CD44 is associated with proliferation, rather than a specific cancer stem cell population, in cultured canine cancer cells.

BACKGROUND: The cancer stem cell hypothesis proposes that tumours are maintained by a population of cancer stem cells (CSC), which must be eradicated to prevent disease relapse after treatment. Cells expressing high levels of CD44 have been identified as candidate CSC in a variety of human tumours. This study sought to investigate CD44 expression and its potential as a CSC marker in canine cancer. METHODS: CD44 expression in several canine cancer cell lines was determined by flow cytometry. Cells with low and high levels of CD44 expression were examined for differences in growth characteristics, colony forming ability, drug sensitivity and cell cycle profile. RESULTS: CD44(High) cells demonstrated enhanced growth and colony forming capacity, under both adherent and low-density serum free ("tumoursphere") conditions. However, no difference in sensitivity to doxorubicin was seen between the two populations. Moreover, whilst most CD44(Low) cells were in resting or G1 growth phase, an increased proportion of CD44(High) cells were in G2M phase of the cell cycle. Upon proliferation in culture, both populations gave rise to progeny with a full spectrum of CD44 expression. CONCLUSION: CD44 expression is associated with proliferation in cultured canine cancer cells, but transient and fluctuating expression may limit its utility as a CSC marker.

Anti-NeuGcGM3 antibodies, actively elicited by idiotypic vaccination in nonsmall cell lung cancer patients, induce tumor cell death by an oncosis-like mechanism.

1E10 is a murine anti-idiotypic mAb specific for an idiotypic mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In melanoma, breast, and lung cancer patients, this anti-idiotypic Ab was able to induce a specific Ab response against N-glycosylated gangliosides, attractive targets for cancer immunotherapy as these glycolipids are not naturally expressed in humans. A clinical study with nonsmall cell lung cancer patients showed encouraging clinical benefits. Immunological studies performed in 20 of these patients suggested a correlation between the induction of Abs against NeuGcGM3 and longer survival times. The induced anti-NeuGcGM3 Abs recognized and directly killed tumor cells expressing the Ag, by a mechanism independent of complement activation. In the present work, we show that this cytotoxicity differs from apoptosis because it is temperature independent, no chromatin condensation or caspase 3 induction are detected, and the DNA fragmentation induced has a different pattern than the one characteristic for apoptosis. It is a very quick process and involves cytosqeleton reorganization. The Abs induce cellular swelling and the formation of big membrane lesions that allow the leakage of cytoplasm and the loss of the cell membrane integrity. All of these characteristics resemble a process of oncotic necrosis. To our knowledge, this is the first report of the active induction in cancer patients of NeuGcGM3-specific Abs able to induce complement independent oncotic necrosis to tumor cells. These results contribute to reinforcing the therapeutic potential of anti-idiotypic vaccines and the importance of NeuGcGM3 ganglioside as antitumor target.

ATTEMPTS system: a macromolecular prodrug strategy for cancer drug delivery.

In order to reduce systemic toxicity and effectively deliver macromolecular drug into tumor cells, a system termed "ATTEMPTS" (antibody targeted, [protamine] triggered, electrically modified prodrug-type strategy) was developed in our laboratory. This approach was adapted from our previously reported heparin/protamine-based system for controlled delivery of protease drugs such as tissue- specific plasminogen activator (tPA). In this "ATTEMPTS" system, the cell-permeable protein drugs are synthesized by conjugating proteins to cell-penetrating peptides (CPPs). Cell penetration ability of such CPP-protein conjugates would initially be disabled, acting as a "prodrug", by forming polyelectrolyte complexes with a functionalized heparin-antibody moiety. The complexes would accumulate in tumor sites by the antibody targeting function, and then the local release of CPP-protein conjugates would be triggered by protamine. We applied this system to the macromolecular anticancer agents, such as the protein drugs (gelonin and asparaginase) as well as the polymerdrugs (polyrotaxane-doxorubicin and polyrotaxane-camptothecin). Both in vitro and preliminary in vivo studies demonstrated the regulable cell penetration behavior based on the competitive ionic interactions between CPP/heparin and heparin/protamine. Thus, this ATTEMPTS approach provides a multi-functionalized system incorporating the features of targeting, prodrug-like, triggerable release, and cell penetration ability for the delivery of macromolecular anticancer agents. A summary of our work on "ATTEMPTS" is presented in this review.

Attenuation of PI3K/Akt-mediated tumorigenic signals through PTEN activation by DNA vaccine-induced anti-ErbB2 antibodies.

By studying BALB/c mice deficient in immune components, we show that the protective immunity to rat ErbB2(+) tumors rests on the Ab response elicited by the electroporation of a DNA vaccine encoding the extracellular and transmembrane domains of rat ErbB2. In vivo, the adoptive transfer of vaccine-elicited anti-rat ErbB2 Abs protected against a challenge of rat ErbB2(+) carcinoma cells (TUBO cells). In vitro, such Abs inhibited TUBO cell growth by impairing cell cycle progression and inducing apoptosis. To correlate intrinsic mechanisms of Ab action with their tumor-inhibitory potential, first we showed that TUBO cells constitutively express phosphorylated transgenic ErbB2/autochthonous ErbB3 heterodimers and exhibit a basal level of Akt phosphorylation, suggesting a constitutive activation of the PI3K/Akt pathway. Treatment with anti-ErbB2 Abs caused a drastic reduction in the basal level of Akt phosphorylation in the absence of an impairment of PI3K enzymatic activity. Notably, the same Ab treatment induced an increase in PTEN phosphatase activity that correlated with a reduced PTEN phosphorylation. In conclusion, vaccine-induced anti-ErbB2 Abs directly affected the transformed phenotype of rat ErbB2(+) tumors by impairing ErbB2-mediated PI3K/Akt signaling.

Cutting edge: permissive MHC class II allele changes the pattern of antitumor immune response resulting in failure of tumor rejection.

We studied the growth of transgenic adenocarcinoma of mouse prostate (TRAMP)-C1 tumor cells expressing human prostate-specific Ag (PSA) in HLA-DRB1*1501 (DR2b) transgenic mice. TRAMP-PSA tumors were frequently rejected by HLA-DR2b(-) mice but had increased incidence in HLA-DR2b(+) littermates. The levels of PSA-specific CD8 T cell responses were significantly higher in the HLA-DR2b(-) mice that rejected TRAMP-PSA tumors compared with HLA-DR2b(+) tumor-bearing littermates. In contrast, Ab responses to PSA were strong in HLA-DR2b(+) mice bearing TRAMP-PSA tumors and were virtually undetectable in HLA-DR2b(-) littermates. The analysis of CD4 T cell responses to PSA revealed the presence of several CD4 T cell epitopes in HLA-DR2b(+) mice but failed to identify strong I-A(b)-restricted epitopes in HLA-DR2b(-) mice. Our data demonstrate that the expression of a permissive HLA class II allele can change the pattern of the immune response to a tumor Ag, resulting in the failure of tumor rejection.

Tumor cell killing mechanisms of epidermal growth factor receptor (EGFR) antibodies are not affected by lung cancer-associated EGFR kinase mutations.

The epidermal growth factor receptor (EGFR) serves as a molecular target for novel cancer therapeutics such as tyrosine kinase inhibitors (TKI) and EGFR Abs. Recently, specific mutations in the EGFR kinase domain of lung cancers were identified, which altered the signaling capacity of the receptor and which correlated with clinical response or resistance to TKI therapy. In the present study, we investigated the impact of such EGFR mutations on antitumor cell activity of EGFR Abs. Thus, an EGFR-responsive cell line model was established, in which cells with tumor-derived EGFR mutations (L858R, G719S, delE746-A750) were significantly more sensitive to TKI than wild-type EGFR-expressing cells. A clinically relevant secondary mutation (T790M) abolished TKI sensitivity. Significantly, antitumor effects of EGFR Abs, including signaling and growth inhibition and Ab-dependent cellular cytotoxicity, were not affected by any of these mutations. Somatic tumor-associated EGFR kinase mutations, which modulate growth inhibition by TKI, therefore do not impact the activity of therapeutic Abs in vitro.

The tumour suppressor hSNF5/INI1 controls the differentiation potential of malignant rhabdoid cells.

Malignant rhabdoid tumours (MRT) are highly aggressive cancers of early childhood that arise in different organs or tissues. The unifying criterion for these tumours is the presence of inactivating mutations of the hSNF5/INI1 tumour suppressor gene which encodes a core subunit of the chromatin remodelling SWI/SNF complex. Using a variety of markers we analysed the phenotypic traits of MON and DEV cell lines derived respectively from an undifferentiated abdominal MRT and from a brain MRT. DEV cells express spontaneously a wide range of neural and glial markers. It can be induced to differentiate into the neural lineage following hSNF5/INI1 expression with appearance of neurite processes, strong increase of neural markers and decrease of glial markers. A less pronounced neural differentiation is also observed with MON cells, which possess more primitive polyphenotypic features with positivity for markers from the three embryonic layers. Finally, we show that the neural differentiation of rat PC12 cells in the presence of nerve growth factor (NGF) is strongly impaired when hSNF5/INI1 expression is inhibited by RNA interference. Altogether these results indicate that hSNF5/INI1 is an essential subunit for SWI/SNF-dependant induction of neural differentiation programs. Further experiments should enable documentation of whether it provides instructive or permissive signals for differentiation.

The role of human glioma-infiltrating microglia/macrophages in mediating antitumor immune responses.

Little is known about the immune performance and interactions of CNS microglia/macrophages in glioma patients. We found that microglia/macrophages were the predominant immune cell infiltrating gliomas ( approximately 1% of total cells); others identified were myeloid dendritic cells (DCs), plasmacytoid DCs, and T cells. We isolated and analyzed the immune functions of CD11b/c+CD45+ glioma-infiltrating microglia/macrophages (GIMs) from postoperative tissue specimens of glioma patients. Although GIMs expressed substantial levels of Toll-like receptors (TLRs), they did not appear stimulated to produce pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin 1, or interleukin 6), and in vitro, lipopolysaccharides could bind TLR-4 but could not induce GIM-mediated T-cell proliferation. Despite surface major histocompatibility complex class II expression, they lacked expression of the costimulatory molecules CD86, CD80, and CD40 critical for T-cell activation. Ex vivo, we demonstrate a corresponding lack of effector/activated T cells, as glioma-infiltrating CD8+ T cells were phenotypically CD8+CD25-. By contrast, there was a prominent population of regulatory CD4 T cells (CD4+CD25+FOXP3+) infiltrating the tumor. We conclude that while GIMs may have a few intact innate immune functions, their capacity to be stimulated via TLRs, secrete cytokines, upregulate costimulatory molecules, and in turn activate antitumor effector T cells is not sufficient to initiate immune responses. Furthermore, the presence of regulatory T cells may also contribute to the lack of effective immune activation against malignant human gliomas.

Targeted apoptosis activation with GrB/scFvMEL modulates melanoma growth, metastatic spread, chemosensitivity, and radiosensitivity.

GrB/scFvMEL, a fusion protein composed of human granzyme B (GrB) and the single-chain antibody scFvMEL, targets melanoma gp240 antigen and exerts impressive cytotoxic effects by inducing apoptosis. We evaluated the effects of GrB/scFvMEL on chemotherapy, radiation therapy, metastasis in vitro, and the growth of human melanoma A375 xenograft tumors in nude mice. GrB/scFvMEL showed synergistic cytotoxicity when coadministered with doxorubicin, vincristine or cisplatin, and additive effects, in combination with etoposide or cytarabine. Optimal cytotoxic effects were obtained when cells were treated first with GrB/scFvMEL followed by exposure to the agent (rather than the reverse). Pretreatment of A375 cells with GrB/scFvMEL significantly sensitized melanoma cells to ionizing radiation assessed using a clonogenic survival assay. Subtoxic doses of GrB/scFvMEL inhibited the invasion of A375 cells into Matrigel. GrB/scFvMEL (37.5 mg/kg) was administered intravenously to nude mice bearing A375 tumors. Saline-treated tumors increased 24-fold, whereas tumors treated with GrB/scFvMEL showed a significant tumor growth delay increasing four-fold. Tumor tissue displayed an increase in apoptotic nuclei compared to control. Thus, the targeted delivery of GrB to tumors may have a significant potential for cancer treatment. Targeted therapeutic agents specifically designed to impact cellular apoptotic pathways may represent a novel class of therapeutic agents.

Antibodies against tumor cell glycolipids and proteins, but not mucins, mediate complement-dependent cytotoxicity.

One of several effector mechanisms thought to contribute to Ab efficacy against cancer is complement-dependent cytotoxicity (CDC). Serological analysis of a series of clinical trials conducted over a 10-year period suggested that six vaccines containing different glycolipids induced Abs mediating CDC whereas four vaccines containing carbohydrate or peptide epitopes carried almost exclusively by mucin molecules induced Abs that did not mediate CDC. To explore this further, we have now compared cell surface reactivity using flow cytometry assays (FACS), complement-fixing ability, and CDC activity of a panel of mAbs and immune sera from these trials on the same two tumor cell lines. Abs against glycolipids GM2, globo H and Lewis Y, protein KSA (epithelial cell adhesion molecule, also known as EpCAM) and mucin Ags Tn, sialylated Tn, Thomsen Friedenreich (TF), and MUC1 all reacted comparably by FACS with tumor cells expressing these Ags. Compared with the strong complement binding and CDC with Abs against glycolipids and KSA, complement binding was diminished with Abs against mucin Ags and no CDC was detected. A major difference between these two groups of Ags is proximity to the cell membrane. Glycolipids and globular glycoproteins extend less than 100 A from the cell membrane while mucins extend up to 5000 A. Although complement activation at sites remote from the cell membrane has long been known as a mechanism for resistance from complement lysis in bacteria, it is identified here for the first time as a factor which may contribute to resistance from CDC against cancer cells.

TCR vaccines against a murine T cell lymphoma: a primary role for antibodies of the IgG2c class in tumor protection.

Tumor-associated proteins can act as effective immunotherapeutic targets. Immunization with tumor TCR protein conjugated to the immunogenic protein keyhole limpet hemocyanin (KLH) protects mice from tumor challenge with the murine T cell lymphoma C6VL. The immune mechanisms responsible for this tumor protection are of interest for designing more effective vaccine strategies. Previous studies using depletion experiments had suggested a CD8-mediated component of protection induced by TCR-KLH vaccines. In this study we used CD8alpha knockout, micro MT, and FcgammaR knockout mice to investigate the relative roles of CD8+ T cells and Ab in protective immunity induced by TCR-KLH immunization. We found that CD8+ T cells are not required for tumor protection, although they may contribute to protection. Vaccine-induced Abs are sufficient to mediate protection against this murine T cell lymphoma through an FcR-dependent mechanism. This was confirmed with Ab transfers, which protect challenged mice. Additionally, recombinase-activating gene 1(-/-) splenocytes can mediate Ab-dependent cellular cytotoxicity against this tumor in the presence of bound anti-TCR Abs. IFN-gamma knockout mice demonstrated a requirement for IFN-gamma, probably via generation of IgG2c Abs, in vaccine-induced tumor protection. IFN-gamma knockout mice were not protected by immunization and had a severe impairment in IgG2c Ab production in response to immunization. Although mock-depleted anti-TCR Abs could transfer tumor protection, IgG2c-deficient anti-TCR Abs were unable to transfer tumor protection to wild-type mice. These results suggest that TCR-KLH vaccine-induced tumor protection in the C6VL system is primarily attributable to the induction of IgG2c Abs and humoral immunity.

Anti-tumor immunoglobulin M increases lung metastasis in an experimental model of malignant melanoma.

Cancer metastasis involves distinct steps that depend on complicated tumor-host interactions. The hematogenous dissemination of tumor cells may be facilitated by factors that promote the arrest and adherence of cancer cells in capillaries. We examined whether anti-tumor monoclonal immunoglobulin M (IgM) antibodies promoted the hematogenous dissemination of B16 melanoma cells in syngeneic mice. IgM monoclonal antibodies were generated that selectively bind to B16 melanoma cells as compared to syngeneic fibroblasts, lymphocytes or Lewis lung carcinoma cells. Incubation of B16-BL6 or B16-F0 melanoma cells with these IgM anti-tumor antibodies significantly increased the number of lung colonies as compared with control antibodies. Moreover, intraperitoneal injection of specific antibody also significantly increased lung colonization. All anti-tumor antibodies promoted the aggregation of B16 melanoma cells. A chemically generated immunoglobulin G (IgG)-like fragment of an anti-tumor IgM antibody displayed greatly reduced tumor aggregation and, in contrast to intact IgM, did not significantly increase lung colonization of B16 melanoma cells. Neither intact IgM nor the IgG-like fragment enhanced the in vitro invasiveness of B16 melanoma cells across Matrigel-coated membranes. Our results, therefore, suggest that besides their beneficial anti-tumor effects, anti-tumor IgM antibodies may also promote the hematogenous dissemination of cancer cells.

Nonredundant roles of antibody, cytokines, and perforin in the eradication of established Her-2/neu carcinomas.

Since the mechanisms by which specific immunity destroys Her-2/neu carcinoma cells are highly undetermined, these were assessed in BALB/c mice vaccinated with plasmids encoding extracellular and transmembrane domains of the protein product (p185(neu)) of the rat Her-2/neu oncogene shot into the skin by gene gun. Vaccinated mice rejected a lethal challenge of TUBO carcinoma cells expressing p185(neu). Depletion of CD4 T cells during immunization abolished the protection, while depletion of CD8 cells during the effector phase halved it, and depletion of polymorphonuclear granulocytes abolished all protection. By contrast, Ig mu-chain gene KO mice, as well as Fcgamma receptor I/III, beta-2 microglobulin, CD1, monocyte chemoattractant protein 1 (MCP1), IFN-gamma, and perforin gene KO mice were protected. Only mice with both IFN-gamma and perforin gene KOs were not protected. Although immunization also cured all BALB/c mice bearing established TUBO carcinomas, it did not cure any of the perforin KO or perforin and IFN-gamma KO mice. Few mice were cured that had knockouts of the gene for Ig mu-chain, Fcgamma receptor I/III, IFN-gamma, or beta-2 microglobulin. Moreover, vaccination cured half of the CD1 and the majority of the MCP1 KO mice. The eradication of established p185(neu) carcinomas involves distinct mechanisms, each endowed with a different curative potential.

Immunoengineering: a credible mechanism for CAMPATH-1H action in bone marrow and organ transplantation and the implications for treatment of the immune dysfunction AIDS.

Immunoengineering is a term coined to represent the mostly future ability to use or target the immune system's natural components, with emphasis on the regulatory components, to up or down regulate the immune system's attack against specific proteins associated with an unwanted pathology or immune occurrence. It will constitute manipulating parts of the immune system, mostly those specific for the disease associated antigen(s) and generally of a regulatory nature, in various immunological locale or the whole body compartment, to achieve a disease free state for the patient. The number of practical applications awaiting the mastery of immune components as regulatory therapeutics is enormous and immunoengineering should provide treatments in a wide range of disease categories. HIV is a disease where this discipline could provide a quick cure, even eradication of the virus. A potential cheap solution to HIV infection, based on using immunoengineering and adaptable to the infrastructure problems of the Third World is highlighted in the following because of the health emergency that exists in the Third World.

Autoimmune and antitumor consequences of antibodies against antigens shared by normal and malignant tissues.

There is now a considerable body of information documenting the autoimmune consequences of antibodies induced by growing malignancies, or by passively administered and actively induced antibodies, in cancer patients against antigens shared by normal and malignant tissues. This provides a rich source of information addressing the consequences of autoantibodies against a range of antigens. Antibodies against cell-surface or intracellular antigens in the central nervous system (CNS) or on epithelial surfaces of normal tissues do not generally result in autoimmunity, but the same types and titers of antibodies against cell surface antigens in the subepidermal skin, peripheral nerves, blood, or vascular sites such as the spleen and bone marrow readily induce autoimmunity. The blood brain barrier of the CNS and apical antigen expression and the basement membrane in epithelial tissues, may protect these sites from antibody induced damage. Cancer cells, however, are protected by neither unidirectional antigen expression nor basement membranes. Vaccine induced antibodies against a variety of cancer cell surface antigens have been associated with prevention of tumor recurrence in preclinical models and in vaccinated cancer patients, in the absence of demonstrable autoimmunity. This forms the basis for a series of ongoing Phase III trials with single or polyvalent antigen cancer vaccines designed for optimal antibody induction.

The CD45 tyrosine phosphatase regulates Campath-1H (CD52)-induced TCR-dependent signal transduction in human T cells.

Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo. The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.

CD4+ lymphocytes provide MUC1-specific tumor immunity in vivo that is undetectable in vitro and is absent in MUC1 transgenic mice.

A C57BL/6 mouse transgenic for human MUC1 (MUC1.Tg) was developed to evaluate MUC1-specific tumor immunity in an animal that expresses MUC1 as a normal self protein. Previous studies showed that MUC1.Tg mice, challenged with syngeneic tumors expressing MUC1 (B16.MUC1), developed progressively growing MUC1-positive tumors, whereas wild-type C57BL/6 (wt) mice developed MUC1-negative tumors at a significantly slower rate. The results of a limiting dilution CTL frequency assay were not informative, in that similar numbers of MUC1-specific CTL precursors (CTL) were detected in MUC1.Tg and wt mice. Tumor immunity in vivo was characterized by an adoptive transfer method to evaluate the degree of MUC1 or non-MUC1 tumor immunity in wt or MUC1.Tg mice. The results revealed that wt mice developed protective tumor immunity mediated by MUC1-specific CD4+ lymphocytes, while MUC1.Tg mice were functionally tolerant to MUC1 in vivo. The potential of adoptive immunotherapy to provide immunity to tumors expressing MUC1 and to produce undesirable autoimmunity in recipient MUC1.Tg mice expressing MUC1 as a self Ag was evaluated. Adoptive transfer of immune cells from wt mice primed in vivo with B16.MUC1 tumor cells into MUC1.Tg recipients resulted in significant increases in the survival of MUC1.Tg recipients compared with unmanipulated control MUC .Tg mice challenged with B16.MUC1 tumor cells. This response was specific for MUC1 since control tumors developed at equivalent rates in recipient or control MUC1.Tg mice. No gross or histologic evidence of autoimmunity was observed in recipient MUC1.Tg mice, indicating that tumor immune responses mediated by MUC1-specific CD4+ lymphocytes spare nontransformed epithelia-expressing MUC1.