miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

Dual-specificity phosphatase 8 (DUSP8) plays an important role as a selective c-Jun N-terminal kinase (JNK) phosphatase in mitogen-activated protein kinase (MAPK) signaling. In this study, we found that DUSP8 is silenced by miR-147b in patients with lung adenocarcinoma (LUAD), which correlates with poor overall survival. Overexpression of DUSP8 resulted in a tumor-suppressive phenotype in vitro and in vivo experimental models, whereas silencing DUSP8 with a siRNA approach abrogated the tumor-suppressive properties. We found that miR-147b is a posttranscriptional regulator of DUSP8 that is highly expressed in patients with LUAD and is associated with lower survival. NanoString analysis revealed that the MAPK signaling pathway is mainly affected by overexpression of miR-147b, leading to increased proliferation and migration and decreased apoptosis in vitro. Moreover, induction of miR-147b promotes tumor progression in vitro and in vivo experimental models. Knockdown of miR-147b restored DUSP8, decreased tumor progression in vitro, and increased apoptosis via JNK phosphorylation. These results suggest that miR-147b plays a key role in regulating MAPK signaling in LUAD. The link between DUSP8 and miR-147b may provide novel approaches for the treatment of lung cancer.

The HDAC2-SP1 Axis Orchestrates Protumor Macrophage Polarization.

Tumor-associated macrophages (TAM), including antitumor M1-like TAMs and protumor M2-like TAMs, are transcriptionally dynamic innate immune cells with diverse roles in lung cancer development. Epigenetic regulators are key in controlling macrophage fate in the heterogeneous tumor microenvironment. Here, we demonstrate that the spatial proximity of HDAC2-overexpressing M2-like TAMs to tumor cells significantly correlates with poor overall survival of lung cancer patients. Suppression of HDAC2 in TAMs altered macrophage phenotype, migration, and signaling pathways related to interleukins, chemokines, cytokines, and T-cell activation. In coculture systems of TAMs and cancer cells, suppressing HDAC2 in TAMs resulted in reduced proliferation and migration, increased apoptosis of cancer cell lines and primary lung cancer cells, and attenuated endothelial cell tube formation. HDAC2 regulated the M2-like TAM phenotype via acetylation of histone H3 and transcription factor SP1. Myeloid cell-specific deletion of Hdac2 and pharmacologic inhibition of class I HDACs in four different murine lung cancer models induced the switch from M2-like to M1-like TAMs, altered infiltration of CD4+ and CD8+ T cells, and reduced tumor growth and angiogenesis. TAM-specific HDAC2 expression may provide a biomarker for lung cancer stratification and a target for developing improved therapeutic approaches. SIGNIFICANCE: HDAC2 inhibition reverses the protumor phenotype of macrophages mediated by epigenetic modulation induced by the HDAC2-SP1 axis, indicating a therapeutic option to modify the immunosuppressive tumor microenvironment.

Fibrocytes boost tumor-supportive phenotypic switches in the lung cancer niche via the endothelin system.

Fibrocytes are bone marrow-derived monocytic cells implicated in wound healing. Here, we identify their role in lung cancer progression/ metastasis. Selective manipulation of fibrocytes in mouse lung tumor models documents the central role of fibrocytes in boosting niche features and enhancing metastasis. Importantly, lung cancer patients show increased number of circulating fibrocytes and marked fibrocyte accumulation in the cancer niche. Using double and triple co-culture systems with human lung cancer cells, fibrocytes, macrophages and endothelial cells, we substantiate the central features of cancer-supporting niche: enhanced cancer cell proliferation and migration, macrophage activation, augmented endothelial cell sprouting and fibrocyte maturation. Upregulation of endothelin and its receptors are noted, and dual endothelin receptor blockade suppresses all cancer-supportive phenotypic alterations via acting on fibrocyte interaction with the cancer niche. We thus provide evidence for a crucial role of fibrocytes in lung cancer progression and metastasis, suggesting targets for treatment strategies.

Interferon Regulatory Factor 9 Promotes Lung Cancer Progression via Regulation of Versican.

Transcription factors can serve as links between tumor microenvironment signaling and oncogenesis. Interferon regulatory factor 9 (IRF9) is recruited and expressed upon interferon stimulation and is dependent on cofactors that exert in tumor-suppressing or oncogenic functions via the JAK-STAT pathway. IRF9 is frequently overexpressed in human lung cancer and is associated with decreased patient survival; however, the underlying mechanisms remain to be elucidated. Here, we used stably transduced lung adenocarcinoma cell lines (A549 and A427) to overexpress or knockdown IRF9. Overexpression led to increased oncogenic behavior in vitro, including enhanced proliferation and migration, whereas knockdown reduced these effects. These findings were confirmed in vivo using lung tumor xenografts in nude mice, and effects on both tumor growth and tumor mass were observed. Using RNA sequencing, we identified versican (VCAN) as a novel downstream target of IRF9. Indeed, IRF9 and VCAN expression levels were found to be correlated. We showed for the first time that IRF9 binds at a newly identified response element in the promoter region of VCAN to regulate its transcription. Using an siRNA approach, VCAN was found to enable the oncogenic properties (proliferation and migration) of IRF9 transduced cells, perhaps with CDKN1A involvement. The targeted inhibition of IRF9 in lung cancer could therefore be used as a new treatment option without multimodal interference in microenvironment JAK-STAT signaling.

Microglia/macrophages express alternative proangiogenic factors depending on granulocyte content in human glioblastoma.

Myeloid cells are an inherent part of the microenvironment of glioblastoma multiforme (GBM). There is growing evidence for their participation in mechanisms of tumor escape, especially in the development of resistance following initially promising anti-VEGF/VEGFR treatment. Thus, we sought to define the capability of myeloid cells to contribute to the expression of proangiogenic molecules in human GBM. We investigated GBM specimens in comparison with anaplastic astrocytoma (WHO grade III) and epilepsy patient samples freshly obtained from surgery. Flow cytometric analyses revealed two distinct CD11b+ CD45+ cell populations in GBM tissues, which were identified as microglia/macrophages and granulocytes. Due to varied granulocyte influx, GBM samples were subdivided into groups with low (GBM-lPMNL) and high (GBM-hPMNL) numbers of granulocytes (polymorphonuclear leukocytes; PMNL), which were related to activation of the microglia/macrophage population. Microglia/macrophages of the GBM-lPMNL group were similar to those of astrocytoma specimens, but those of GBM-hPMNL tissues revealed an altered phenotype by expressing high levels of CD163, TIE2, HIF1α, VEGF, CXCL2 and CD13. Although microglia/macrophages represented the main source of alternative proangiogenic factors, additionally granulocytes participated by production of IL8 and CD13. Moreover, microglia/macrophages of the GBM-hPMNL specimens were highly associated with tumor blood vessels, accompanied by remodeling of the vascular structure. Our data emphasize that tumor-infiltrating myeloid cells might play a crucial role for limited efficacy of anti-angiogenic therapy bypassing VEGF-mediated pathways through expression of alternative proangiogenic factors. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CCR2 of Tumor Microenvironmental Cells Is a Relevant Modulator of Glioma Biology.

Glioblastoma multiforme (GBM) shows a high influx of tumor-associated macrophages (TAMs). The CCR2/CCL2 pathway is considered a relevant signal for the recruitment of TAMs and has been suggested as a therapeutic target in malignant gliomas. We found that TAMs of human GBM specimens and of a syngeneic glioma model express CCR2 to varying extents. Using a Ccr2-deficient strain for glioma inoculation revealed a 30% reduction of TAMs intratumorally. This diminished immune cell infiltration occurred with augmented tumor volumes likely based on increased cell proliferation. Remaining TAMs in Ccr2-/- mice showed comparable surface marker expression patterns in comparison to wildtype mice, but expression levels of inflammatory transcription factors (Stat3, Irf7, Cox2) and cytokines (Ifnß, Il1ß, Il12α) were considerably affected. Furthermore, we demonstrated an impact on blood vessel integrity, while vascularization of tumors appeared similar between mouse strains. The higher stability and attenuated leakiness of the tumor vasculature imply improved sustenance of glioma tissue in Ccr2-/- mice. Additionally, despite TAMs residing in the perivascular niche in Ccr2-/- mice, their pro-angiogenic activity was reduced by the downregulation of Vegf. In conclusion, lacking CCR2 solely on tumor microenvironmental cells leads to enhanced tumor progression, whereby high numbers of TAMs infiltrate gliomas independently of the CCR2/CCL2 signal.

Fibroblast Growth Factor-14 Acts as Tumor Suppressor in Lung Adenocarcinomas.

Investigation of the molecular dynamics in lung cancer is crucial for the development of new treatment strategies. Fibroblast growth factor (FGF) 14 belongs to the FGF family, which might play a crucial role in cancer progression. We analyzed lung adenocarcinoma (LUAC) patients samples and found that FGF14 was downregulated, correlating with reduced survival and oncogenic mutation status. FGF14 overexpression in lung cancer cell lines resulted in decreased proliferation, colony formation, and migration, as well as increased expression of epithelial markers and a decreased expression of mesenchymal markers, indicating a mesenchymal to epithelial transition in vitro. We verified these findings using small interfering RNA against FGF14 and further confirmed the suppressive effect of FGF14 in a NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ immunodeficient xenograft tumor model. Moreover, FGF14 overexpressing tumor cell RNA sequencing data suggests that genes affected by FGF14 were related to the extracellular matrix, playing a role in proliferation and migration. Notably, newly identified FGF14 target genes, adenosine deaminase RNA specific B1 (ADARB1), collagen and calcium-binding epidermal growth factor domain-containing protein 1 (CCBE1), α1 chain of collagen XI (COL11A1), and mucin 16 (MUC16) expression was negatively correlated with overall survival when FGF14 was downregulated in LUAC. These findings led us to suggest that FGF14 regulates proliferation and migration in LUAC.

The Multi-Modal Effect of the Anti-fibrotic Drug Pirfenidone on NSCLC.

Although immune checkpoint and targeted therapies offer remarkable benefits for lung cancer treatment, some patients do not qualify for these regimens or do not exhibit consistent benefit. Provided that lung cancer appears to be driven by transforming growth factor beta signaling, we investigated the single drug potency of Pirfenidone, an approved drug for the treatment of lung fibrosis. Five human lung cancer cell lines and one murine line were investigated for transforming growth factor beta inhibition via Pirfenidone by using flow cytometry, In-Cell western analysis, proliferation assays as well as comprehensive analyses of the transcriptome with subsequent bioinformatics analysis. Overall, Pirfenidone induced cell cycle arrest, down-regulated SMAD expression and reduced proliferation in lung cancer. Furthermore, cell stress pathways and pro-apoptotic signaling may be mediated by reduced expression of Survivin. A murine subcutaneous model was used to assess the in vivo drug efficacy of Pirfenidone and showed reduced tumor growth and increased infiltration of T cells and NK cells. This data warrant further clinical evaluation of Pirfenidone with advanced non-small cell lung cancer. The observed in vitro and in vivo effects point to a substantial benefit for using Pirfenidone to reactivate the local immune response and possible application in conjunction with current immunotherapies.

Repurposing Thioridazine (TDZ) as an anti-inflammatory agent.

Nuclear factor-kB (NF-kB) is a crucial transcription factor in the signal transduction cascade of the inflammatory signaling. Activation of NF-κB depends on the phosphorylation of IκBα by IκB kinase (IKKß) followed by subsequent ubiquitination and degradation. This leads to the nuclear translocation of the p50- p65 subunits of NF-κB, and further triggers pro-inflammatory cytokine gene expression. Thus, in the need of a more effective therapy for the treatment of inflammatory diseases, specific inhibition of IKKß represents a rational alternative strategy to the current therapies. A computer-aided drug identification protocol was followed to identify novel IKKß inhibitors from a database of over 1500 Food and Drug Administration (FDA) drugs. The best scoring compounds were compared with the already known high-potency IKKß inhibitors for their ability to bind and inhibit IKKß by evaluating their docking energy. Finally, Thioridazinehydrochloride (TDZ), a potent antipsychotic drug against Schizophrenia was selected and its efficiency in inhibiting IκBα protein degradation and NF-κB activation was experimentally validated. Our study has demonstrated that TDZ blocks IκBα protein degradation and subsequent NF-κB activation to inhibit inflammation. Thus, it is a potential repurposed drug against inflammation.

VEGF as a modulator of the innate immune response in glioblastoma.

VEGF is an important factor in tumor vascularization and used as target for anti-angiogenic treatment strategies in glioma. In this study, we demonstrate for the first time that VEGF is a modulator of the innate immune response with suppressive effects on the immunologic and pro-angiogenic function of microglia/macrophages in a glioblastoma rodent model. High level of VEGF led to threefold enlarged tumor volumes and a pronounced remodeling of the vascular structure along with a reduced infiltration of microglia/macrophages by approximately 50%. Remaining microglia/macrophages showed an enhanced rate of apoptosis as well as significant downregulation of the VEGF-receptor, VEGFR2, and others such as CXCR4. Consequently, we determined a substantially impaired migration of these microglia/macrophages to VEGF and SDF1α in vitro. Furthermore, we observed an increased presentation of the surface molecules MHCI and MHCII on microglia/macrophages from VEGF-overexpressing gliomas that are essential for activation of the adaptive immune system. In contrast, the expression of pro-inflammatory and suppressive cytokines, associated with the innate immune response, were mainly downregulated. Remarkably, the abundance of VEGF provoked less accumulation of microglia/macrophages within the perivascular niche and concomitantly reduced the release of pro-angiogenic factors, like VEGF, suggesting a possible regulatory feedback mechanism. Thus, the quantity of VEGF in the glioma microenvironment seems to be crucial for the participation of microglia/macrophages on tumor progression and should be considered for developing novel therapeutic approaches.

Redirecting tumor-associated macrophages to become tumoricidal effectors as a novel strategy for cancer therapy.

Cancer research in recent decades has highlighted the potential influence of the tumor microenvironment on the progression and metastasis of most known cancer types. Within the established microenvironment, tumor-associated macrophages (TAMs) are one of the most abundant and crucial non-neoplastic cell types. The polarization of macrophages into tumor-suppressive M1 or tumor-promoting M2 types is a fundamental event in the establishment of the tumor microenvironment. Although ample evidence indicates that TAMs are primarily M2 polarized, the mechanisms responsible for the regulation and maintenance of M1 and M2 polarization imbalance remain unclear. The manipulation of this critical axis through three main approaches may provide new strategies for cancer therapy - (I) specific interference with M2-like TAM survival or inhibiting their signaling cascades, (II) repression of macrophage recruitment to tumors, and (III) repolarization of tumor-promoting M2-like TAMs to a tumoricidal M1-like phenotype. This review summarizes current strategies for cancer intervention via manipulation of macrophage polarization, with particular focus on composition of the tumor microenvironment and its influence on cancer progression and metastasis. It is clear that additional fundamental and preclinical research is required to confirm the efficacy and practicality of this novel and promising strategy for treating cancer.

Myeloid cells expressing high level of CD45 are associated with a distinct activated phenotype in glioma.

Glioblastoma multiforme is characterized by high accumulation of microglia/macrophages. The function of these tumor-infiltrating myeloid cells is not sufficiently elucidated. Therefore, a better understanding of the precise immune cell composition and function in brain tumors is required. In rodent glioma models, two different myeloid cell populations exist, determined by the expression level of CD45, namely CD11b+CD45low and CD11b+CD45high. Previous analyses of cytokine and marker expression profiles were almost exclusively performed on the entire myeloid cell fraction. Consequently, described pro- and anti-tumoral characteristics were not assigned to the evident subpopulations. In the present study, we used a syngeneic glioblastoma mouse model and subsequent flow cytometric analyses to demonstrate the distinct properties of CD11b+CD45high and the CD11b+CD45low cells. First, the majority of CD11b+CD45high cells expressed high level of GR1 and around 6% of IL10 representing in part features of myeloid-derived suppressor cells, while the CD11b+CD45low fraction displayed no upregulation of these molecules. Second, we detected that specifically the CD11b+CD45high population showed antigen-presenting, co-stimulatory, and inflammatory features. Here, we identified up to 80% of MHCII and approximately 50% of CD86 and TNFα-expressing cells. Investigation of MHCI and CD80 revealed a moderate upregulation. By contrast, in the CD11b+CD45low cell fraction, merely MHCII and TNFα were marginally overexpressed. In summary, these data emphasize the specific phenotype of CD11b+CD45high cells in glioma with suppressive as well as pro-inflammatory characteristics whereas the CD11b+CD45low cells were almost unaffected. Hence, primarily, the subpopulation consisting of CD45high-expressing cells is activated by the tumor and should be considered as therapeutic target.

Resident microglia rather than peripheral macrophages promote vascularization in brain tumors and are source of alternative pro-angiogenic factors.

Myeloid cells are an essential part of the glioblastoma microenvironment. However, in brain tumors the function of these immune cells is not sufficiently clarified. In our study, we investigated differential pro-angiogenic activities of resident microglia and peripheral macrophages and their impact on glioma vascularization and progression. Our data demonstrate stable accumulation of microglia/macrophages during tumor growth. These cells often interact with tumor blood vessels correlating with vascular remodeling. Here, we identified resident microglia as well as peripheral macrophages as part of the perivascular niche, primarily contacting endothelial cells. We found overexpression of a variety of pro-angiogenic molecules within freshly isolated microglia/macrophages from glioma. CXCL2, until now a poorly described chemokine, was strongly up-regulated and showed better angiogenic activity than VEGF in vitro. Blocking the CXCL2-CXCR2 signaling pathway resulted in considerably diminished glioma sizes. Additionally, the importance of microglia/macrophages in tumor angiogenesis was confirmed by depletion of these cells in vivo. Vessel density decreased by 50% leading to significantly smaller tumor volumes. Remarkably, selective reduction of resident microglia affected tumoral vessel count comparable to ablation of the whole myeloid cell fraction. These results provide evidence that resident microglia are the crucial modulatory cell population playing a central role in regulation of vascular homeostasis and angiogenesis in brain tumors. Thus, resident microglia represent an alternative source of pro-angiogenic growth factors and cytokines.

Microglia inflict delayed brain injury after subarachnoid hemorrhage.

Inflammatory changes have been postulated to contribute to secondary brain injury after aneurysmal subarachnoid hemorrhage (SAH). In human specimens after SAH as well as in experimental SAH using mice, we show an intracerebral accumulation of inflammatory cells between days 4 and 28 after the bleeding. Using bone marrow chimeric mice allowing tracing of all peripherally derived immune cells, we confirm a truly CNS-intrinsic, microglial origin of these immune cells, exhibiting an inflammatory state, and rule out invasion of myeloid cells from the periphery into the brain. Furthermore, we detect secondary neuro-axonal injury throughout the time course of SAH. Since neuronal cell death and microglia accumulation follow a similar time course, we addressed whether the occurrence of activated microglia and neuro-axonal injury upon SAH are causally linked by depleting microglia in vivo. Given that the amount of neuronal cell death was significantly reduced after microglia depletion, we conclude that microglia accumulation inflicts secondary brain injury after SAH.

Resident microglia, and not peripheral macrophages, are the main source of brain tumor mononuclear cells.

Gliomas consist of multiple cell types, including an abundant number of microglia and macrophages, whereby their impact on tumor progression is controversially discussed. To understand their unique functions and consequently manipulate either microglia or macrophages in therapeutic approaches, it is essential to discriminate between both cell populations. Because of the lack of specific markers, generally total body irradiated chimeras with labeled bone marrow cells were used to identify infiltrated cells within the brain. However, total body irradiation (TBI) affects the blood-brain barrier integrity, which in turn potentially facilitates immune cell infiltration. In this study, changes on the blood-brain barrier were avoided using head-protected irradiation (HPI). Head protection and total body irradiated chimeras exhibited similar reconstitution levels of the myeloid cell lineage in the blood, enabling the comparable analyses of brain infiltrates. We demonstrate that the HPI model impeded a massive unspecific influx of donor-derived myeloid cells into naive as well as tumor-bearing brains. Moreover, experimental artifacts such as an enlarged distribution of infiltrated cells and fourfold increased tumor volumes are prevented in head-protected chimeras. In addition, our data evidenced for the first time that microglia are able to up-regulate CD45 and represent an inherent part of the CD45(high) population in the tumor context. All in all, HPI allowed for the unequivocal distinction between microglia and macrophages without alterations of tumor biology and consequently permits a detailed and realistic description of the myeloid cell composition in gliomas.