Loss of CX3CR1 increases accumulation of inflammatory monocytes and promotes gliomagenesis

The most abundant populations of non-neoplastic cells in the glioblastoma (GBM) microenvironment are resident microglia, macrophages and infiltrating monocytes from the blood circulation. The mechanisms by which monocytes infiltrate into GBM, their fate following infiltration, and their role in GBM growth are not known. Here we tested the hypothesis that loss of the fractalkine receptor CX3CR1 in microglia and monocytes would affect gliomagenesis. Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice. Loss of Cx3cr1 did not affect accumulation of microglia/macrophages in peri-tumoral areas, but instead indirectly promoted the trafficking of CD11b⁺CD45^hiCX3CR1^lowLy-6C^hiLy-6G⁻F4/80^−/low circulating inflammatory monocytes into the CNS, resulting in their increased accumulation in the perivascular area. Cx3cr1-deficient microglia/macrophages and monocytes demonstrated upregulation of IL1β expression that was inversely proportional to Cx3cr1 gene dosage. The Proneural subgroup of the TCGA GBM patient dataset with high IL1β expression showed shorter survival compared to patients with low IL1β. IL1β promoted tumor growth and increased the cancer stem cell phenotype in murine and human Proneural glioma stem cells (GSCs). IL1β activated the p38 MAPK signaling pathway and expression of monocyte chemoattractant protein (MCP-1/CCL2) by tumor cells. Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration. These data suggest that enhancing signaling to CX3CR1 or inhibiting IL1β signaling in intra-tumoral macrophages can be considered as potential strategies to decrease the tumor-promoting effects of monocytes in Proneural GBM.     

Circulating T regulatory cells migration and phenotype in glioblastoma patients: an in vitro study

Glioblastoma multiforme (GBM) is the most aggressive primary human brain tumor. The relatively high amount of T regulatory lymphocytes present in the tumor, contributes to the establishment of an immunosuppressive microenvironment. Samples of peripheral blood were collected from GBM patients and healthy controls and a purified population of Treg (CD4⁺/CD25^bright) was isolated using flow cytometric cell sorting. Treg migrating capacities toward human glioma cell line conditioned medium were evaluated through an in vitro migration test. Our data show that supernatants collected from GBM cell lines were more attractant to Treg when compared to complete standard medium. The addition of an anti-CCL2 antibody to conditioned medium decreased conditioned medium-depending Treg migration, suggesting that CCL2 (also known as Monocyte Chemoattractant Protein, MCP-1) is implicated in the process. The number of circulating CD4⁺/μL or Treg/μL was similar in GBM patients and controls. Specific Treg markers (FOXP3; CD127; Helios; GITR; CTLA4; CD95; CCR2, CCR4; CCR7) were screened in peripheral blood and no differences could be detected between the two populations. These data confirm that the tumor microenvironment is attractive to Treg, which tend to migrate toward the tumor region changing the immunological response. Though we provide evidence that CCL2 is implicated in Treg migration, other factors are needed as well to provide such effect.     

Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma

Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4⁺ HNSCC cells, but not CCR4^? cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206⁺ M2‐like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206^high M2‐like macrophages increased the cell motility of CCR4⁺ HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4⁺ cancer cells, but not CCR4^? cells, metastasized to lymph nodes which contained CCL22 producing M2‐like macrophages. These results demonstrate that lymph node metastasis of CCR4⁺ HNSCC is promoted by CCL22 in an autocrine or M2‐like macrophage‐dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.     

Propentofylline decreases tumor growth in a rodent model of glioblastoma multiforme by a direct mechanism on microglia

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer, with a median survival of less than 2 years after diagnosis. The tumor microenvironment plays a critical role in tumor invasion and progression. Microglia and infiltrating macrophages are the most abundant immune cells in the tumor. In the present study, we demonstrate that systemic propentofylline (PPF), an atypical methylxanthine with central nervous system (CNS) glial modulating and anti-inflammatory actions, significantly decreased tumor growth in a CNS-1 rat model of GBM by targeting microglia and not tumor cells. Rats received tumor injections of 1 × 10(5) CNS-1 cells in the right striatum with daily intraperitonial injections of PPF (50 mg/kg) or saline beginning the day of tumor injection. PPF did not cause apoptosis or decrease proliferation of CNS-1 tumor cells. Furthermore, we demonstrate, using in vitro methods, that PPF decreased microglial migration toward CNS-1 tumor cells and decreased MMP-9 expression. The effects of PPF were shown to be specific to microglia and not peripheral macrophages. These results support a differential functional role of resident microglia and infiltrating macrophages in the brain tumor environment. Our data highlight microglia as a crucial target for future therapeutic development and present PPF as a possible drug for treatment of human GBM.     

Curcumin changes the polarity of tumor‐associated microglia and eliminates glioblastoma

Glioblastoma (GBM) is one of the most pernicious forms of cancer and currently chances of survival from this malady are extremely low. We have used the noninvasive strategy of intranasal (IN) delivery of a glioblastoma‐directed adduct of curcumin (CC), CC‐CD68Ab, into the brain of mouse GBM GL261‐implanted mice to study the effect of CC on tumor remission and on the phenotype of the tumor‐associated microglial cells (TAMs). The treatment caused tumor remission in 50% of GL261‐implanted GBM mice. A similar rescue rate was also achieved through intraperitoneal infusion of a lipid‐encapsulated formulation of CC, Curcumin Phytosome, into the GL261‐implanted GBM mice. Most strikingly, both forms of CC elicited a dramatic change in the tumor‐associated Iba1+ TAMs, suppressing the tumor‐promoting Arginase1^high, iNOS^low M2‐type TAM population while inducing the Arginase1^low, iNOS^high M1‐type tumoricidal microglia. Concomitantly, we observed a marked induction and activation of microglial NF‐kB and STAT1, which are known to function in coordination to cause induction of iNOS. Therefore, our novel findings indicate that appropriately delivered CC can directly kill GBM cells and also repolarize the TAMs to the tumoricidal M1 state.     

Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain

Background

To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo.

Methods

Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels.

Results

Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA.

Conclusions

Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs.     

The CCR4 as a novel-specific molecular target for immunotherapy in Hodgkin lymphoma.

Here, we report that tumor cells from some patients (23.8%) with Hodgkin lymphoma (HL) are positive for CC chemokine receptor 4 (CCR4). We therefore tested the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to enhance antibody-dependent cellular cytotoxicity (ADCC), as a novel immunotherapy for refractory HL. KM2760 demonstrated a promising antitumor activity in the CCR4-positive HL-bearing mouse model in the therapeutic setting. Although KM2760 did not induce any ADCC mediated by mouse natural killer (NK) cells, it significantly enhanced phagocytosis mediated by mouse monocytes/macrophages against the CCR4-positive HL cell line in vitro. Together with the findings that KM2760 did not exhibit any complement-dependent cytotoxicity or direct antiproliferation activity in vitro, these data indicated that KM2760 exerted its robust in vivo antitumor activity via monocytes/macrophages in mice. In the human system, KM2760 enhanced phagocytic activity mediated by monocytes/macrophages. Furthermore, it induced robust ADCC mediated by NK cells against the CCR4-positive HL cell line in vitro. Thus, it is conceivable that KM2760 would have much more potent antitumor activity in humans than in mice. Collectively, this study strongly indicates that anti-CCR4 mAb could be a novel treatment modality for patients with CCR4-positive HL.     

IFN-γ–STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in vivo

The induction of antitumor effector T cells in the tumor microenvironment is a crucial event for cancer immunotherapy. Neurokinin receptor 2 (NK2R), a G protein-coupled receptor for neurokinin A (NKA), regulates diverse physiological functions. However, the precise role of NKA–NK2R signaling in antitumor immunity is unclear. Here, we found that an IFN-γ–STAT1 cascade augmented NK2R expression in CD8⁺ T cells, and NK2R-mediated NKA signaling was involved in inducing antitumor effector T cells in vivo. The administration of a synthetic analog of double-stranded RNA, polyinosinic–polycytidylic acid (poly I:C), into a liver cancer mouse model induced type I and type II IFNs and significantly suppressed the tumorigenesis of Hepa1-6 liver cancer cells in a STAT1-dependent manner. The reduction in tumor growth was diminished by the depletion of CD8⁺ T cells. IFN-γ stimulation significantly induced NK2R and tachykinin precursor 1 (encodes NKA) gene expression in CD8⁺ T cells. NKA stimulation combined with anti-CD3 monoclonal antibody (mAb) treatment significantly augmented IFN-γ and granzyme B production by CD8⁺ T cells compared with the anti-CD3 mAb alone in vitro. ERK1/2 phosphorylation and IκBα degradation in activated CD8⁺ T cells were suppressed under NK2R deficiency. Finally, we confirmed that tumor growth was significantly increased in NK2R-deficient mice compared with that in wild-type mice, and the antitumor effects of poly I:C were abolished by NK2R absence. These findings suggest that IFN-γ–STAT1-mediated NK2R expression is involved in the induction of antitumor effector T cells in the tumor microenvironment, which contributes to the suppression of cancer cell tumorigenesis in vivo. In this study, we revealed that IFN-γ–STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8⁺ T cells in the tumor microenvironment, which contributes to suppressing the tumorigenesis of liver cancer cells in vivo.     

Inhibition of glioma progression by a newly discovered CD38 inhibitor

Glioma, the most common cancer of the central nervous system, has very poor prognosis and no effective treatment. It has been shown that activated microglia/macrophages in the glioma tumor microenvironment support progression. Hence, inhibition of the supporting effect of these cells may constitute a useful therapeutic approach. Recently, using a syngeneic mouse glioma progression model, we showed that the ectoenzyme CD38 regulated microglia activation and, in addition, that the loss of CD38 from the tumor microenvironment attenuated glioma progression and prolonged the life span of the tumor‐bearing mice. These studies, which employed wild‐type (WT) and Cd38^?/? C57BL/6J mice, suggest that inhibition of CD38 in glioma microenvironment may be used as a new therapeutic approach to treat glioma. Our study tested this hypothesis. Initially, we found that the natural anthranoid, 4,5‐dihydroxyanthraquinone‐2‐carboxylic acid (rhein), and its highly water‐soluble tri‐potassium salt form (K‐rhein) are inhibitors of CD38 enzymatic (nicotinamide adenine dinucleotide glycohydrolase) activity (IC₅₀ = 1.24 and 0.84 μM, respectively, for recombinant mouse CD38). Treatment of WT, but not Cd38^?/? microglia with rhein and K‐rhein inhibited microglia activation features known to be regulated by CD38 (lipopolysaccharide/IFN‐γ‐induced activation, induced cell death and NO production). Furthermore, nasal administration of K‐rhein into WT, but not Cd38^?/? C57BL/6J, mice intracranially injected with GL261 cells substantially and significantly inhibited glioma progression. Hence, these results serve as a proof of concept, demonstrating that targeting CD38 at the tumor microenvironment by small‐molecule inhibitors of CD38, for example, K‐rhein, may serve as a useful therapeutic approach to treat glioma.     

Plasticity in Tumor-Promoting Inflammation: Impairment of Macrophage Recruitment Evokes a Compensatory Neutrophil Response

Previous studies in the K14-HPV/E₂ mouse model of cervical carcinogenesis demonstrated that infiltrating macrophages are the major source of matrix metalloproteinase 9 (MMP-9), a metalloprotease important for tumor angiogenesis and progression. We observed increased expression of the macrophage chemoattractant, CCL2, and its receptor, CCR2, concomitant with macrophage influx and MMP-9 expression. To study the role of CCL2-CCR2 signaling in cervical tumorigenesis, we generated CCR2-deficient K14-HPV/E₂ mice. Cervixes of CCR2-null mice contained significantly fewer macrophages. Surprisingly, there was only a modest delay in time to progression from dysplasia to carcinoma in the CCR2-deficient mice, and no difference in end-stage tumor incidence or burden. Moreover, there was an unexpected persistence of MMP-9 activity, associated with increased abundance of MMP-9⁺ neutrophils in tumors from CCR2-null mice. In vitro bioassays revealed that macrophages produce soluble factor(s) that can suppress neutrophil dynamics, as evidenced by reduced chemotaxis in response to CXCL8, and impaired invasion into three-dimensional tumor masses grown in vitro. Our data suggest a mechanism whereby CCL2 attracts proangiogenic CCR2⁺ macrophages with the ancillary capability to limit infiltration by neutrophils. If such tumor-promoting macrophages are suppressed, MMP-9⁺ neutrophils are then recruited, providing alternative paracrine support for tumor angiogenesis and progression.     

Thrombin-processed Ecrg4 recruits myeloid cells and induces antitumorigenic inflammation

Background

Extensive infiltration of brain tumors by microglia and macrophages is a hallmark of tumor progression, and yet the overall tumor microenvironment is characterized by an immunosuppressive phenotype. Here we identify esophageal cancer-related gene 4 (Ecrg4) as a novel thrombin-processed monocyte chemoattractant that recruits myeloid cells, promotes their activation, and leads to a blockade of tumor progression.

Methods

Both xenograft glioma and syngeneic glioma models were used to measure orthotopic tumor progression and overall survival. Flow cytometry and immunohistochemical analyses were performed to assess myeloid cell localization, recruitment, and activation.

Results

Ecrg4 promotes monocyte recruitment and activation of microglia in a T-/B-cell–independent mechanism, which leads to a reduction in glioma tumor burden and increased survival. Mutational analysis reveals that the biological activity of Ecrg4 is dependent on a thrombin-processing site at the C-terminus, inducing monocyte invasion in vivo and in vitro. Furthermore, tumor-induced myeloid cell recruitment is impaired in Ecrg4 knockout mice, leading to increased tumor burden and decreased survival.

Conclusions

Together, these results identify Ecrg4 as a paracrine factor that activates microglia and is chemotactic for monocytes, with potential as an antitumor therapeutic.     

Suppression of Tregs by anti‐glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon‐α gene therapy for pancreatic cancer

We have reported that interferon (IFN)‐α can attack cancer cells by multiple antitumor mechanisms including the induction of direct cancer cell death and the enhancement of an immune response in several pancreatic cancer models. However, an immunotolerant microenvironment in the tumors is often responsible for the failure of the cancer immunotherapy. Here we examined whether the suppression of regulatory T cells (Tregs) within tumors can enhance an antitumor immunity induced by an intratumoral IFN‐α gene transfer. First we showed that an intraperitoneal administration of an agonistic anti‐glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model. The anti‐GITR mAb was then combined with the intratumoral injection of the IFN‐α‐adenovirus vector. The treatment with the antibody synergistically augmented the antitumor effect of IFN‐α gene therapy not only in the vector‐injected tumors but also in the vector‐uninjected tumors. Immunostaining showed that the anti‐GITR mAb decreased Foxp3⁺ cells infiltrating in the tumors, while the intratumoral IFN‐α gene transfer increased CD4⁺ and CD8⁺ T cells in the tumors. Therefore, the combination therapy strongly inclined the immune balance of the tumor microenvironment in an antitumor direction, leading to a marked systemic antitumor effect. The CCR5 expression on Tregs was downregulated in the antibody‐treated mice, which may explain the decrease of tumor‐infiltrating Tregs. The combination of Treg‐suppression by GITR mAb and the tumor immunity induction by IFN‐α gene therapy could be a promising therapeutic strategy for pancreatic cancer.     

Tumor associated CD70 expression is involved in promoting tumor migration and macrophage infiltration in GBM

Tumor migration/metastasis and immunosuppression are major obstacles in effective cancer therapy. Incidentally, these 2 hurdles usually coexist inside tumors, therefore making therapy significantly more complicated, as both oncogenic mechanisms must be addressed for successful therapeutic intervention. Our recent report highlights that the tumor expression of a TNF family member, CD70, is correlated with poor survival for primary gliomas. In this study, we investigated how CD70 expression by GBM affects the characteristics of tumor cells and the tumor microenvironment. We found that the ablation of CD70 in primary GBM decreased CD44 and SOX2 gene expression, and inhibited tumor migration, growth and the ability to attract monocyte‐derived M2 macrophages in vitro. In the tumor microenvironment, CD70 was associated with immune cell infiltrates, such as T cells; myeloid‐derived suppressor cells; and monocytes/macrophages based on the RNA‐sequencing profile. The CD163+ macrophages were far more abundant than T cells were. This overwhelming level of macrophages was identified only in GBM and not in low‐grade gliomas and normal brain specimens, implying their tumor association. CD70 was detected only on tumor cells, not on macrophages, and was highly correlated with CD163 gene expression in primary GBM. Additionally, the co‐expression of the CD70 and CD163 genes was found to correlate with decreased survival for patients with primary GBM. Together, these data suggest that CD70 expression is involved in promoting tumor aggressiveness and immunosuppression via tumor‐associated macrophage recruitment/activation. Our current efforts to target this molecule using chimeric antigen receptor T cells hold great potential for treating patients with GBM.