Glioblastoma multiforme is one of the most common human brain tumors. The tumor is generally highly infiltrative, making it extremely difficult to treat by surgical resection or radiotherapy. This feature contributes to recurrence and a very poor prognosis. Few anticancer drugs have been shown to alter rapid tumor growth and none are ultimately effective. Oncolytic vectors have been employed as a treatment alternative based on the ability to tailor virus replication to tumor cells. The human neurotropic herpes simplex virus (HSV) is especially attractive for development of oncolytic vectors (oHSV) because this virus is highly infectious, replicates rapidly and can be readily modified to achieve vector attenuation in normal brain tissue. Tumor specificity can be achieved by deleting viral genes that are only required for virus replication in normal cells and permit mutant virus replication selectively in tumor cells. The anti-tumor activity of oHSV can be enhanced by arming the vector with genes that either activate chemotherapeutic drugs within the tumor tissue or promote anti-tumor immunity. In this review, we describe current designs of oHSV and the experience thus far with their potential utility for glioblastoma therapy. In addition, we discuss the impediments to vector effectiveness and describe our view of future developments in vector improvement.
gene therapy; glioblastoma; HSV oncolytic vector
The role of the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO1), in tumor escape and metastasis formation was analyzed using two pairs of Ido1+ and Ido1− murine breast cancer cell lines. Ido1 expression in 4T1 cells was knocked down by shRNA, and Ido1 expression in NT-5 cells was upregulated by stable transfection. Growth of Ido1− tumors and spontaneous metastasis formation were inhibited in immunocompetent mice. A higher level of cytotoxic T lymphocytes was generated by spleen cells from mice bearing Ido1− tumors than Ido1+ tumors. Tumor and metastatic growth was enhanced in immunodeficient mice, confirming an intensified immune response in the absence of Ido1 expression. However, Ido1+ tumors grow faster than Ido1− tumors in immunodeficient SCID/beige mice (lacking T, B, and NK cells) suggesting that some Ido1-controlled nonimmunological mechanisms may be involved in tumor cell growth regulation. In vitro experiments demonstrated that downregulation of Ido1 in tumor cells was associated with decreased cell proliferation, increased apoptosis, and changed expression of cell cycle regulatory genes, whereas upregulation of Ido1 in the cells had the opposite effects. Taken together, our findings indicate that Ido1 expression could exert immunological and nonimmunological effects in murine breast tumor cells.
Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of γ34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of α47. Infection with oHSV G47△ (ICP6-, γ34.5-, α47-) not only killed GBMSCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Δ significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis.
Immune escape is a fundamental trait of cancer in which the Th1-type cytokine interferon- γ (IFN-γ) seems to play a key role. Among other tumoricidal biochemical pathways, IFN-γ induces the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) in a variety of cells including macrophages, dendritic cells (DCs) and tumor cells. IDO activity has been shown to reflect the extent and the course in a plethora of malignancies including prostate, colorectal, pancreatic, cervical, endometrial, gastric, lung, bladder, ovarian, esophageal and renal cell carcinomas, glioblastomas, mesotheliomas, and melanomas. Furthermore IDO activity during malignant tumor diseases seems to be part of the tumoricidal immune defense strategy, which in the long run is detrimental to the host, when tryptophan deprivation and production of pro-apoptotic tryptophan catabolites counteract T-cell responsiveness.
IDO; Tryptophan; Malignant tumor disease
This article covers what is currently known about the role of the enzyme indoleamine 2,3-dioxygenase (IDO) in cancer-related immunosuppression and the clinical research on IDO inhibitors. A PUBMED search was performed using the terms IDO, indoleamine 2,3-dioxygenase, 1-MT. IDO is an inducible enzyme that catalyzes the rate-limiting first step in tryptophan catabolism. This enzyme is overexpressed in response to IFNγ in a variety of different malignancies. IDO causes immunosuppression through breakdown of tryptophan in the tumor microenvironment and tumor-draining lymph nodes. The depletion of tryptophan and toxic catabolites renders effector T cells inactive and dendritic cells immunosuppressive. Preclinical data suggest that IDO inhibition can delay tumor growth, enhance dendritic cell vaccines, and synergize with chemotherapy through immune-mediated mechanisms. The lead IDO inhibitor, D-1-methyl-tryptophan (D-1-MT), was selected for phase I trials and seems to have immune modulating activity. Subsequently, another isoform of IDO, IDO2, was discovered and found to be the target of D-1-MT. Multiple single-nucleotide polymorphisms in IDO2 affecting its catalytic activity may serve as a pharmacogenetic predictive biomarker for D-1-MT. The IDO pathway is an important mechanism of tumor-related immunosuppression and blocking it could improve cancer immunotherapy outcomes. Clinical development of D-1-MT and other IDO inhibitors as systemic immunomodulators to be combined with other immune modulators, vaccines, and chemotherapy are ongoing.
immunotherapy; indoleamine-pyrrole 2,3-dioxygenase; 1-methyl-tryptophan
Oncolytic virotherapy has shown substantial promises as an alternative therapeutic modality for solid tumors in both preclinical studies and clinical trials. The main therapeutic activity of virotherapy derives from the direct lytic effect associated with virus replication and the induction of host immune responses to the infected tumor cells. Here we show that some human and murine tumor cell lines are highly resistant to the lytic effect of a type II herpes simplex virus-derived oncolytic virus, FusOn-H2, which was constructed by deleting the N-terminal region of the ICP10 gene. However, these tumor cells still respond exceptionally well to FusOn-H2 virotherapy in vivo. Histological examination of the treated tumors revealed that, in contrast to tumors supporting FusOn-H2 replication, implants of these highly resistant lines showed massive infiltration of neutrophils after virotherapy. Further analysis showed that there is a correlation between an intrinsically strong interferon response activity and the recruitment of neutrophils in these tumors. These results suggest that an innate immune response mainly represented by neutrophils may be part of the virotherapy-mediated antitumor mechanism in these tumors.
oncolytic virus; cancer virotherapy; herpes simplex virus; neutrophil infiltration; innate immunity
The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. However, the inhibitory effects of IDO on the CD8+ tumour-infiltrating lymphocytes (CD8+ TILs) in esophageal squamous cell carcinoma (ESCC) have not been clarified yet. Here, we found that the level of IDO expression in ESCC tumor specimens correlated with a reduction in the number of CD8+ TILs. Patients with high IDO expression and a low number of CD8+ TILs had significantly impaired overall survival time. IDO expression and functional enzyme activity in ESCC cell lines could be induced by IFNγ. When exposed to the milieu generated by IDO-expressing Eca109 cells, the CD8+ TILs were suppressed in proliferation, and their cytolytic functions against target tumor cells were lost. These results suggested that impairing CD8+ TIL functions by IDO expressed in ESCC possibly contributed to the finding that patients with higher IDO expression have more aggressive disease progression and shorter overall survival time.
The prognosis of patients diagnosed with malignant gliomas including glioblastoma multiforme (GBM) is poor and there is an urgent need to develop and translate novel therapies into the clinic. Neural stem cells display remarkable tropism toward GBMs and thus may provide a platform to deliver oncolytic agents to improve survival. First we provide a brief review of clinical trials that have used intra-tumoral herpes simplex virus thymidine kinase (HSV/tk) gene therapy to treat brain tumors. Then, we review recent evidence that neural stem cells can be used to deliver HSV/tk to GBMs in animal models. While previous clinical trials used viruses or non-migratory vector-producing cells to deliver HSV/tk, the latter approaches were not effective in humans, primarily because of satellite tumor cells that escaped surgical resection and survived due to low efficiency delivery of HSV/tk. To enhance delivery of HSV/tk to kill gliomas cells, recent animal studies have focused on the ability of neural stem cells, transduced with HSV/tk, to migrate efficiently and selectively to regions occupied by GBM cells. This approach holds the promise of targeting GBM cells that have infiltrated the brain well beyond the original site of the tumor epicenter.
Glioblastoma multiforme; tropism; neural stem cells; bystander effect; Herpes Simplex Virus/thymidine kinase
Glioblastoma multiforme (GBM) is an aggressive adult brain tumor with a poor prognosis. One hallmark of GBM is the accumulation of immunosuppressive and tumor-promoting CD4+FoxP3+GITR+ regulatory T cells (Tregs). Here, we investigated the role of indoleamine 2,3 dioxygenase (IDO) in brain tumors and the impact on Treg recruitment.
To determine the clinical relevance of IDO expression in brain tumors, we first correlated patient survival to the level of IDO expression from resected glioma specimens. We also used novel orthotopic and transgenic models of glioma to study how IDO affects Tregs. The impact of tumor-derived and peripheral IDO expression on Treg recruitment, GITR expression and long-term survival was determined.
Downregulated IDO expression in glioma predicted a significantly better prognosis in patients. Co-incidently, both IDO -competent and -deficient mice showed a survival advantage bearing IDO-deficient brain tumors, when compared to IDO-competent brain tumors. Moreover, IDO-deficiency was associated with a significant decrease in brain-resident Tregs, both in orthotopic and transgenic mouse glioma models. IDO-deficiency was also associated with lower GITR expression levels on Tregs. Interestingly, the long-term survival advantage conferred by IDO-deficiency was lost in T cell-deficient mice.
These clinical and pre-clinical data confirm that IDO expression increases the recruitment of immunosuppressive Tregs which leads to tumor outgrowth. In contrast, IDO deficiency decreases Treg recruitment and enhances T cell-mediated tumor rejection. Thus, the data suggest a critical role for IDO-mediated immunosuppression in glioma and supports the continued investigation of IDO-Treg interactions in the context of brain tumors.
immunosuppression; tryptophan; glioblastoma; metabolism; immunotherapy
We exploited the differential activation of hypoxia-inducible factor (HIF)-dependent gene expression in tumors versus normal tissue for the design of a targeted oncolytic Herpes simplex virus type-1 (HSV-1). A gene that is essential for viral replication, ICP4, was placed under the regulation of a HIF-responsive promoter and then introduced into the thymidine kinase locus (UL23) of HSV d120 which contains partial deletions in the two endogenous ICP4 genes. Recombinant HIF-HSV were isolated and their derivation from d120 was verified by expression of a truncated, nonfunctional form of ICP4 protein. Disruption of the UL23 locus was confirmed by loss of thymidine kinase expression and resistance to acyclovir. Unexpectedly, HIF-HSV expressed ICP4 and induced tumor cell lysis at similar levels under normoxia and hypoxia. The lack of HIF-dependent ICP4 transgene expression by HIF-HSV was due to two factors that have not previously been reported- reversion of the ICP4 gene region to its wild-type configuration and increased HIF-transcriptional activity under normoxia when cells were infected with any strain of HSV-1. The findings that an oncolytic HSV-1 is genetically unstable and can activate a tumor-related promoter in a non-specific manner have important implications for any proposed use of this virus in cancer therapy.
hypoxia; hypoxia-inducible factor (HIF); herpes simplex virus; HSV; oncolytic; ICP4
Hypoxia plays a critical role in the tumor microenvironment of high-grade gliomas by promoting the glioma stem cell (GSC)-like phenotype, which displays resistance to standard therapies. We tested three glioblastoma multiforme xenograft lines (xenolines) against γ134.5-deleted recombinant oncolytic herpes simplex virus (oHSV) C101 under 1% (hypoxia) and 20.8% (normoxia) oxygen tension for effects on oHSV infectivity, replication, and cytotoxicity in all tumor cells and CD133+ GSCs. Expression levels of CD133, a putative GSC marker, and CD111 (nectin-1), an adhesion molecule that is the most efficient method for HSV entry, increased significantly under hypoxia in all three xenolines. Despite increased CD111 expression under hypoxic conditions, oHSV infectivity, cytotoxicity and viral recovery were not improved or were diminished in all three xenolines under hypoxia. In contrast, wild-type HSV-1 equally infected xenoline cells in normoxia and hypoxia, suggesting that the 34.5 mutation plays a role in the decreased C101 infectivity in hypoxia. Importantly, CD133+ cells were not more resistant to oHSV than CD133- tumor cells regardless of oxygen tension. Furthermore, CD133 expression decreased as viral dose increased in two of the xenolines suggesting that up-regulation of CD133 in hypoxia was not the cause of reduced viral efficacy. Our findings that oHSV infectivity and cytotoxicity were diminished under hypoxia in several GBM xenolines likely have important implications for clinical applications of oHSV therapies, especially considering the vital role of hypoxia in the microenvironment of GBM tumors.
The ICP34.5 protein of herpes simplex virus (HSV) is involved in many aspects of viral pathogenesis; promoting neurovirulence, inhibiting interferon-induced shutoff of protein synthesis, interacting with PCNA and TBK1, inhibiting dendritic cell (DC) maturation, and binding to Beclin 1 to interfere with autophagy. Because of its key role in neuropathogenicity, the γ34.5 gene is deleted in all oncolytic HSVs (oHSVs) currently in clinical trial for treating malignant gliomas. Unfortunately, deletion of γ34.5 attenuates virus replication in cancer cells, especially human glioblastoma stem cells (GSCs). To develop new oHSVs for use in the brain and that replicate in GSCs, we explored the effect of deleting the γ34.5 Beclin 1 binding domain (BBD). To ensure cancer selectivity and safety, we inactivated the ICP6 gene (UL39, large subunit of ribonucleotide reductase), constructing ICP6 mutants with different γ34.5 genotypes: Δ68HR-6, intact γ34.5; Δ68H-6, γ34.5 BBD deleted; and 1716-6, γ34.5 deleted. Multimutated Δ68H-6 exhibited minimal neuropathogenicity in HSV-1-susceptible mice, as opposed to Δ68H and Δ68HR-6. It replicated well in human glioma cell lines and GSCs, effectively killing cells in vitro and prolonging survival of mice bearing orthotopic brain tumors. In contrast, 1716 and 1716-6 barely replicated in GSCs. Infection of glioma cells with Δ68H-6 and 1716-6 induced autophagy and increased phosphorylation of eIF2α, while inhibition of autophagy, by Beclin 1 short hairpin RNA (shRNA) knockdown or pharmacological inhibition, had no effect on virus replication or phosphorylated eIF2α (p-eIF2α) levels. Thus, Δ68H-6 represents a new oHSV vector that is safe and effective against a variety of brain tumor models.
Immune escape, the ability of tumor cells to avoid tumor-specific immune responses, occurs during the development and progression of several types of human malignancies, including colorectal cancer (CRC). Indoleamine 2,3-dioxygenase (IDO), the tryptophan catabolic enzyme, plays a significant role in regulating the immune response and provides tumor cells with a potent tool to evade the immune system. In the present study, we examined the effects of (-)-epigallocatechin gallate (EGCG), the major catechin in green tea, on the inhibition of IDO expression induced by interferon (IFN)-γ in human CRC cells. We found that IFN-γ increased the expression levels of IDO protein and mRNA in HT29 and SW837 CRC cell lines. Treatment of SW837 cells with EGCG significantly decreased IFN-γ-induced expression of IDO protein and mRNA in a dose-dependent manner. Enzymatic activity of IDO, determined by the concentration of L-kynurenine in the culture medium, was also significantly inhibited by EGCG treatment. Phosphorylation of signal transducer and activator of transcription 1 (STAT1) induced by IFN-γ was also significantly inhibited by EGCG. Reporter assays indicated that EGCG inhibited the transcriptional activities of IDO promoters, IFN-stimulated response element and IFN-γ activation sequence, activated by STAT1 phosphorylation. These findings suggest that EGCG may exert antitumor effects on CRC, at least in part, by inhibiting the expression and function of IDO through the suppression of STAT1 activation. EGCG may, thus, serve as a potential agent for antitumor immunotherapy and be useful in the chemoprevention and/or treatment of CRC.
indoleamine 2; 3-dioxygenase; (-)-epigallocatechin gallate; colorectal cancer cells; interferon-γ; signal transducer and activator of transcription 1
Tumor virotherapy has been and continues to be used in clinical trials. One barrier to effective viral oncolysis, consisting of the interferon (IFN) response induced by viral infection, is inhibited by valproic acid (VPA) and other histone deacetylase inhibitors (HDACi). Innate immune cell recruitment and activation have been shown to be deleterious to the efficacy of oncolytic herpes simplex virus (oHSV) infection, and in this report we demonstrate that VPA limits this deleterious response. VPA, administered prior to oHSV inoculation in an orthotopic glioblastoma mouse model, resulted in a decline in NK and macrophage recruitment into tumor-bearing brains at 6 and 24 h post-oHSV infection. Interestingly, there was a robust rebound of recruitment of these cells at 72 h post-oHSV infection. The observed initial decline in immune cell recruitment was accompanied by a reduction in their activation status. VPA was also found to have a profound immunosuppressive effect on human NK cells in vitro. NK cytotoxicity was abrogated following exposure to VPA, consistent with downmodulation of cytotoxic gene expression of granzyme B and perforin at the mRNA and protein levels. In addition, suppression of gamma IFN (IFN-γ) production by VPA was associated with decreased STAT5 phosphorylation and dampened T-BET expression. Despite VPA-mediated immune suppression, mice were not at significantly increased risk for HSV encephalitis. These findings indicate that one of the avenues by which VPA enhances oHSV efficacy is through initial suppression of immune cell recruitment and inhibition of inflammatory cell pathways within NK cells.
The inflammatory enzyme indoleamine 2, 3-dioxygenase (IDO) participates in immune tolerance and promotes immune escape of IDO+ tumors. A recent hypothesis suggested that IDO may contribute to the differentiation of new T regulatory cells (Tregs) from naive CD4+ T cells. In this study we investigated the role of IDO in induction of immunosuppression in breast cancer by increasing the apoptosis of T cells and the proportion of Tregs.
An IDO expression plasmid was constructed and Chinese hamster ovary (CHO) cells were stably transfected with human IDO. Purified CD3+ T cells were isolated from the peripheral blood monouclear cells of breast cancer patients. After co-culturing IDO expressing or untransfected (control) CHO cells with T cells, T cells apoptosis were determined by flow cytometry analysis and annexin-V and PI staining. The proportion of the regulatory T cell (Tregs [CD4 + CD25 + CD127-]) subset was measured by flow cytometry analysis. T cells total RNA and cellular protein samples were isolated for detecting Foxp3 gene and protein expression.
IDO transgenic CHO cells yielded high levels of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in 79.07 ± 8.13% of CD3+T cells after co-cultured with IDO+ CHO cells for 3 days and the proportion of CD4 + CD25 + CD127- T cells increased from 3.43 ± 1.07% to 8.98 ± 1.88% (P < 0.05) as well. The specific inhibitor of IDO,1-MT efficiently reversed enhancement of T cells apoptosis and amplification of Tregs in vitro. Increased expression of Foxp3, a key molecular marker of Tregs, was confirmed by RT-PCR, real-time RT-PCR and Western blot analysis at the same time.
These results suggest that IDO helps to create a tolerogenic milieu in breast tumors by directly inducing T cell apoptosis and enhancing Treg-mediated immunosuppression.
Indoleamine-Pyrrole 2; 3-Dioxygenase; breast neoplasms; immune tolerance; CHO Cells; regulatory T-Lymphocytes
The role of the immune response to oncolytic Herpes Simplex viral (oHSV) therapy for glioblastoma is controversial. Within hours of oHSV infection of human or syngeneic glioblastoma in mice, activated natural killer (NK) cells are recruited to the site of infection. This response significantly diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on NK cell natural cytotoxicity receptors (NCR) NKp30 and NKp46, whose ligands were up-regulated in oHSV-infected glioblastoma cells. HSV titers and oHSV efficacy were increased in Ncr1−/− mice and in a Ncr1−/− NK cell adoptive transfer model of glioma, respectively. These in vitro and in vivo (mouse) results demonstrate that glioblastoma virotherapy is partly limited by an antiviral NK cell response involving specific NCRs, uncovering novel potential targets to enhance cancer virotherapy.
Herpes simplex virus; gene therapy; oncolytic virus; brain tumor; microglia; macrophages
Inflammation stimulates immunity but can create immune privilege in some settings. Here, we show that cutaneous Leishmania major infection stimulated expression of the immune regulatory enzyme indoleamine 2,3 dioxygenase (IDO) in local lymph nodes. Induced IDO attenuated the T cell stimulatory functions of dendritic cells and suppressed local T cell responses to exogenous and nominal parasite antigens. IDO ablation reduced local inflammation and parasite burdens, as did pharmacologic inhibition of IDO in mice with established infections. IDO ablation also enhanced local expression of proinflammatory cytokines and induced some CD4+ T cells to express interleukin (IL) 17. These findings showed that IDO induced by L. major infection attenuated innate and adaptive immune responses. Thus, IDO acts as a molecular switch regulating host responses, and IDO inhibitor drugs are a potential new approach to enhance host immunity to established leishmania infections.
Regulatory T cells, Tregs, are a subset of lymphocytes that have immunosuppressive attributes. They are elevated in blood of glioblastoma patients and within this tumor's tissue itself. Indoleamine 2,3-dioxygenase, IDO, converts tryptophan to kynurenine. IDO activity enhances Treg formation by pathways that are unknown. Experimentally, inhibition of IDO decreases Treg function and number in rodents. The common anti-viral agent acyclovir inhibits IDO. Acyclovir may thereby decrease Treg function in glioblastoma. If it can be confirmed that Treg counts are elevated in glioblastoma patients' tumor tissue, and if we can document acyclovir's lowering of tissue Treg counts by a small trial of acyclovir in pre-operative glioblastoma patients, a trial of acyclovir effect on survival should be done given the current poor prognosis of glioblastoma and the well-established safety and low side effect burden of acyclovir.
Indoleamine 2,3-dioxygenase (IDO), a catabolizing enzyme of tryptophan, is supposed to play a role in tumor immune escape. Its expression in solid tumors has not yet been well elucidated: IDO can be expressed by the tumor cells themselves, or by ill-defined infiltrating cells, possibly depending on tumor type. We have investigated IDO expression in 25 cases of non-small cell lung cancer (NSCLC). Using histochemistry and immunohistochemistry, we found that IDO was expressed not by tumor cells, but by normal cells infiltrating the peritumoral stroma. These cells were neither macrophages nor dendritic cells, and were identified as eosinophil granulocytes. The amount of IDO-positive eosinophils varied in different cases, ranging from a few cells to more than 50 per field at x200 magnification. IDO protein in NSCLC was enzymatically active. Therefore, at least in NSCLC cases displaying a large amount of these cells in the inflammatory infiltrate, IDO-positive eosinophils could exert an effective immunosuppressive action. On analyzing the 17 patients with adequate follow-up, a significant relationship was found between the amount of IDO-positive infiltrate and overall survival. This finding suggests that the degree of IDO-positive infiltrate could be a prognostic marker in NSCLC.
Non-small cell lung cancer; indoleamine 2,3-dioxygenase; eosinophil granulocytes; immune escape; prognostic marker
One of the putative mechanisms of tumor immune escape is based on the hypothesis that carcinomas actively create an immunosuppressed state via the expression of indoleamine 2,3-dioxygenase (IDO), both in the cancer cells and in the immune cells among the tumor-draining lymph nodes (TDLN). In an attempt to verify this hypothesis, the patterns of expression of IDO in the cancer cells and the immune cells among colon cancers were examined.
Seventy-one cases of pathologically-confirmed colon cancer tissues matched with adjacent non-cancerous tissues, lymph node metastases, and TDLN without metastases were collected at the Sun Yat-sen Cancer Center between January 2000 and December 2000. The expression of IDO and Bin1, an IDO regulator, was determined with an immunohistochemical assay. The association between IDO or Bin1 expression and TNM stages and the 5-year survival rate in colon cancer patients was analyzed.
IDO and Bin1 were detected in the cytoplasm of cancer cells and normal epithelium. In primary colon cancer, the strong expression of IDO existed in 9/71 cases (12.7%), while the strong expression of Bin1 existed in 33/71 cases (46.5%). However, similar staining of IDO and Bin1 existed in the adjacent non-cancerous tissues. Among the 41 cases with primary colon tumor and lymph node metastases, decreased expression of IDO was documented in the lymph node metastases. Furthermore, among the TDLN without metastases, a higher density of IDO+cells was documented in 21/60 cases (35%). Both univariate and multivariate analyses revealed that the density of IDO+cells in TDLN was an independent prognostic factor. The patients with a higher density of IDO+cells in TDLN had a lower 5-year survival rate (37.5%) than the cells with a lower density (73.1%).
This study demonstrated paradoxical patterns of expression of IDO in colon cancer. The high density IDO+cells existed in TDLN and IDO was down-regulated in lymph nodes with metastases, implying that IDO in tumor and immune cells functions differently.
Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host innate and adaptive antiviral immunity against hepatitis B virus (HBV) infection in vivo. In an effort to elucidate the antiviral mechanism of these cytokines, 37 IFN-stimulated genes (ISGs), which are highly inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon overexpression in human hepatoma cells. One ISG candidate, indoleamine 2,3-dioxygenase (IDO), an IFN-γ-induced enzyme catalyzing tryptophan degradation, efficiently reduced the level of intracellular HBV DNA without altering the steady-state level of viral RNA. Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV replication, and tryptophan supplementation in culture completely restored HBV replication in IDO-expressing cells, indicating that the antiviral effect elicited by IDO is mediated by tryptophan deprivation. Interestingly, IDO-mediated tryptophan deprivation preferentially inhibited viral protein translation and genome replication but did not significantly alter global cellular protein synthesis. Finally, tryptophan supplementation was able to completely restore HBV replication in IFN-γ- but not IFN-α-treated cells, which strongly argues that IDO is the primary mediator of IFN-γ-elicited antiviral response against HBV in human hepatocyte-derived cells.
Indoleamine 2,3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. In cultured cells, the induction of IDO leads to depletion of tryptophan and tryptophan starvation. Recent studies suggest that modulation of tryptophan concentration via IDO plays a fundamental role in innate immune responses. Induction of IDO by interferon-γ in macrophages and dendritic cells results in tryptophan depletion and suppresses the immune-mediated activation of fibroblasts and T, B, and natural killer cells. To assess the role of IDO in collagen-induced arthritis (CIA), a model of rheumatoid arthritis characterized by a primarily Th1-like immune response, activity of IDO was inhibited by 1-methyl-tryptophan (1-MT) in vivo. The results showed significantly increased incidence and severity of CIA in mice treated with 1-MT. Activity of IDO, as determined by measuring the levels of kynurenine/tryptophan ratio in the sera, was increased in the acute phase of arthritis and was higher in collagen-immunized mice that did not develop arthritis. Treatment with 1-MT resulted in an enhanced cellular and humoral immune response and a more dominant polarization to Th1 in mice with arthritis compared with vehicle-treated arthritic mice. The results demonstrated that development of CIA was associated with increased IDO activity and enhanced tryptophan catabolism in mice. Blocking IDO with 1-MT aggravated the severity of arthritis and enhanced the immune responses. These findings suggest that IDO may play an important and novel role in the negative feedback of CIA and possibly in the pathogenesis of rheumatoid arthritis.
Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.
Although both the alkylating agent temozolomide (TMZ) and oncolytic viruses hold promise for treating glioblastoma, which remains uniformly lethal, the effectiveness of combining the two treatments and the mechanism of their interaction on cancer stem cells are unknown.
We investigated the efficacy of combining TMZ and the oncolytic herpes simplex virus (oHSV) G47Δ in killing glioblastoma stem cells (GSCs), using Chou–Talalay combination index analysis, immunocytochemistry and fluorescence microscopy, and neutral comet assay. The role of treatment-induced DNA double-strand breaks, activation of DNA damage responses, and virus replication in the cytotoxic interaction between G47Δ and TMZ was examined with a panel of pharmacological inhibitors and short-hairpin RNA (shRNA)–mediated knockdown of DNA repair pathways. Comparisons of cell survival and virus replication were performed using a two-sided t test (unpaired). The survival of athymic mice (n = 6–8 mice per group) bearing GSC-derived glioblastoma tumors treated with the combination of G47Δ and TMZ was analyzed by the Kaplan–Meier method and evaluated with a two-sided log-rank test.
The combination of G47Δ and TMZ acted synergistically in killing GSCs but not neurons, with associated robust induction of DNA damage. Pharmacological and shRNA-mediated knockdown studies suggested that activated ataxia telangiectasia mutated (ATM) is a crucial mediator of synergy. Activated ATM relocalized to HSV DNA replication compartments where it likely enhanced oHSV replication and could not participate in repairing TMZ-induced DNA damage. Sensitivity to TMZ and synergy with G47Δ decreased with O6-methylguanine-DNA-methyltransferase (MGMT) expression and MSH6 knockdown. Combined G47Δ and TMZ treatment extended survival of mice bearing GSC-derived intracranial tumors, achieving long-term remission in four of eight mice (median survival = 228 days; G47Δ alone vs G47Δ + TMZ, hazard ratio of survival = 7.1, 95% confidence interval = 1.9 to 26.1, P = .003) at TMZ doses attainable in patients.
The combination of G47Δ and TMZ acts synergistically in killing GSCs through oHSV-mediated manipulation of DNA damage responses. This strategy is highly efficacious in representative preclinical models and warrants clinical translation.
1-methyl-D-tryptophan (1-D-MT) is currently being used in clinical trials in patients with relapsed or refractory solid tumors with the aim of inhibiting indoleamine-2,3-dioxygenase (IDO)-mediated tumor immune escape. IDO is expressed in tumors and tumor-draining lymph nodes and degrades tryptophan (trp) to create an immunsuppressive micromilieu both by depleting trp and by accumulating immunosuppressive metabolites of the kynurenine (kyn) pathway. Here we show that proliferation of alloreactive T-cells cocultured with IDO1-positive human cancer cells paradoxically was inhibited by 1-D-MT. Surprisingly incubation with 1-D-MT increased kyn production of human cancer cells. Cell-free assays revealed that 1-D-MT did not alter IDO1 enzymatic activity. Instead, 1-D-MT induced IDO1 mRNA and protein expression through pathways involving p38 MAPK and JNK signalling. Treatment of cancer patients with 1-D-MT has transcriptional effects that may promote rather than suppress anti-tumor immune escape by increasing IDO1 in the cancer cells. These off-target effects should be carefully analyzed in the ongoing clinical trials with 1-D-MT.