Anne-Marie Engel

MCA Sarcomas Induced in scid Mice are More Immunogenic than MCA Sarcomas Induced in Congenic, Immunocompetent Mice

With the aim of studying possible T‐cell mediated selection of the cells in growing tumours, 108 mice of the C.B‐17 strain, either immunocompetent C.B‐17 mice or histocompatible immunodeficient C.B‐17 severe combined immune deficiency (scid) were treated with 3‐methylcholanthrene (MCA) in two different dosages. A total of 51 tumours were obtained, 44 of which were established as uncloned tumour cell lines, and used for further study. Tumour incidence correlated with carcinogen‐dosage in that more tumours developed in groups treated with a high MCA dose than in groups treated with a low MCA dose, but not with immune status of the tumour host. No significant difference in the level of MHC class I molecule expression was found between the two groups of tumours. The rate of rejection after transplantation to syngeneic immunocompetent hosts was significantly higher for the scid tumours than for the non‐scid tumours. The authors suggest that this reflects an immunoselection performed by T cells in the immunocompetent host in which the tumour originated, which has eliminated highly immunogenic tumour cells, leaving non‐immunogenic tumour cells to grow.

The role of cytotoxic T-lymphocytes in the prevention and immune surveillance of tumors— lessons from normal and immunodeficient mice

The idea of immunological surveillance against cancer has existed for nearly 100 years but as no conclusive evidence has yet been published the importance of the cellular immune defense in the detection and removal of incipient or existing tumors is still a hotly debated subject. However, in order to select a relevant immunotherapeutic strategy in the treatment of cancer, a fundamental understanding of the basic immunologic conditions under which a tumor develops and exists is a prerequisite. Therefore, a murine model was set up that we hoped would enable us to confirm or reject the theory of immunological surveillance. A large panel of methylcholanthrene induced tumors was established in T-cell immunodeficient nude mice and congenic normal mice to study the influence of the immune system on developing tumors. As nude mice developed tumors fastest and with the highest incidence, we concluded that in this model the immune system constituted a ‘tumor-suppressive factor’ delaying and sometimes abrogating tumor growth, i.e. performing immune surveillance. Immunogenicity of the tumors was assessed by transplantation back to normal histocompatible mice. Tumors originating from the immunodeficient nude mice turned out to be far more immunogenic than tumors from normal mice, resulting in a high rejection rate. CD8+cytotoxic T cells were found to be indispensable for this rejection, leading to the conclusion that the cytotoxic T cells perform immune selection in normal mice, eliminating immunogenic tumor cell variants in the incipient tumor.In this review, we discuss the difficulties facing immunotherapy when conclusions are drawn from the presented observations and hypotheses.

Methylcholanthrene-induced sarcomas in nude mice have short induction times and relatively low levels of surface MHC class I expression.

In order to study the role of the T‐cell‐mediated immune defense in tumor development, a total of 93 sarcomas were induced using different doses (8 μg (0.1%), 40 μg (0.5%) and 400 μg (5%)) of 3‐methylcholanthrene in athymic nude Balb/c mice and phenotypically normal immunocompetent Balb/c mice. A shorter tumor induction time and a higher tumor incidence after treatment with low doses of methylcholanthrene were seen in nude mice than in immunocompetent mice, indicating that they have a lower resistance to the carcinogen. Contrary to expectations we found that the MHC class I expression of tumors from nude mice was lower than that of tumors from normal mice. Higher surface expression of MHC class I was demonstrated on high dose tumors from normal mice than on low dose tumors from normal mice. The cellular composition of the individual tumors raised in nude mice was more heterogeneous with respect to MHC class I expression. Since the mice differ genetically only with respect to the nu gene, these results indicate that a lack of T‐cell‐mediated defense mechanisms may confer upon the bearer a lower resistance to 3‐methylcholanthrene and a different MHC profile of the ensuing tumor.

A quantitative ELISA-based co-culture angiogenesis and cell proliferation assay

Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well‐tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335–44) to a quantitative ELISA‐based fibroblast and endothelial cell co‐culture angiogenesis assay. In each well of a 96‐microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co‐culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well‐known angiogenesis inhibitors on HUVEC tube formation.

Interferon-gamma-induced MHC class I expression and defects in Jak/Stat signalling in methylcholanthrene-induced sarcomas.

Seventy‐eight uncloned tumour cell lines, each established from a primary sarcoma induced with methylcholanthrene in immunocompetent nu/+ BALB/c and C.B.‐17 mice or in immunodeficient nu/nu BALB/c and severe combined immunodeficient (SCID) mice, were examined for sensitivity to interferon‐γ (IFN‐γ) as measured by tumour cell augmentation of major histocompatibility complex (MHC) class I expression. The tumour cells were cultured with IFN‐γ and their expression of Kd, Dd and Ld was measured by fluorescence‐activated cell sorter analysis. All but three of the 78 tumour lines up‐regulated Kd, Dd and Ld to a variable degree in response to IFN‐γ, indicating that IFN‐γ resistance is not a common property of these sarcomas. The tumour cell lines varied greatly in their MHC class I expression before as well as after IFN‐γ stimulation. There was a tendency towards a higher MHC expression after IFN‐γ stimulation in tumour lines from immunocompetent mice compared to immunodeficient mice, but no common maximum MHC class I expression level was found for the 78 tumour cell lines. Three of the tumour lines, all from immunodeficient mice, completely failed to respond to IFN‐γ by up‐regulating MHC class I expression. The same three also displayed absence of IFN‐γ‐induced Stat1β tyrosine phosphorylation and low Stat1α tyrosine phosphorylation, indicating a defect in the signal transduction pathway affecting phosphorylation of Stat1. These findings strongly suggest a link between defects in Stat1 phosphorylation and the failure to up‐regulate MHC class I. In all tumour lines tested, the Stat1 Western blotting revealed a 78 kDa protein (p78) not previously described.

CD8+ T cells are crucial for the ability of congenic normal mice to reject highly immunogenic sarcomas induced in nude mice with 3-methylcholanthrene

An attempt was made to identify the selection pressures put upon a growing tumour by CD8+ T cells. To this end tumours induced with 3‐methylcholanthrene in T cell‐deficient nude mice and in congenic T cell‐competent nu/+ mice were transplanted to nu/+ recipients. The rejection rate of the sarcomas from nude mice was almost twice that of the sarcomas from nu/+ mice. Depletion of CD8+ T cells from nu/+ recipients prior to transplantation made them accept nude tumours that were consistently rejected by untreated nu/+ recipients. These findings suggest that a methylcholanthrene sarcoma during its growth in a T cell‐competent host adapts to the T cell system through a selective elimination of highly immunogenic tumour cells that are susceptible to CD8+ T cell‐mediated lysis.

Influence of Levamisole and Other Angiogenesis Inhibitors on Angiogenesis and Endothelial Cell Morphology in Vitro

Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity in vivo since it inhibits ECs in vitro with a morphology resembling that obtained with antibodies to VEGF.

The Susceptibility to Cytotoxic T Lymphocyte Mediated Lysis of Chemically Induced Sarcomas from Immunodeficient and Normal Mice

A panel of sarcomas induced with 3‐methylcholanthrene in normal and immunodeficient mice was studied for their capacity to present antigen by the endogenous, MHC class I restricted pathway. Lymphocytic choriomeningitis virus was used to infect cultured tumour cells, and the infected cells were tested for susceptibility to cytolysis by virus specific cytotoxic T cells. Tumour cells originating from tumours induced in immunocompetent C.B.‐17 mice presented virus antigen more efficiently than tumour cells from immunodeficient SCID mice. No significant difference in virus antigen presentation was found between tumours from nude and nu/+ BALB/c mice. The sensitivity of target cells from the individual tumours to cytotoxic T lymphocyte (CTL) mediated lysis correlated negatively with their sensitivity to natural killer (NK) cell mediated lysis. There was a positive correlation between the sensitivity to CTL mediated lysis and surface expression of the MHC class I molecule Ld of the tumour cells. Tumour cells incapable of in vitro presentation of viral antigen to specific cytotoxic T cells originated from tumours known from previous experiments to be readily accepted after transplantation to immunocompetent, histocompatible recipients.

Molecular aberrations in the MHC class I-restricted pathway for antigen presentation in methylcholanthrene sarcomas from nude mice: discrepancies between MHC mRNA and surface protein

In a previous study, we demonstrated that eight sarcomas induced by chemical carcinogenesis in nude mice were rejected by syngeneic immunocompetent recipients at a much higher rate than eight sarcomas induced with the same method in syngeneic immmunocompetent mice. In the present study, we investigated these 16 sarcomas for structural and quantitative aberrations in components of the MHC class I‐restricted antigen‐processing and ‐presentation pathway. Considerable discrepancies between mRNA levels and cell surface protein expression of MHC class I (Kd, Dd and Ld) molecules were observed almost exclusively in the tumours derived from nude mice. Several of the nude mouse‐derived tumours also displayed incongruent levels of heavy chain mRNA and β2‐microglobulin mRNA. These findings are taken as indications of abnormal regulation of gene transcription in nude mouse tumours, and if this abnormal regulation extends to the entire genome, it may explain the pronounced immunogenicity of these tumours. Proteasome composition, heat shock protein expression, TAP‐molecule inducibility and intercellular adhesion molecule‐1 expression were investigated in the same tumours. We found no indications of structural defects or quantitative differences in these molecules between the two groups of tumours.

Synthesis and Anti-Angiogenic Effect of Conjugates Between Serum Albumin and Non-Steroidal Anti-Inflammatory Drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit tumor growth and angiogenesis. Covalent linkage of naproxen to human serum albumin (HSA) has been shown to target it efficiently to the liver and this may potentially be exploited for liver-selective inhibition of angiogenesis. With the aim of investigating the anti-angiogenic efficiency of NSAID-HSA conjugates in vitro, three NSAIDs, aspirin, ibuprofen, and naproxen were conjugated to HSA using different concentrations of their N-hydroxysuccinimide esters. Conjugation ratios from 10 to 50 were achieved and the conjugates retained a growth inhibitory effect on endothelial cells at or above the level of the non-conjugated NSAIDs in an in vitro angiogenesis assay.