cells ALA was an adequate stimulator. The high-metastatic Esb lymphoma cells accumulated comparatively more porphyrin than the low-metastatic Eb cell line. Maximal porphyrin accumulation produced mortality rates of more than 99% after 10 min of photoactivation of the three leukemic lines. Thymidine incorporation was inhibited by the photodynamic effect depending on porphyrin concentration. These results confirm the photodynamic ability of endogenous porphyrins to inactivate cancer cells of different origins.

Immunobiology and Immunotherapy of Cancer Metastases Ten-Year Studies in an Animal Model Resulting the Design of an Immunotherapy Procedure now under Clinical Testing

AbstractFailure of cancer therapy can most often be attributed to metastasis, which changes a localised disease into a systemic one. Animal models have contributed to improved understanding of metastasis formation, dissemination and treatment, and in this review immunological findings from the highly metastatic murine ESb lymphoma model are presented. Insights gained from this research can be applied to the design of new immunotherapy protocols for the treatment of many human cancers. The development and application of active specific immunotherapy (ASI) are described.

Microvascular injury in pathogenesis of interferon-induced necrosis of subcutaneous tumors in mice.

DBA/2 mice were injected sc with cells from the highly malignant Friend erythroleukemia cell (FLC) 3Cl8 subline, which is resistant to mouse interferon alpha/beta, or with the ESb lymphoma. When interferon alpha/beta was injected intratumorally or peritumorally, tumor growth was markedly suppressed, and established vascularized tumor nodules became progressively necrotic. Tumor necrosis was of the coagulation type that usually results from deprivation of blood flow. Morphologic examination of approximately 1,000 blood vessel profiles and approximately 2,000 endothelial cells in 1-micron Epon sections of sc 3C18 FLC tumors showed that interferon treatment resulted in rapid and pronounced vascular endothelial cell damage that preceded tumor necrosis. No inflammatory cell infiltrate was observed. Our results suggest that interferon alpha/beta exerted an antitumor effect in these tumor models by damaging tumor blood vessels, causing disruption of tumor blood flow, which led to ischemic tumor necrosis.

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.

Detection of Tumour‐Associated Transplantation Antigens by Syngeneic Cytotoxic T Lymphocytes after Fusion of Tumour Cell Membrane Vesicles to Recipient Cells

Sendai virus envelope-mediated fusion was used to transfer plasma membrane vesicles of ESb lymphoma cells (H-2d) to syngeneic or allogeneic recipient cells. After fusion, ESb-specific cell surface determinants were detectable on the recipient cells, as revealed by indirect immunofluorescence and cytofluorographic analysis. Fused cells were also tested as targets in 51Cr-release assays, using syngeneic DBA/2 anti-ESb- or anti-H-2d-specific T lymphocytes (CTL). Fused recipient cells became susceptible to lysis by both types of CTL, indicating that both tumour-specific and H-2d determinants were inserted on fusion. The experimental approach presented could be of help as an assay system for the molecular identification of tumour-specific transplantation antigens.

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.