Steffen Aulwurm

SD-208, a Novel Transforming Growth Factor β Receptor I Kinase Inhibitor, Inhibits Growth and Invasiveness and Enhances Immunogenicity of Murine and Human Glioma Cells In vitro and In vivo

The cytokine transforming growth factor (TGF)-β, by virtue of its immunosuppressive and promigratory properties, has become a major target for the experimental treatment of human malignant gliomas. Here we characterize the effects of a novel TGF-β receptor (TGF-βR) I kinase inhibitor, SD-208, on the growth and immunogenicity of murine SMA-560 and human LN-308 glioma cells in vitro and the growth of and immune response to intracranial SMA-560 gliomas in syngeneic VM/Dk mice in vivo. SD-208 inhibits the growth inhibition of TGF-β–sensitive CCL64 cells mediated by recombinant TGF-β1 or TGF-β2 or of TGF-β–containing glioma cell supernatant at an EC50 of 0.1 μmol/L. SD-208 blocks autocrine and paracrine TGF-β signaling in glioma cells as detected by the phosphorylation of Smad2 or TGF-β reporter assays and strongly inhibits constitutive and TGF-β–evoked migration and invasion, but not viability or proliferation. Peripheral blood lymphocytes or purified T cells, cocultured with TGF-β–releasing LN-308 glioma cells in the presence of SD-208, exhibit enhanced lytic activity against LN-308 targets. The release of interferon γ and tumor necrosis factor α by these immune effector cells is enhanced by SD-208, whereas the release of interleukin 10 is reduced. SD-208 restores the lytic activity of polyclonal natural killer cells against glioma cells in the presence of recombinant TGF-β or of TGF-β–containing glioma cell supernatant. The oral bioavailability of SD-208 was verified by demonstrating the inhibition of TGF-β–induced Smad phosphorylation in spleen and brain. Systemic SD-208 treatment initiated 3 days after the implantation of SMA-560 cells into the brains of syngeneic VM/Dk mice prolongs their median survival from 18.6 to 25.1 days. Histologic analysis revealed no difference in blood vessel formation, proliferation, or apoptosis. However, animals responding to SD-208 showed an increased tumor infiltration by natural killer cells, CD8 T cells, and macrophages. These data define TGF-β receptor I kinase inhibitors such as SD-208 as promising novel agents for the treatment of human malignant glioma and other conditions associated with pathological TGF-β activity.

Monocyte chemoattractant protein–1 increases microglial infiltration and aggressiveness of gliomas

Macrophages are thought to represent a first line of defense in anti‐tumor immunity. Despite infiltration by microglial cells, however, malignant gliomas are still highly aggressive tumors. We here identify monocyte chemoattractant protein‐1 (MCP‐1) as a critical chemoattractant for glioma‐infiltrating microglial cells. MCP‐1–transfected rat CNS‐1 gliomas were massively infiltrated by microglial cells. Whereas MCP‐1 did not promote the growth of CNS‐1 cells in vitro, intracerebral CNS‐1–transfected tumors grew more aggressively than control‐transfected tumors. This provides the first functional evidence that MCP‐1 recruits microglial cells to gliomas and promotes their growth in vivo. Microglial cells may support rather than suppress glioma growth.

Processing of Immunosuppressive Pro-TGF-β1,2 by Human Glioblastoma Cells Involves Cytoplasmic and Secreted Furin-Like Proteases

TGF-β is a putative mediator of immunosuppression associated with malignant glioma and other types of cancer. Subtilisin-like proprotein convertases such as furin are thought to mediate TGF-β processing. Here we report that human malignant glioma cell lines express furin mRNA and protein, exhibit furin-like protease (FLP) activity, and release active furin into the cell culture supernatant. FLP activity is not modulated by exogenous TGF-β or neutralizing TGF-β Abs. Exposure of LN-18 and T98G glioma cell lines to the furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, inhibits processing of the TGF-β1 and TGF-β2 precursor molecules and, consequently, the release of mature bioactive TGF-β molecules. Ectopic expression of PDX, a synthetic antitrypsin analog with antifurin activity, in the glioma cells inhibits FLP activity, TGF-β processing, and TGF-β release. Thus, subtilisin-like proprotein convertases may represent a novel target for the immunotherapy of malignant glioma and other cancers or pathological conditions characterized by enhanced TGF-β bioactivity.

Therapeutic anti-tumor immunity triggered by injections of immunostimulating single-stranded RNA

Stabilized synthetic RNA oligonucleotides (ORN) and protected messenger RNA (mRNA) were recently discovered to possess an immunostimulatory capacity through their recognition by TLR 7 and 8. We wanted to find out whether this danger signal is capable of triggering anti‐tumor immunity when injected locally into an established tumor. Using the mouse glioma tumor cell line SMA‐560 in syngenic VM/Dk mice, we were able to show that intra‐tumor injections of protamine‐stabilized mRNA do indeed induce tumor regression and long‐term anti‐tumor immunity. Residual RNA‐injected tumors show CD8 infiltration. Distant injections of protamine‐protected mRNA and intra‐tumor injection of naked mRNA also result in anti‐tumor immunity. Although they are strong danger signals, RNA are labile molecules with a short half‐life: they do not trigger side effects such as long‐term, uncontrolled immunostimulation evidenced by splenomegaly in CpG DNA‐treated mice. In conclusion, RNA molecules are potent and safe danger signals that are relevant for active immunotherapy strategies aimed at the eradication of solid tumors.

Inhibiting TGF-β signaling restores immune surveillance in the SMA-560 glioma model

Transforming growth factor-beta (TGF-beta) is a proinvasive and immunosuppressive cytokine that plays a major role in the malignant phenotype of gliomas. One novel strategy of disabling TGF-beta activity in gliomas is to disrupt the signaling cascade at the level of the TGF-beta receptor I (TGF-betaRI) kinase, thus abrogating TGF-beta-mediated invasiveness and immune suppression. SX-007, an orally active, small-molecule TGF-betaRI kinase inhibitor, was evaluated for its therapeutic potential in cell culture and in an in vivo glioma model. The syngeneic, orthotopic glioma model SMA-560 was used to evaluate the efficacy of SX-007. Cells were implanted into the striatum of VM/Dk mice. Dosing began three days after implantation and continued until the end of the study. Efficacy was established by assessing survival benefit. SX-007 dosed at 20 mg/kg p.o. once daily (q.d.) modulated TGF-beta signaling in the tumor and improved the median survival. Strikingly, approximately 25% of the treated animals were disease-free at the end of the study. Increasing the dose to 40 mg/kg q.d. or 20 mg/kg twice daily did not further improve efficacy. The data suggest that SX-007 can exert a therapeutic effect by reducing TGF-beta-mediated invasion and reversing immune suppression. SX-007 modulates the TGF-beta signaling pathway and is associated with improved survival in this glioma model. Survival benefit is due to reduced tumor invasion and reversal of TGF-beta-mediated immune suppression, allowing for rejection of the tumor. Together, these results suggest that treatment with a TGF-betaRI inhibitor may be useful in the treatment of glioblastoma.

Immune stimulatory effects of CD70 override CD70-mediated immune cell apoptosis in rodent glioma models and confer long-lasting antiglioma immunity in vivo

CD70 (CD27 ligand) promotes the expansion of primed lymphocytes by enhancing cell survival. Surprisingly, we previously observed that CD70 aberrantly expressed on human glioma cells promoted immune cell apoptosis and inhibited alloreactive lysis. Here we report that ectopic expression of CD70 in mouse glioma cells enhances apoptosis of T, B and NK cells in coculture, but nevertheless promotes glioma cell lysis by NK cells in vitro. In nude mice, CD70 expression in SMA‐560 gliomas delays the glioma growth upon subcutaneous (s.c.) or intracerebral (i.c.) inoculation, suggesting a role for CD70/CD27‐dependent NK cell activity in tumor surveillance. In syngeneic immunocompetent VM/Dk mice, CD70 allows the rejection of s.c. and i.c. implanted SMA‐560 tumors. The tumorigenicity of CD70‐expressing glioma cells is abrogated when TGF‐β signaling is blocked. Moreover, mice surviving the s.c. CD70 glioma challenge subsequently also reject wild‐type glioma cells administered i.c. Similarly, CD70‐expressing GL‐261 gliomas are rejected in syngeneic C57BL/6 mice, while glioma growth is restored in C57BL/6 CD27−/− mice, suggesting that the CD70/CD27 interaction recruits a tumor‐specific T‐cell repertoire and induces tumor‐specific memory. Altogether, these observations indicate that the net effect of aberrant CD70 expression in gliomas is immune stimulatory rather than immune paralytic and encourage its application in tumor immunotherapy.

APO010, a synthetic hexameric CD95 ligand, induces human glioma cell death in vitro and in vivo.

Death receptor targeting has emerged as one of the promising novel approaches of cancer therapy. The activation of one such prototypic death receptor, CD95 (Fas/APO-1), has remained controversial because CD95 agonistic molecules have exhibited either too strong toxicity or too little activity. The natural CD95 ligand (CD95L) is a cytokine, which needs to trimerize to mediate a cell death signal. Mega-Fas-Ligand, now referred to as APO010, is a synthetic hexameric CD95 agonist that exhibits strong antitumor activity in various tumor models. Here, we studied the effects of APO010 in human glioma models in vitro and in vivo. Compared with a cross-linked soluble CD95L or a CD95-agonistic antibody, APO010 exhibited superior activity in glioma cell lines expressing CD95 and triggered caspase-dependent cell death. APO010 reduced glioma cell viability in synergy when combined with temozolomide. The locoregional administration of APO010 induced glioma cell death in vivo and prolonged the survival of tumor-bearing mice. A further exploration of APO010 as a novel antiglioma agent is warranted.

Regeneration and Tolerance Factor: A Novel Mediator of Glioblastoma-Associated Immunosuppression

Regeneration and tolerance factor (RTF) was originally identified in placenta where it is thought to be essential for fetal allograft survival. Here we report that RTF mRNA and protein are also expressed in human glioma cells in vitro and in vivo. Suppression of RTF expression by RNA interference promotes the lysis of glioma cells by natural killer (NK) and T cells in vitro. Moreover, RTF-depleted glioma cells are less tumorigenic than control cells in nude mice in vivo. Depletion of NK cells in these animals abolished this effect. RTF is thus a novel aberrantly expressed molecule which confers immune privilege to human malignant gliomas.

Soluble CD70: A novel immunotherapeutic agent for experimental glioblastoma: Laboratory investigation

OBJECT Given the overall poor outcome with current treatment strategies in malignant gliomas, immunotherapy has been considered a promising experimental approach to glioblastoma for more than 2 decades. A cell surface molecule, CD70, may induce potent antitumor immune responses via activation of the costimulatory receptor CD27 expressed on immune effector cells. There is evidence that a soluble form of CD70 (sCD70) may exhibit biological activity, too. A soluble costimulatory ligand is attractive because it may facilitate immune activation and may achieve a superior tissue distribution. METHODS To test the antiglioma effect of sCD70, the authors genetically modified SMA-560 mouse glioma cells to secrete the extracellular domain of CD70. They assessed the immunogenicity of the transfected cells in cocultures with immune effector cells by the determination of immune cell proliferation and the release of interferon-gamma. Syngeneic VM/Dk mice were implanted orthotopically with control or sCD70-releasing glioma cells to determine a survival benefit mediated by sCD70. Depletion studies were performed to identify the cellular mediators of prolonged survival of sCD70-releasing glioma-bearing mice. RESULTS The authors found that ectopic expression of sCD70 enhanced the proliferation and interferon-gamma release of syngeneic splenocytes in vitro. More importantly, sCD70 prolonged the survival of syngeneic VM/Dk mice bearing intracranial SMA-560 gliomas. The survival rate at 60 days increased from 5 to 45%. Antibody-mediated depletion of CD8-positive T cells abrogates the survival advantage conferred by sCD70. CONCLUSIONS These data suggest that sCD70 is a potent stimulator of antiglioma immune responses that depend critically on CD8-positive T cells. Soluble CD70 could be a powerful adjuvant for future immunotherapy trials for glioblastoma.

MIP-1α Antagonizes the Effect of a GM-CSF-Enhanced Subcutaneous Vaccine in a Mouse Glioma Model

Subcutaneous vaccination using granulocyte–macrophage colony-stimulating factor (GM-CSF)-transduced glioma cells substantially prolongs survival in the mouse GL261 glioma model. To potentiate the efficacy of GM-CSF-based vaccination, syngeneic C57BL/6 mice bearing pre-implanted intracerebral GL261 gliomas were vaccinated twice subcutaneously with various combinations of glioma cells retrovirally engineered to release GM-CSF, interleukin (IL)-4 or macrophage inflammatory protein (MIP)-1α. More than 80% of the animals vaccinated with GM-CSF-secreting or GM-CSF- and IL-4-secreting cells were long-term survivors (>120 days). Their survival was significantly prolonged compared with animals vaccinated with wild-type cells, which died after a median survival time of 30 days. The combination of IL-4 with GM-CSF did not provide a survival advantage over GM-CSF alone, regardless of whether the animals carried a small or large intracranial tumor load. Further, when the animals were vaccinated with a mixture of GM-CSF-, IL-4- and MIP-1α-secreting cells, the median survival was 37 days, and only 22% of the animals in this group were long-term survivors, similar to the vaccination effect of non-modified glioma cells. Thus, unexpectedly, the co-expression of MIP-1α, which was meant to attract T cells for stimulation by GM-CSF- and IL-4-stimulated dendritic cells, nullified the induction of an immune response against the GL261 glioma by a GM-CSF- and IL-4-expressing subcutaneous vaccine.