Jason Miska

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, 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 Tregs and MDSCs are recruited in various tumors are 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 MDSCs 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 Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671-82. ©2016 AACR.

Dendritic Cell Based Vaccines that Utilize Myeloid Rather than Plasmacytoid Cells Offer a Superior Survival Advantage in Malignant Glioma

Dendritic cells (DCs) are professional APCs that are traditionally divided into two distinct subsets, myeloid DC (mDCs) and plasmacytoid DC (pDCs). pDCs are known for their ability to secrete large amounts of IFN-α. Apart from IFN-α production, pDCs can also process Ag and induce T cell immunity or tolerance. In several solid tumors, pDCs have been shown to play a critical role in promoting tumor immunosuppression. We investigated the role of pDCs in the process of glioma progression in the syngeneic murine model of glioma. We show that glioma-infiltrating pDCs are the major APC in glioma and are deficient in IFN-α secretion (p < 0.05). pDC depletion leads to increased survival of the mice bearing intracranial tumor by decreasing the number of regulatory T cells (Tregs) and by decreasing the suppressive capabilities of Tregs. We subsequently compared the ability of mDCs and pDCs to generate effective antiglioma immunity in a GL261-OVA mouse model of glioma. Our data suggest that mature pDCs and mDCs isolated from naive mice can be effectively activated and loaded with SIINFEKL Ag in vitro. Upon intradermal injection in the hindleg, a fraction of both types of DCs migrate to the brain and lymph nodes. Compared to mice vaccinated with pDC or control mice, mice vaccinated with mDCs generate a robust Th1 type immune response, characterized by high frequency of CD4+T-bet+ T cells and CD8+SIINFEKEL+ T cells. This robust antitumor T cell response results in tumor eradication and long-term survival in 60% of the animals (p < 0.001).

βIII-Tubulin Regulates Breast Cancer Metastases to the Brain

Brain metastases occur in about 10% to 30% of breast cancer patients, which culminates in a poor prognosis. It is, therefore, critical to understand the molecular mechanisms underlying brain metastatic processes to identify relevant targets. We hypothesized that breast cancer cells must express brain-associated markers that would enable their invasion and survival in the brain microenvironment. We assessed a panel of brain-predominant markers and found an elevation of several neuronal markers (βIII-tubulin, Nestin, and AchE) in brain metastatic breast cancer cells. Among these neuronal predominant markers, in silico analysis revealed overexpression of βIII-tubulin (TUBB3) in breast cancer brain metastases (BCBM) and its expression was significantly associated with distant metastases. TUBB3 knockdown studies were conducted in breast cancer models (MDA-Br, GLIM2, and MDA-MB-468), which revealed significant reduction in their invasive capabilities. MDA-Br cells with suppressed TUBB3 also demonstrated loss of key signaling molecules such as β3 integrin, pFAK, and pSrc in vitro. Furthermore, TUBB3 knockdown in a brain metastatic breast cancer cell line compromised its metastatic ability in vivo, and significantly improved survival in a brain metastasis model. These results implicate a critical role of TUBB3 in conferring brain metastatic potential to breast cancer cells. Mol Cancer Ther; 14(5); 1152–61. ©2015 AACR.

Intratumoral oncolytic adenoviral treatment modulates the glioma microenvironment and facilitates systemic tumor-antigen-specific T cell therapy.

Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor and is associated with poor survival. Virotherapy is a promising candidate for the development of effective, novel treatments for GBM. Recent studies have underscored the potential of virotherapy in enhancing antitumor immunity despite the fact that its mechanisms remain largely unknown. Here, using a syngeneic GBM mouse model, we report that intratumoral virotherapy significantly modulates the tumor microenvironment. We found that intratumoral administration of an oncolytic adenovirus, AdCMVdelta24, decreased tumor-infiltrating CD4+ Foxp3+ regulatory T cells (Tregs) and increased IFNγ-producing CD8+ T cells in treated tumors, even in late stage disease in which a highly immunosuppressive tumor microenvironment is considered to be a significant barrier to immunotherapy. Importantly, intratumoral AdCMVdelta24 treatment augmented systemically transferred tumor-antigen-specific T cell therapy. Furthermore, mechanistic studies showed (1) downregulation of Foxp3 in Tregs that were incubated with media conditioned by virus-infected tumor cells, (2) downregulation of indoleamine 2,3 dioxygenase 1 (IDO) in glioma cells upon infection by AdCMVdelta24, and (3) reprograming of Tregs from an immunosuppressive to a stimulatory state. Taken together, our findings demonstrate the potency of intratumoral oncolytic adenoviral treatment in enhancing antitumor immunity through the regulation of multiple aspects of immune suppression in the context of glioma, supporting further clinical development of oncolytic adenovirus-based immune therapies for malignant brain cancer.

Fatty Acid Uptake in T Cell Subsets Using a Quantum Dot Fatty Acid Conjugate

Fatty acid (FA) metabolism directly influences the functional capabilities of T cells in tumor microenvironments. Thus, developing tools to interrogate FA-uptake by T cell subsets is important for understanding tumor immunosuppression. Herein, we have generated a novel FA-Qdot 605 dye conjugate with superior sensitivity and flexibility to any of the previously commercially available alternatives. For the first time, we demonstrate that this nanoparticle can be used as a specific measure of fatty acid uptake by T cells both in-vitro and in-vivo. Flow cytometric analysis shows that both the location and activation status of T cells determines their FA uptake. Additionally, CD4+ Foxp3+ regulatory T cells (Tregs) uptake FA at a higher rate than effector T cell subsets, supporting the role of FA metabolism for Treg function. Furthermore, we are able to simultaneously detect glucose and fatty acid uptake directly within the tumor microenvironment. Cumulatively, our results suggest that this novel fluorescent probe is a powerful tool to understand FA utilization within the tumor, thereby providing an unprecedented opportunity to study T cell FA metabolism in-vivo.

A Comparative Study of Replication-Incompetent and -Competent Adenoviral Therapy-Mediated Immune Response in a Murine Glioma Model

Oncolytic virotherapy is a treatment approach with increasing clinical relevance, as indicated by the marked survival benefit seen in animal models and its current exploration in human patients with cancer. The use of an adenovirus vector for this therapeutic modality is common, has significant clinical benefit in animals, and its efficacy has recently been linked to an anti-tumor immune response that occurs following tumor antigen presentation. Here, we analyzed the adaptive immune system’s response following viral infection by comparing replication-incompetent and replication-competent adenoviral vectors. Our findings suggest that cell death caused by replication-competent adenoviral vectors is required to induce a significant anti-tumor immune response and survival benefits in immunocompetent mice bearing intracranial glioma. We observed significant changes in the repertoire of immune cells in the brain and draining lymph nodes and significant recruitment of CD103+ dendritic cells (DCs) in response to oncolytic adenoviral therapy, suggesting the active role of the immune system in anti-tumor response. Our data suggest that the response to oncolytic virotherapy is accompanied by local and systemic immune responses and should be taken in consideration in the future design of the clinical studies evaluating oncolytic virotherapy in patients with glioblastoma multiforme (GBM).

Tamoxifen overrides autophagy inhibition in Beclin-1-deficient glioma cells and their resistance to adenovirus-mediated oncolysis via upregulation of PUMA and BAX

Autophagy is an evolutionarily conserved process regulating cellular homeostasis via digestion of dysfunctional proteins and whole cellular organelles by mechanisms, involving their enclosure into double-membrane vacuoles that are subsequently fused to lysosomes. Glioma stem cells utilize autophagy as a main mechanism of cell survival and stress response. Most recently, we and others demonstrated induction of autophagy in gliomas in response to treatment with chemical drugs, such as temozolomide (TMZ) or oncolytic adenoviruses (Ads). As autophagy has been implicated in the mechanism of Ad-mediated cell killing, autophagy deficiency in some glioma tumors could be the reason for their resistance to oncolysis. Despite the observed connection, the exact relationship between autophagy-activating cell signaling and adenoviral infection remains unclear. Here, we report that inhibition of autophagy in target glioma cells induces their resistance to killing by oncolytic agent CRAd-S-5/3. Furthermore, we found that downregulation of autophagy inducer Beclin-1 inhibits replication-competent Ad-induced oncolysis of human glioma by suppressing cell proliferation and inducing premature senescence. To overcome the autophagy-deficient state of such glioma cells and restore their susceptibility to oncolytic Ad infection, we propose treating glioma tumors with an anticancer drug tamoxifen (TAM) as a means to induce apoptosis in Ad-targeted cancer cells via upregulation of BAX/PUMA genes. In agreement with the above hypothesis, our data suggest that TAM improves susceptibility of Beclin-1-deficient glioma cells to CRAd-S-5/3 oncolysis by means of activating autophagy and pro-apoptotic signaling pathways in the target cancer cells.

A Dendritic Cell-Targeted Adenoviral Vector Facilitates Adaptive Immune Response Against Human Glioma Antigen (CMV-IE) and Prolongs Survival in a Human Glioma Tumor Model

Antitumor immunotherapeutic strategies represent an especially promising set of approaches with rapid translational potential considering the dismal clinical context of high-grade gliomas. Dendritic cells (DCs) are the body’s most professional antigen-presenting cells, able to recruit and activate T cells to stimulate an adaptive immune response. In this regard, specific loading of tumor-specific antigen onto dendritic cells potentially represents one of the most advanced strategies to achieve effective antitumor immunization. In this study, we developed a DC-specific adenoviral (Ad) vector, named Ad5scFvDEC205FF, targeting the DC surface receptor, DEC205. In vitro analysis shows that 60% of DCs was infected by this vector while the infectivity of other control adenoviral vectors was less than 10%, demonstrating superior infectivity on DCs. Moreover, an average of 14% of DCs were infected by Ad5scFvDEC205FF-GFP, while less than 3% of non-DCs were infected following in vivo administration, demonstrating highly selective in vivo DC infection. Importantly, vaccination with this vehicle expressing human glioma-specific antigen, Ad5scFvDEC205FF-CMV-IE, shows a prolonged survival benefit in GL261CMV-IE-implanted murine glioma models (p < 0.0007). Furthermore, when rechallenged, cancerous cells were completely rejected. In conclusion, our novel, viral-mediated, DC-based immunization approach has the significant therapeutic potential for patients with high-grade gliomas.

Initiation of inflammatory tumorigenesis by CTLA4 insufficiency due to type 2 cytokines

Genetically predisposed CTLA4 insufficiency in humans is associated with gastric cancer development, which is paradoxical to the prototypical role of CTLA4 in suppressing antitumor immunity. CTLA4 is a critical immune checkpoint against autoimmune disorders. Autoimmunity has been implicated in protumor or antitumor activities. Here, we show that CTLA4 insufficiency initiates de novo tumorigenesis in the mouse stomach through inflammation triggered by host-intrinsic immune dysregulation rather than microbiota, with age-associated progression to malignancy accompanied by epigenetic dysregulation. The inflammatory tumorigenesis required CD4 T cells, but not the TH1 or TH17 subsets. Deficiencies in IL-4 and IL-13 or IL-4 receptor &agr; broke the link between inflammation and initiation of tumorigenesis. This study establishes the causality of CTLA4 insufficiency in gastric cancer and uncovers a role of type 2 inflammation in initiating gastric epithelial transformation. These findings suggest possible improvement of immune therapies by blocking tumorigenic type 2 inflammation while preserving antitumor type 1 immunity.

The Role of Regulatory T Cells and Indoleamine-2,3-dioxygenase in Brain Tumor Immunosuppression

Malignant glioma induced immunosuppression is one of the major hurdles in the way of designing successful antiglioma therapy. Several factors, such as inhibitory dendritic cells (DCs), regulatory T cells (Tregs), inhibitory enzymes, and cytokines contribute to orchestrate a very redundant and potent immunosuppressive microenvironment. Two of the most influential and hence most studied players associated with glioma immunosuppression are Treg and indoleamine-2,3-dioxygenase (IDO), an enzyme, expressed by glioma cells and inhibitory plasmacytoid DCs. Several preclinical studies have shown that targeting Tregs and IDO is a successful and well-tolerated strategy of inducing potent antiglioma response. Based on these results, strategies of Treg depletion and targeting IDO have been tried in clinical settings. In this chapter, we review the literature summarizing the role of Tregs and IDO in glioma immunosuppression and outline the preclinical works, which led to the development of several successful clinical trials to overcome glioma immunosuppression and boost antiglioma immunotherapy.