Mechanisms of immune modulation in the tumor microenvironment and implications for targeted therapy.

The efficacy of cancer therapies is limited to a great extent by immunosuppressive mechanisms within the tumor microenvironment (TME). Numerous immune escape mechanisms have been identified. These include not only processes associated with tumor, immune or stromal cells, but also humoral, metabolic, genetic and epigenetic factors within the TME. The identification of immune escape mechanisms has enabled the development of small molecules, nanomedicines, immune checkpoint inhibitors, adoptive cell and epigenetic therapies that can reprogram the TME and shift the host immune response towards promoting an antitumor effect. These approaches have translated into series of breakthroughs in cancer therapies, some of which have already been implemented in clinical practice. In the present article the authors provide an overview of some of the most important mechanisms of immunosuppression within the TME and the implications for targeted therapies against different cancers.

RNA Sequencing Identifies Novel NRG1 Fusions in Solid Tumors that Lack Co-Occurring Oncogenic Drivers.

NRG1 gene fusions are rare, therapeutically relevant, oncogenic drivers that occur across solid tumor types. To understand the landscape of NRG1 gene fusions, 4397 solid tumor formalin-fixed, paraffin-embedded samples consecutively tested by comprehensive genomic and immune profiling during standard care were analyzed. Nineteen NRG1 fusions were found in 17 unique patients, across multiple tumor types, including non-small-cell lung (n = 7), breast (n = 2), colorectal (n = 3), esophageal (n = 2), ovarian (n = 1), pancreatic (n = 1), and unknown primary (n = 1) carcinomas, with a cumulative incidence of 0.38%. Fusions were identified with breakpoints across four NRG1 introns spanning 1.4 megabases, with a mixture of known (n = 8) and previously unreported (n = 11) fusion partners. Co-occurring driver alterations in tumors with NRG1 fusions were uncommon, except colorectal carcinoma, where concurrent alterations in APC, BRAF, and ERBB2 were present in a subset of cases. The overall lack of co-occurring drivers highlights the importance of identifying NRG1 gene fusions, as these patients are unlikely to harbor other targetable alterations. In addition, RNA sequencing is important to identify NRG1 gene fusions given the variety of fusion partners and large genomic areas where breakpoints can occur.

High-throughput screens identify a lipid nanoparticle that preferentially delivers mRNA to human tumors in vivo.

Lipid nanoparticles (LNPs) are a clinically relevant way to deliver therapeutic mRNA to hepatocytes in patients. However, LNP-mRNA delivery to end-stage solid tumors such as head and neck squamous cell carcinoma (HNSCC) remains more challenging. While scientists have used in vitro assays to evaluate potential nanoparticles for HNSCC delivery, high-throughput delivery assays performed directly in vivo have not been reported. Here we use a high-throughput LNP assay to evaluate how 94 chemically distinct nanoparticles delivered nucleic acids to HNSCC solid tumors in vivo. DNA barcodes were used to identify LNPHNSCC, a novel LNP for systemic delivery to HNSCC solid tumors. Importantly, LNPHNSCC retains tropism to HNSCC solid tumors while minimizing off-target delivery to the liver.

Evaluating antitumor activity of Escherichia coli purine nucleoside phosphorylase against head and neck patient-derived xenografts.

BACKGROUND: Purine nucleoside phosphorylase (PNP) gene transfer represents a promising approach to treatment of head and neck malignancies. We tested recombinant adenovirus already in phase I/II clinical testing and leading-edge patient-derived xenografts (PDX) as a means to optimize this therapeutic strategy. METHODS: Our experiments investigated purine base cytotoxicity, PNP enzyme activity following treatment of malignant tissue, tumor mass regression, viral receptor studies, and transduction by tropism-modified adenovirus. RESULTS: Replication deficient vector efficiently transduced PDX cells and mediated significant anticancer effect following treatment with fludarabine phosphate in vivo. Either 6-methylpurine or 2-fluoroadenine (toxic molecules generated by the PNP approach) ablated head and neck cancer cell proliferation. High levels of adenovirus-3 specific receptors were detected in human tumor models, and vector was evaluated that utilizes this pathway. CONCLUSIONS: Our studies provide the scientific foundation necessary to improve PNP prodrug cleavage and advance a new treatment for head and neck cancer.

Development of a novel purification protocol to isolate and identify brain microglia.

Microglia, the tissue-resident macrophage of the central nervous system (CNS), play a paramount role in brain health and disease status. Here, we describe a novel method for enriching and isolating primary microglia from mouse brain tissue. This isolation method yields a high number of cells from either young or adult mice, and importantly, maintains the health and function of the cells for subsequent cell culture. We also describe flow cytometry methods using novel cell surface markers, including CX3CR1 and Siglec-H, to specifically label microglia while avoiding other bone marrow and/or non-CNS derived macrophages and monocytes, which has been historically difficult to achieve. As microglia are crucial in multiple aspects of biology, such as in normal brain development/function, immune response, neurodegeneration, and cancer, this isolation technique could greatly benefit a wide range of studies in human CNS biology, health, and disease mechanisms. Being able to isolate a largely pure population of microglia could also allow for a more comprehensive understanding of their functional dynamics and role in disease mechanisms, advancement of potential biomarkers, and development of novel therapeutic targets to improve prognosis and quality of life in multiple diseases.

Changes in the tumor microenvironment and outcome for TME-targeting therapy in glioblastoma: A pilot study.

Glioblastoma (GBM) is a hypervascular and aggressive primary malignant tumor of the central nervous system. Recent investigations showed that traditional therapies along with antiangiogenic therapies failed due to the development of post-therapy resistance and recurrence. Previous investigations showed that there were changes in the cellular and metabolic compositions in the tumor microenvironment (TME). It can be said that tumor cell-directed therapies are ineffective and rethinking is needed how to treat GBM. It is hypothesized that the composition of TME-associated cells will be different based on the therapy and therapeutic agents, and TME-targeting therapy will be better to decrease recurrence and improve survival. Therefore, the purpose of this study is to determine the changes in the TME in respect of T-cell population, M1 and M2 macrophage polarization status, and MDSC population following different treatments in a syngeneic model of GBM. In addition to these parameters, tumor growth and survival were also studied following different treatments. The results showed that changes in the TME-associated cells were dependent on the therapeutic agents, and the TME-targeting therapy improved the survival of the GBM bearing animals. The current GBM therapies should be revisited to add agents to prevent the accumulation of bone marrow-derived cells in the TME or to prevent the effect of immune-suppressive myeloid cells in causing alternative neovascularization, the revival of glioma stem cells, and recurrence. Instead of concurrent therapy, a sequential strategy would be better to target TME-associated cells.

Critical immunosuppressive effect of MDSC­derived exosomes in the tumor microenvironment.

Myeloid­derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME). Along with the role of MDSC immunosuppression and antitumor immunity, MDSCs facilitate tumor growth, differentiation, and metastasis in several ways that are yet to be explored. Like any other cell type, MDSCs also release a tremendous number of exosomes, or nanovesicles of endosomal origin, that participate in intercellular communications by dispatching biological macromolecules. There have been no investigational studies conducted to characterize the role of MDSC­derived exosomes (MDSC exo) in modulating the TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant level of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed a higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those in the spleen or bone marrow. Our in vitro data suggest that MDSC exo are capable of hyper­activating or exhausting CD8 T­cells and induce reactive oxygen species production that elicits activation­induced cell death. We confirmed the depletion of CD8 T­cells in vivo by treating mice with MDSC exo. We also observed a reduction in pro­inflammatory M1­macrophages in the spleen of those animals. Our results indicate that the immunosuppressive and tumor­promoting functions of MDSCs are also implemented by MDSC­derived exosomes which would open up a new avenue of MDSC research and MDSC­targeted therapy.

Induction of DNMT3B by PGE2 and IL6 at Distant Metastatic Sites Promotes Epigenetic Modification and Breast Cancer Colonization.

Current cancer treatments are largely based on the genetic characterization of primary tumors and are ineffective for metastatic disease. Here we report that DNA methyltransferase 3B (DNMT3B) is induced at distant metastatic sites and mediates epigenetic reprogramming of metastatic tumor cells. Multiomics analysis and spontaneous metastatic mouse models revealed that DNMT3B alters multiple pathways including STAT3, NFκB, PI3K/Akt, ß-catenin, and Notch signaling, which are critical for cancer cell survival, apoptosis, proliferation, invasion, and colonization. PGE2 and IL6 were identified as critical inflammatory mediators in DNMT3B induction. DNMT3B expression levels positively correlated with human metastatic progression. Targeting IL6 or COX-2 reduced DNMT3B induction and improved chemo or PD1 therapy. We propose a novel mechanism linking the metastatic microenvironment with epigenetic alterations that occur at distant sites. These results caution against the "Achilles heel" in cancer therapies based on primary tumor characterization and suggests targeting DNMT3B induction as new option for treating metastatic disease. SIGNIFICANCE: These findings reveal that DNMT3B epigenetically regulates multiple pro-oncogenic signaling pathways via the inflammatory microenvironment at distant sites, cautioning the clinical approach basing current therapies on genetic characterization of primary tumors.

Oncoprotein GT198 vaccination delays tumor growth in MMTV-PyMT mice.

Targeting early lesion in breast cancer is more therapeutically effective. We have previously identified an oncoprotein GT198 (PSMC3IP) in human breast cancer. Here we investigated GT198 in MMTV-PyMT mouse mammary gland tumors and found that GT198 is a shared early lesion in both species. Similar to human breast cancer even before a tumor appears, cytoplasmic GT198 is overexpressed in mouse tumor stroma including pericyte stem cells, descendent adipocytes, fibroblasts, and myoepithelial cells. Using recombinant GT198 protein as an antigen, we vaccinated MMTV-PyMT mice and found that the GT198 vaccine delayed mouse tumor growth and reduced lung metastasis. The antitumor effects were linearly correlated with vaccinated mouse serum titers of GT198 antibody, which recognized cell surface GT198 protein on viable tumor cells confirmed by FACS. Furthermore, GT198+ tumor cells isolated from MMTV-PyMT tumor induced faster tumor growths than GT198- cells when re-implanted into normal FVB/N mice. Together, this first study of GT198 vaccine in mouse showed its effectiveness in antitumor and anti-metastasis. The finding supports GT198 as a potential target in human immunotherapy since GT198 defect is shared in both human and mouse.

CD73 on cancer-associated fibroblasts enhanced by the A2B-mediated feedforward circuit enforces an immune checkpoint.

CD73, an ecto-5'-nucleotidase (NT5E), serves as an immune checkpoint by generating adenosine (ADO), which suppresses immune activation through the A2A receptor. Elevated CD73 levels in tumor tissues correlate with poor clinical outcomes. However, the crucial source of CD73 activity within the tumor microenvironment remains unspecified. Here, we demonstrate that cancer-associated fibroblasts (CAFs) constitute the prominent CD73hi population in human colorectal cancers (CRCs) and two CD73- murine tumor models, including a modified CRC. Clinically, high CAF abundancy in CRC tissues correlates strongly with elevated CD73 activity and poor prognosis. Mechanistically, CAF-CD73 expression is enhanced via an ADO-A2B receptor-mediated feedforward circuit triggered by tumor cell death, which enforces the CD73-checkpoint. Simultaneous inhibition of A2A and A2B pathways with CD73-neutralization synergistically enhances antitumor immunity in CAF-rich tumors. Therefore, the strategic and effective targeting of both the A2B-mediated ADO-CAF-CD73 feedforward circuit and A2A-mediated immune suppression is crucial for improving therapeutic outcomes.

Differential in vivo biodistribution of 131I-labeled exosomes from diverse cellular origins and its implication for theranostic application.

Exosomes are critical mediators of intercellular crosstalk and are regulator of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosomes research has been thwarted by our limited knowledge of the most efficient isolation method and their in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 µm syringe filter and 100 k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes. We also demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.

CXCR2-Expressing Tumor Cells Drive Vascular Mimicry in Antiangiogenic Therapy-Resistant Glioblastoma.

BACKGROUND: Glioblastoma (GBM) was shown to relapse faster and displayed therapeutic resistance to antiangiogenic therapies (AATs) through an alternative tumor cell-driven mechanism of neovascularization called vascular mimicry (VM). We identified highly upregulated interleukin 8 (IL-8)-CXCR2 axis in tumor cells in high-grade human glioma and AAT-treated orthotopic GBM tumors. METHODS: Human GBM tissue sections and tissue array were used to ascertain the clinical relevance of CXCR2-positive tumor cells in the formation of VM. We utilized U251 and U87 human tumor cells to understand VM in an orthotopic GBM model and AAT-mediated enhancement in VM was modeled using vatalanib (anti-VEGFR2) and avastin (anti-VEGF). Later, VM was inhibited by SB225002 (CXCR2 inhibitor) in a preclinical study. RESULTS: Overexpression of IL8 and CXCR2 in human datasets and histological analysis was identified as a bonafide candidate to validate VM through in vitro and animal model studies. AAT-treated tumors displayed a higher number of CXCR2-positive GBM-stem cells with endothelial-like phenotypes. Stable knockdown of CXCR2 expression in tumor cells led to decreased tumor growth as well as incomplete VM structures in the animal models. Similar data were obtained following SB225002 treatment. CONCLUSIONS: The present study suggests that tumor cell autonomous IL-8-CXCR2 pathway is instrumental in AAT-mediated resistance and VM formation in GBM. Therefore, CXCR2 can be targeted through SB225002 and can be combined with standard therapies to improve the therapeutic outcomes in clinical trials.

Delineating Pro-Angiogenic Myeloid Cells in Cancer Therapy.

Recent evidence suggests that myeloid cells are critical in cancer development and therapy resistance processes. Pharmacological targeting of tumor-associated myeloid cells is an emerging approach among upcoming immune therapies. Surprisingly, myeloid cells are heterogeneous, including a subset of the myeloid cell displaying angiogenic properties in solid tumors. There is an urgent need to delineate angiogenic myeloid cell populations in order to facilitate specific targeting of protumor myeloid cells among heterogeneous pool. This review article is intended to compile all the relevant information in the literature for improved understanding of angiogenic myeloid cells and their role in tumor refractoriness to cancer therapy.

miR-130a and miR-145 reprogram Gr-1+CD11b+ myeloid cells and inhibit tumor metastasis through improved host immunity.

Tumor-derived soluble factors promote the production of Gr-1+CD11b+ immature myeloid cells, and TGFß signaling is critical in their immune suppressive function. Here, we report that miR-130a and miR-145 directly target TGFß receptor II (TßRII) and are down-regulated in these myeloid cells, leading to increased TßRII. Ectopic expression of miR-130a and miR-145 in the myeloid cells decreased tumor metastasis. This is mediated through a downregulation of type 2 cytokines in myeloid cells and an increase in IFNγ-producing cytotoxic CD8 T lymphocytes. miR-130a- and miR-145-targeted molecular networks including TGFß and IGF1R pathways were correlated with higher tumor stages in cancer patients. Lastly, miR-130a and miR-145 mimics, as well as IGF1R inhibitor NT157 improved anti-tumor immunity and inhibited metastasis in preclinical mouse models. These results demonstrated that miR-130a and miR-145 can reprogram tumor-associated myeloid cells by altering the cytokine milieu and metastatic microenvironment, thus enhancing host antitumor immunity.

Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization.

Inflammation is an important mediator of secondary neurological injury after traumatic brain injury (TBI). Endocannabinoids, endogenously produced arachidonate based lipids, have recently emerged as powerful anti-inflammatory compounds, yet the molecular and cellular mechanisms underlying these effects are poorly defined. Endocannabinoids are physiological ligands for two known cannabinoid receptors, CB1R and CB2R. In the present study, we hypothesized that selective activation of CB2R attenuates neuroinflammation and reduces neurovascular injury after TBI. Using a murine controlled cortical impact (CCI) model of TBI, we observed a dramatic upregulation of CB2R within infiltrating myeloid cells beginning at 72 h. Administration of the selective CB2R agonist, GP1a (1-5 mg/kg), attenuated pro-inflammatory M1 macrophage polarization, increased anti-inflammatory M2 polarization, reduced edema development, enhanced cerebral blood flow, and improved neurobehavioral outcomes after TBI. In contrast, the CB2R antagonist, AM630, worsened outcomes. Taken together, our findings support the development of selective CB2R agonists as a therapeutic strategy to improve TBI outcomes while avoiding the psychoactive effects of CB1R activation.

Major Challenges and Potential Microenvironment-Targeted Therapies in Glioblastoma.

Glioblastoma (GBM) is considered one of the most malignant, genetically heterogeneous, and therapy-resistant solid tumor. Therapeutic options are limited in GBM and involve surgical resection followed by chemotherapy and/or radiotherapy. Adjuvant therapies, including antiangiogenic treatments (AATs) targeting the VEGF-VEGFR pathway, have witnessed enhanced infiltration of bone marrow-derived myeloid cells, causing therapy resistance and tumor relapse in clinics and in preclinical models of GBM. This review article is focused on gathering previous clinical and preclinical reports featuring major challenges and lessons in GBM. Potential combination therapies targeting the tumor microenvironment (TME) to overcome the myeloid cell-mediated resistance problem in GBM are discussed. Future directions are focused on the use of TME-directed therapies in combination with standard therapy in clinical trials, and the exploration of novel therapies and GBM models for preclinical studies. We believe this review will guide the future of GBM research and therapy.

Anti-Jagged Immunotherapy Inhibits MDSCs and Overcomes Tumor-Induced Tolerance.

Myeloid-derived suppressor cells (MDSC) are a major obstacle to promising forms of cancer immunotherapy, but tools to broadly limit their immunoregulatory effects remain lacking. In this study, we assessed the therapeutic effect of the humanized anti-Jagged1/2-blocking antibody CTX014 on MDSC-mediated T-cell suppression in tumor-bearing mice. CTX014 decreased tumor growth, affected the accumulation and tolerogenic activity of MDSCs in tumors, and inhibited the expression of immunosuppressive factors arginase I and iNOS. Consequently, anti-Jagged therapy overcame tumor-induced T-cell tolerance, increased the infiltration of reactive CD8+ T cells into tumors, and enhanced the efficacy of T-cell-based immunotherapy. Depletion of MDSC-like cells restored tumor growth in mice treated with anti-Jagged, whereas coinjection of MDSC-like cells from anti-Jagged-treated mice with cancer cells delayed tumor growth. Jagged1/2 was induced in MDSCs by tumor-derived factors via NFkB-p65 signaling, and conditional deletion of NFkB-p65 blocked MDSC function. Collectively, our results offer a preclinical proof of concept for the use of anti-Jagged1/2 to reprogram MDSC-mediated T-cell suppression in tumors, with implications to broadly improve the efficacy of cancer therapy. Cancer Res; 77(20); 5628-38. ©2017 AACR.

HET0016 decreases lung metastasis from breast cancer in immune-competent mouse model.

Distant metastasis is the primary cause of death in the majority of the cancer types. Recently, much importance has been given to tumor microenvironment (TME) in the development of invasive malignant tumors, as well as the metastasis potential. The ability of tumor cells to modulate TME and to escape immune-mediated attack by releasing immunosuppressive cytokines has become a hallmark of breast cancer. Our study shows the effect of IV formulation of HET0016 (HPßCD-HET0016) a selective inhibitor of 20-HETE synthesis, administered intravenously in immune-competent in vivo mouse model of murine breast cancer. 4T1 luciferase positive cells were implanted to the mammary fat pad in Balb/c mice. Treatment started on day 15, and was administered for 5 days a week for 3 weeks. The development of metastasis was detected via optical imaging. Blood, spleen, lungs, bone marrow and tumor were collected for flow cytometry, to investigate changes in myeloid-derived suppressive cells (MDSCs) populations and endothelial phenotype. Tumor and lungs were collected for protein analysis. Our results show that HPßCD-HET0016: (1) decreased tumor volume and lung metastasis compared to the vehicle group; (2) reduced migration and invasion of tumor cells and levels of metalloproteinases in the lungs of animals treated with HPßCD-HET0016 via PI3K/AKT pathway; and (3) decreased expression of pro-inflammatory cytokines, growth factors and granulocytic MDSCs population in the lung microenvironment in treated animals. Thus, HPßCD-HET0016 showed potential in treating lung metastasis in a preclinical mouse model and needs further investigations on TME.