Matthew J. Cotter

HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment.

The hypoxic environment of tumors dictates the phenotype of local myeloid-derived suppressor cells (MDSCs) via HIF-1a expression; hypoxia converts splenic MDSCs from specific into nonspecific suppressors.

Mechanism Regulating Reactive Oxygen Species in Tumor-Induced Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) are a major component of the immune suppressive network described in cancer and many other pathological conditions. Recent studies have demonstrated that one of the major mechanisms of MDSC-induced immune suppression is mediated by reactive oxygen species (ROS). However, the mechanism of this phenomenon remained unknown. In this study, we observed a substantial up-regulation of ROS by MDSC in all of seven different tumor models and in patients with head and neck cancer. The increased ROS production by MDSC is mediated by up-regulated activity of NADPH oxidase (NOX2). MDSC from tumor-bearing mice had significantly higher expression of NOX2 subunits, primarily p47phox and gp91phox, compared with immature myeloid cells from tumor-free mice. Expression of NOX2 subunits in MDSC was controlled by the STAT3 transcription factor. In the absence of NOX2 activity, MDSC lost the ability to suppress T cell responses and quickly differentiated into mature macrophages and dendritic cells. These findings expand our fundamental understanding of the biology of MDSC and may also open new opportunities for therapeutic regulation of these cells in cancer.

Anti-inflammatory Triterpenoid Blocks Immune Suppressive Function of MDSCs and Improves Immune Response in Cancer

Purpose: Myeloid-derived suppressor cells (MDSC) are one of the major factors responsible for immune suppression in cancer. Therefore, it would be important to identify effective therapeutic means to modulate these cells. Experimental Design: We evaluated the effect of the synthetic triterpenoid C-28 methyl ester of 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO-Me; bardoxolone methyl) in MC38 colon carcinoma, Lewis lung carcinoma, and EL-4 thymoma mouse tumor models, as well as blood samples from patients with renal cell cancer and soft tissue sarcoma. Samples were also analyzed from patients with pancreatic cancer treated with CDDO-Me in combination with gemcitabine. Results: CDDO-Me at concentrations of 25 to 100 nmol/L completely abrogated immune suppressive activity of MDSC in vitro. CDDO-Me reduced reactive oxygen species in MDSCs but did not affect their viability or the levels of nitric oxide and arginase. Treatment of tumor-bearing mice with CDDO-Me did not affect the proportion of MDSCs in the spleens but eliminated their suppressive activity. This effect was independent of antitumor activity. CDDO-Me treatment decreased tumor growth in mice. Experiments with severe combined immunodeficient–beige mice indicated that this effect was largely mediated by the immune system. CDDO-Me substantially enhanced the antitumor effect of a cancer vaccines. Treatment of pancreatic cancer patients with CDDO-Me did not affect the number of MDSCs in peripheral blood but significantly improved the immune response. Conclusions: CDDO-Me abrogated the immune suppressive effect of MDSCs and improved immune responses in tumor-bearing mice and cancer patients. It may represent an attractive therapeutic option by enhancing the effect of cancer immunotherapy. Clin Cancer Res; 16(6); 1812–23

Combination of External Beam Radiotherapy (EBRT) With Intratumoral Injection of Dendritic Cells as Neo-Adjuvant Treatment of High-Risk Soft Tissue Sarcoma Patients

PURPOSE The goal of this study was to determine the effect of combination of intratumoral administration of dendritic cells (DC) and fractionated external beam radiation (EBRT) on tumor-specific immune responses in patients with soft-tissue sarcoma (STS). METHODS AND MATERIAL Seventeen patients with large (>5 cm) high-grade STS were enrolled in the study. They were treated in the neoadjuvant setting with 5,040 cGy of EBRT, split into 28 fractions and delivered 5 days per week, combined with intratumoral injection of 10(7) DCs followed by complete resection. DCs were injected on the second, third, and fourth Friday of the treatment cycle. Clinical evaluation and immunological assessments were performed. RESULTS The treatment was well tolerated. No patient had tumor-specific immune responses before combined EBRT/DC therapy; 9 patients (52.9%) developed tumor-specific immune responses, which lasted from 11 to 42 weeks. Twelve of 17 patients (70.6%) were progression free after 1 year. Treatment caused a dramatic accumulation of T cells in the tumor. The presence of CD4(+) T cells in the tumor positively correlated with tumor-specific immune responses that developed following combined therapy. Accumulation of myeloid-derived suppressor cells but not regulatory T cells negatively correlated with the development of tumor-specific immune responses. Experiments with (111)In labeled DCs demonstrated that these antigen presenting cells need at least 48 h to start migrating from tumor site. CONCLUSIONS Combination of intratumoral DC administration with EBRT was safe and resulted in induction of antitumor immune responses. This suggests that this therapy is promising and needs further testing in clinical trials design to assess clinical efficacy.

Notch and Wingless Signaling Cooperate in Regulation of Dendritic Cell Differentiation

Dendritic cell (DC) differentiation is regulated by stroma via a network of soluble and cell-bound factors. Notch is one of the major elements of this network. Its role in DC differentiation, however, is controversial. Here, we demonstrate that activation of Notch signaling in hematopoietic progenitor cells (HPCs) promoted differentiation of conventional DCs via activation of the canonical Wingless (Wnt) pathway. Inhibition of the Wnt pathway abrogated the effect of Notch on DC differentiation. The fact that activation of the Wnt pathway in Notch-1-deficient embryonic stem cells restored DC differentiation indicates that Wnt signaling is downstream of the Notch pathway in regulating DC differentiation. Notch signaling activated the Wnt pathway in HPCs via expression of multiple members of the Frizzled family of Wnt receptors, which was directly regulated by the CSL (RPB-Jkappa) transcription factor. Thus, these data suggest a model of DC differentiation via cooperation between Wnt and Notch pathways.

Antiviral Antibodies Target Adenovirus to Phagolysosomes and Amplify the Innate Immune Response

Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-κB-dependent genes, type I IFNs, and caspase-dependent IL-1β maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1β maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

Abstract 2411: Intratumoral dendritic cell vaccine as neo-adjuvant immunotherapy of high-risk soft tissue sarcoma patients undergoing external beam radiation therapy (EBRT): Prospective phase I/II clinical trial

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cell death inducing external beam radiation (EBRT) combined with experimental intratumoral injection of dendritic cells (DC) showed promise by initiating anti-autologous tumor-cell immune responses in mice. Here we performed first clinical trial testing this hypothesis in patients with large high-grade soft tissue sarcomas (STS). This type of cancer has a significant (>50%) risk of progressing to distant metastases. Patients with clinical stage T2N0M0 or T3N0M0 high-grade STS of the extremity/trunk/chest wall were treated with standard neo-adjuvant EBRT 5040 cGy / 180 cGy coordinated with experimental DC therapy consisting of DC progenitor apheresis, ex-vivo expansion and culture, and 4 × intratumoral injections of 10 million DC. Autologous DC product was performed according to standardized GMP laboratory procedures. T cell function was assessed by EliSpot (measuring IFN-γ production) and proliferation (thymidine uptake) in addition to phenotyping of peripheral blood mononuclear cells by flow cytometry. Clinically, targeted accrual was reached with eighteen patients completing neo-adjuvant EBRT with experimental intratumoral DC therapy. All patients have completed full immunologic assessment. Ten patients (56%) were induced to produce significant immune responses against autologous tumor cell lysates or/and survivin antigens as determined using ELISPOT assays or cell proliferation. Interestingly, some of these responses persisted even 30 weeks after start of treatment. Extensive post treatment T cell infiltration was detected within tumors. No clinical toxicity has been observed. Suggestive clinical outcome on 14 patients followed 1 year reveal progress in 1/7 patients with robust response (detected at more than one time point), and 4/7 patients with weak (detected at one time point at the beginning of the study) or no response respectively. Thus, this data is promising for planning future combined treatment approaches as radiation therapy/immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2411.