44 research outputs found
A genetically modified adenoviral vector with a phage display-derived peptide incorporated into fiber fibritin chimera prolongs survival in experimental glioma
The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as “GliomaFF.” We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy
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Ribonucleotide reductase regulatory subunit M2 drives glioblastoma TMZ resistance through modulation of dNTP production
During therapy, adaptations driven by cellular plasticity are partly responsible for driving the inevitable recurrence of glioblastoma (GBM). To investigate plasticity-induced adaptation during standard-of-care chemotherapy temozolomide (TMZ), we performed in vivo single-cell RNA sequencing in patient-derived xenograft (PDX) tumors of GBM before, during, and after therapy. Comparing single-cell transcriptomic patterns identified distinct cellular populations present during TMZ therapy. Of interest was the increased expression of ribonucleotide reductase regulatory subunit M2 (RRM2), which we found to regulate dGTP and dCTP production vital for DNA damage response during TMZ therapy. Furthermore, multidimensional modeling of spatially resolved transcriptomic and metabolomic analysis in patients’ tissues revealed strong correlations between RRM2 and dGTP. This supports our data that RRM2 regulates the demand for specific dNTPs during therapy. In addition, treatment with the RRM2 inhibitor 3-AP (Triapine) enhances the efficacy of TMZ therapy in PDX models. We present a previously unidentified understanding of chemoresistance through critical RRM2-mediated nucleotide production
CCL2 produced by the glioma microenvironment is essential for the recruitment of regulatory T cells and myeloid-derived suppressor cells
In many aggressive cancers, such as glioblastoma multiforme (GBM), progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Treg and MDSC are recruited in various tumors is not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSC in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2 deficient mice failed to maximally accrue Treg and monocytic MDSC. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSC were defective in glioma accumulation. Further, administration of a small molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of GBM, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Lastly, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in GBM patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression
Polyamines Drive Myeloid Cell Survival by Buffering Intracellular pH to Promote Immunosuppression in Glioblastoma
Glioblastoma is characterized by the robust infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs). It is not fully understood how TAMCs survive in the acidic tumor microenvironment to cause immunosuppression in glioblastoma. Metabolic and RNA-seq analysis of TAMCs revealed that the arginine-ornithine-polyamine axis is up-regulated in glioblastoma TAMCs but not in tumor-infiltrating CD8+ T cells. Active de novo synthesis of highly basic polyamines within TAMCs efficiently buffered low intracellular pH to support the survival of these immunosuppressive cells in the harsh acidic environment of solid tumors. Administration of difluoromethylornithine (DFMO), a clinically approved inhibitor of polyamine generation, enhanced animal survival in immunocompetent mice by causing a tumor-specific reduction of polyamines and decreased intracellular pH in TAMCs. DFMO combination with immunotherapy or radiotherapy further enhanced animal survival. These findings indicate that polyamines are used by glioblastoma TAMCs to maintain normal intracellular pH and cell survival and thus promote immunosuppression during tumor evolution
Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation
The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.publishedVersionPeer reviewe
The CXCL16-CXCR6 axis in glioblastoma modulates T-cell activity in a spatiotemporal context
IntroductionGlioblastoma multiforme (GBM) pathobiology is characterized by its significant induction of immunosuppression within the tumor microenvironment, predominantly mediated by immunosuppressive tumor-associated myeloid cells (TAMCs). Myeloid cells play a pivotal role in shaping the GBM microenvironment and influencing immune responses, with direct interactions with effector immune cells critically impacting these processes.MethodsOur study investigates the role of the CXCR6/CXCL16 axis in T-cell myeloid interactions within GBM tissues. We examined the surface expression of CXCL16, revealing its limitation to TAMCs, while microglia release CXCL16 as a cytokine. The study explores how these distinct expression patterns affect T-cell engagement, focusing on the consequences for T-cell function within the tumor environment. Additionally, we assessed the significance of CXCR6 expression in T-cell activation and the initial migration to tumor tissues.ResultsOur data demonstrates that CXCL16 surface expression on TAMCs results in predominant T-cell engagement with these cells, leading to impaired T-cell function within the tumor environment. Conversely, our findings highlight the essential role of CXCR6 expression in facilitating T-cell activation and initial migration to tumor tissues. The CXCL16-CXCR6 axis exhibits dualistic characteristics, facilitating the early stages of the T-cell immune response and promoting T-cell infiltration into tumors. However, once inside the tumor, this axis contributes to immunosuppression.DiscussionThe dual nature of the CXCL16-CXCR6 axis underscores its potential as a therapeutic target in GBM. However, our results emphasize the importance of carefully considering the timing and context of intervention. While targeting this axis holds promise in combating GBM, the complex interplay between TAMCs, microglia, and T cells suggests that intervention strategies need to be tailored to optimize the balance between promoting antitumor immunity and preventing immunosuppression within the dynamic tumor microenvironment
The genomic substrate for adaptive radiation in African cichlid fish
Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification
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T-Cell Mediated Mechanisms of Autoimmunity, Anti-Tumor Immunity and Tumorigenesis
The difficulty in identifying a tumor-specific antigen (TSA) in a majority of tumors poses a clinical problem in treating cancer with immunotherapy. To date, the majority of tumor antigens identified are not tumor-specific, but are rather largely “self” in nature, and the destruction of these tissues appear to be similar to autoimmune damage of normal tissues. Indeed, clinical studies of successful immune checkpoint therapies like such as anti-CTLA4 and anti-PD1/PD-L1 antibody treatment revealed a strong association of autoimmune side effects with therapeutic benefit. In these cases the anti-tumor effect seems to be mediated by, in part, an autoimmune response. The potential interrelatedness of autoimmunity and anti-tumor immunity indicates that the autoimmune process can be used as a potential tool for the destruction of tumor tissue. Further evidence is derived from genetic polymorphisms that alter CTLA4 expression in humans. CTLA4 polymorphisms that cause a reduction in its expression are associated with both a significant increase in autoimmune risk and a decrease in the risk of certain cancers. However, quite perplexingly, the same CTLA4 polymorphisms also are associated with increased risk of gastrointestinal carcinogenesis. These previous studies suggest that regulators of autoimmunity have both pro- and anti-tumor capability depending on the context in which it is studied. To address these questions, we examined the mechanisms of the critical immune checkpoint regulator CTLA4 in the context of autoimmunity, anti-tumor immunity, and tumorigenesis. We first characterized the behavior of autoimmune cells directly in the target tissue utilizing intravital microscopy. By using the anterior chamber of the eye as a “window” to the live tissues, we could visualize the behavior of TCR restricted (BDC2.5) autoimmune T-effector (Teff) and T-regulatory (Treg) cells in protection and rejection of islet grafts. Teff and Treg directly contact one another, and this contact is essential in protecting islet grafts from autoimmune destruction. In a separate model, we demonstrate islet grafts that are directly adjacent to target islets proliferate rapidly as a result of CD8+ mediated destruction of the target. Both of these observations are critical to our understanding of autoimmunity. To study the potential role of autoimmune effectors in anti-tumor immunity, we transplanted tumor cells into mice bearing the same autoimmune targets as the endogenous pancreas. After the injection of autoimmune effectors, both the endogenous pancreas and tumor were completely destroyed. The inclusion of a suboptimal amount of Treg restored protection to the tumor but not the endogenous pancreas. Anti-CTLA4 could overcome this protective effect, suggesting that Anti-CTLA4 antibody therapy works, in part, by inducing an autoimmune mediated antitumor response. The data from these experiments suggest the autoimmunity can play a force in anti-tumor immunity, and CTLA4 can regulate this response. Since small changes in CTLA4 expression can dramatically change the outcome of an anti-tumor response, we generated CTLA4 RNAi mice. These mice have reduced CTLA4 expression that is comparable to reductions caused by promoter polymorphisms in humans. Astoundingly, these mice uniformly develop gastric metaplastic pathology by 5 weeks of age; a process initiated by an inflammatory autoimmune response. This pathology persisted until 17-18 months of age, at which point the gastric metaplasia progressed into adenocarcinoma. An age associated increase in tumor protective 5-hmC epigenetic modification occurs in wildtype mice, which did not occur in the CTLA4 RNAi mice; a finding extended to human adenocarcinoma. The data from these experiments demonstrate the complicated and sometimes paradoxical role of T-cells in autoimmunity and tumor immunity. A better understanding of these mechanisms will lead to new insights on how to: restore immunological balance in individuals with autoimmunity; harness the immense power of the immune system to eradicate tumors; and prevent inflammation induced tumorigenesis.</p