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The immune-specialized environment of the healthy brain is tightly regulated to prevent excessive neuroinflammation. However, after cancer development, a tissue-specific conflict between brain-preserving immune suppression and tumor-directed immune activation may ensue. To interrogate potential roles of T cells in this process, we profiled these cells from individuals with primary or metastatic brain cancers via integrated analyses on the single-cell and bulk population levels. Our analysis revealed similarities and differences in T cell biology between individuals, with the most pronounced differences observed in a subgroup of individuals with brain metastasis, characterized by accumulation of CXCL13-expressing CD39+ potentially tumor-reactive T (pTRT) cells. In this subgroup, high pTRT cell abundance was comparable to that in primary lung cancer, whereas all other brain tumors had low levels, similar to primary breast cancer. These findings indicate that T cell-mediated tumor reactivity can occur in certain brain metastases and may inform stratification for treatment with immunotherapy.

Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment. Bulk tumor preservation and dissociation enable multiple analyses of the brain tumor immune microenvironment via immunofluorescent staining and cell sorting followed by transcriptomics, genomics or in vitro culture of specific cell populations.

High-grade gliomas, the most common and aggressive primary brain tumors, are characterized by a complex tumor microenvironment (TME). Among the immune cells infiltrating the glioma TME, tumor-associated microglia and macrophages (TAMs) constitute the major compartment. In patients with gliomas, increased TAM abundance is associated with more aggressive disease. Alterations in TAM phenotypes and functions have been reported in preclinical models of multiple cancers during tumor development and after therapeutic interventions, including radiotherapy and molecular targeted therapies. These findings indicate that it is crucial to evaluate TAM abundance and dynamics over time. Current techniques to quantify TAMs in patients rely mainly on histological staining of tumor biopsies. Although informative, these techniques require an invasive procedure to harvest the tissue sample and typically only result in a snapshot of a small region at a single point in time. Fluorine isotope 19 MRI ( 19 F MRI) represents a powerful means to noninvasively and longitudinally monitor myeloid cells in pathological conditions by intravenously injecting perfluorocarbon-containing nanoparticles (PFC-NP). In this study, we demonstrated the feasibility and power of 19 F MRI in preclinical models of gliomagenesis, breast-to-brain metastasis, and breast cancer and showed that the major cellular source of 19 F signal consists of TAMs. Moreover, multispectral 19 F MRI with two different PFC-NP allowed us to identify spatially and temporally distinct TAM niches in radiotherapy-recurrent murine gliomas. Together, we have imaged TAMs noninvasively and longitudinally with integrated cellular, spatial, and temporal resolution, thus revealing important biological insights into the critical functions of TAMs, including in disease recurrence.

Dendritic cells (DCs) induce peripheral T cell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral T cell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4

Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment.

Brain malignancies encompass a range of primary and metastatic cancers, including low-grade and high-grade gliomas and brain metastases (BrMs) originating from diverse extracranial tumors. Our understanding of the brain tumor microenvironment (TME) remains limited, and it is unknown whether it is sculpted differentially by primary versus metastatic disease. We therefore comprehensively analyzed the brain TME landscape via flow cytometry, RNA sequencing, protein arrays, culture assays, and spatial tissue characterization. This revealed disease-specific enrichment of immune cells with pronounced differences in proportional abundance of tissue-resident microglia, infiltrating monocyte-derived macrophages, neutrophils, and T cells. These integrated analyses also uncovered multifaceted immune cell activation within brain malignancies entailing converging transcriptional trajectories while maintaining disease- and cell-type-specific programs. Given the interest in developing TME-targeted therapies for brain malignancies, this comprehensive resource of the immune landscape offers insights into possible strategies to overcome tumor-supporting TME properties and instead harness the TME to fight cancer.

Glioblastoma is the most dangerous brain cancer. One reason for glioblastoma's aggressiveness are glioblastoma stem‐like cells. To target them, a number of markers have been proposed (CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6). A comprehensive study of co‐expression patterns of them has, however, not been performed so far. Here, we mapped the multidimensional co‐expression profile of these stemness‐associated molecules. Gliomaspheres – an established model of glioblastoma stem‐like cells – were used. Seven different gliomasphere systems were subjected to multicolor flow cytometry measuring the nine markers CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6 all simultaneously based on a novel 9‐marker multicolor panel developed for this study. The viSNE dimensionality reduction algorithm was applied for analysis. All gliomaspheres were found to express at least five different glioblastoma stem‐like cell markers. Multi‐dimensional analysis showed that all studied gliomaspheres consistently harbored a cell population positive for the molecular signature CD44+/CD133+/ITGA6+/CD36+. Glioblastoma patients with an enrichment of this combination had a significantly worse survival outcome when analyzing the two largest available The Cancer Genome Atlas datasets (MIT/Harvard Affymetrix: P = 0.0015, University of North Carolina Agilent: P = 0.0322). In sum, we detected a previously unknown marker combination – demonstrating feasibility, usefulness, and importance of high‐dimensional gliomasphere marker combinatorics.

Myeloid cells, including neutrophils, are important regulators of tumor growth and metastasis. In Science, Engblom et al. (2017) reveal how lung tumors remotely engage bone-resident cells through a relay mechanism that achieves a sustained supply of tumor-promoting neutrophils.

The brain is one of the most complex organs of the human body. For a long time, it was thought that the brain is hermetically sheltered from entry by invaders, including our body's white blood cells. The discovery that different brain cancers contain quite distinct landscapes of immune cells may help us better understand their development and devise novel and effective therapeutic strategies.

Dendritic cell (DC)–mediated inflammation induced via TLRs is promoted by MAPK-activated protein kinase (MK)-2, a substrate of p38 MAPK. In this study we show an opposing role of MK2, by which it consolidates immune regulatory functions in DCs through modulation of p38, ERK1/2-MAPK, and STAT3 signaling. During primary TLR/p38 signaling, MK2 mediates the inhibition of p38 activation and positively cross-regulates ERK1/2 activity, leading to a reduction of IL-12 and IL-1α/β secretion. Consequently, MK2 impairs secondary autocrine IL-1α signaling in DCs, which further decreases the IL-1α/p38 but increases the anti-inflammatory IL-10/STAT3 signaling route. Therefore, the blockade of MK2 activity enables human and murine DCs to strengthen proinflammatory effector mechanisms by promoting IL-1α–mediated Th1 effector functions in vitro. Furthermore, MK2-deficient DCs trigger Th1 differentiation and Ag-specific cytotoxicity in vivo. Finally, wild-type mice immunized with LPS in the presence of an MK2 inhibitor strongly accumulate Th1 cells in their lymph nodes. These observations correlate with a severe clinical course in DC-specific MK2 knockout mice compared with wild-type littermates upon induction of experimental autoimmune encephalitis. Our data suggest that MK2 exerts a profound anti-inflammatory effect that prevents DCs from prolonging excessive Th1 effector T cell functions and autoimmunity.

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year relative survival rate of 8% and is projected to be the second leading cause of cancer death by 2030, underscoring the urgency to develop new strategies to improve current therapeutic modalities for PDAC. Targeting pancreatic cancer stem cells (PCSCs), which are resistant to radiation and chemotherapy, is a promising strategy. A novel approach which can be readily clinically translated is to repurpose disulfiram (DSF), a drug for treating alcoholism, to target PCSCs. Chemoradiation or the combination of chemotherapy agents FOLFIRINOX, currently standard care for PDAC, can increase stemness in some established or primary PDAC cell lines. However, DSF in the presence of exogenously or endogenously supplied copper (Cu), when combined with chemotherapy or chemoradiation, targets both PCSCs and nonstem PDAC cells. Previously, we demonstrated that DSF/Cu effectively targets breast cancer stem cells in the context of fractionated radiation (FIR) by inhibiting the NF-κB–stemness gene pathway. Therefore, the hypothesis that PCSCs can be effectively targeted by incorporating DSF/Cu into the standard chemoradiation regimen consisting of 5-FU and FIR was investigated and found to be effective in vitro in targeting PCSCs, identified as either ALDHbright or CD24+/CD44+/ESA+ or sphere-forming cells, as well as nonstem PDAC cells. In vivo, the combination of IR+5-FU+DSF/Cu was more effective (72.46%) than either IR+5-FU (30.32%) or IR+FOLFIRINOX therapy (43.04%) in inhibiting growth of the mouse Panc02 tumor. These encouraging results provide a solid foundation for clinical trials to improve the outcomes of the current standard chemoradiation therapy regimen for PDAC.

On the occasion of the 75th anniversary of the first volume of “Progress in the Chemistry of Organic Natural Products” and in addition, the publication of Volume 100, a retrospective examination is taken from its beginnings and through the remarkable development of this book series. The starting point is the biography of the founder and long-term editor of the series, Laszlo Zechmeister (1889–1972). Zechmeister succeeded in getting the creme de la creme of the research elite in the area of natural product chemistry to contribute—of which no less than ten authors were to be awarded a Nobel Prize. Additional Nobel Prizes were awarded to the series editors Haworth and Butenandt. Since 1940, when Zechmeister emigrated from Pecs in Hungary to Pasadena in California, the center of gravity in terms of contributed articles gradually shifted from Europe to North America. From 1970 on, when the book series was no longer “Edited by Zechmeister” but rather “Founded by Zechmeister”, more and more of the published contributions originated further afield, including countries in Africa, Asia, Australia and New Zealand, and South America. From the beginning, “Progress in the Chemistry of Organic Natural Products” allotted much space to the application of new findings in regard to instrumental organic analytical techniques, such as chromatography, X-ray diffraction, mass spectrometry, nuclear magnetic resonance spectroscopy, IR-, UV-, vis-, and fluorescence spectroscopy. Upon publication of Volume 99 in 2014, 412 contributions were edited within this book series covering the whole spectrum of organic natural products. All these chapters are discussed briefly within this chronological retrospective, which is dedicated to an acknowledgement of their scientific value. Rapid development is evident for each branch of organic natural product chemistry covered—ranging from carotenoids, tobacco alkaloids, chlorinated compounds, coumarins, and flavones, to steroids and many other compound types.

The PI3K signaling cascade in APCs has been recognized as an essential pathway to initiate, maintain, and resolve immune responses. In this study, we demonstrate that a cell type–specific loss of the PI3K antagonist phosphatase and tensin homolog (PTEN) in myeloid cells renders APCs toward a regulatory phenotype. APCs deficient for PTEN exhibit reduced activation of p38 MAPK and reduced expression of T cell–polarizing cytokines. Furthermore, PTEN deficiency leads to upregulation of markers for alternative activation, such as Arginase 1, with concomitant downregulation of inducible NO synthase in APCs in vitro and in vivo. As a result, T cell polarization was dysfunctional in PTEN−/− APCs, in particular affecting the Th17 cell subset. Intriguingly, mice with cell type–specific deletions of PTEN-targeting APCs were protected from experimental autoimmune encephalomyelitis, which was accompanied by a pronounced reduction of IL-17– and IL-22–producing autoreactive T cells and reduced CNS influx of classically activated monocytes/macrophages. These observations support the notion that activation of the PI3K signaling cascade promotes regulatory APC properties and suppresses pathogenic T cell polarization, thereby reducing the clinical symptoms and pathology of experimental autoimmune encephalomyelitis.

The MAPK signaling pathway represents a central player in inflammatory processes. As a downstream target of p38, MAPK-activated protein kinase 2 (MK2) contributes to signal transduction regulating the expression and translation of various targets, among them several cytokines such as tumor necrosis factor (TNF) α. While MK2 was shown to promote an inflammatory macrophage phenotype, we have reported its Th1-attenuating function in dendritic cells (DCs). These observations of differential regulatory functions prompted us to investigate MK2 in the context of DCs and other myeloid cells in the tumor microenvironment. Murine DCs lacking MK2 activity exhibit an enhanced potential to differentiate Th1 cells upon Toll-like receptor (TLR) ligation in vitro and in vivo. In line with this finding, we observe significantly reduced B16.F10 melanoma growth in CD11cCre-MK2fl/fl mice upon DC activation by administration of lipopolysaccharide (LPS) together with whole tumor cell lysate as compared to wildtype littermate controls. Interestingly, CD11cCre-MK2fl/fl mice show an overall reduced myeloid cell tumor infiltration. Looking at MK2 expression in DCs isolated from orthotopic B16.F10 tumor-bearing mice, we find elevated MK2 levels in tumor-resident as opposed to splenic DCs, which has also been confirmed in a model of intracranial glioma (GL-261). This MK2 up-regulation in glioma-infiltrating DCs is associated with an enhanced expression of IL-10. Furthermore, tumor-resident myeloid-derived suppressor cells (MDSCs) show elevated MK2 expression, further underlining its potential involvement in immunosuppressive mechanisms in the myeloid lineage. In DCs we have observed cross-regulation of MAPK signaling by MK2, which promotes ERK1/2 while attenuating p38 activation and is further implicated to enhance STAT3 phosphorylation – all together contributing to a tolerogenic DC phenotype. Therefore we are now looking to unravel mechanisms by which MK2 impacts on signaling networks involved in myeloid immunosuppression in the tumor microenvironment. Our data contrast the previously described role of MK2 in pro-inflammatory mechanisms of the p38 signaling route and suggest an additional immunosuppressive feedback function in myeloid cells, which might be exploited by tumor cells to escape immune recognition and elimination. These indications are particularly interesting with regard to glioma, since the glioma microenvironment has been shown to be rich in various cytokines promoting favourable conditions for tumor growth. Finally, MK2 represents a promising target for combination therapy, as it has been reported to mediate chemo- as well as radioresistance in different types of tumors. Nevertheless, its function in tumor-infiltrating immune cells remains to be elucidated. We therefore aim to contribute to a deeper understanding of the complex interplay between extra- and intracellular signaling molecules regulating tumor as well as immune cell phenotypes. Citation Format: Klara Soukup, Angela Halfmann, Fiona Poyer, Katharina Martin, Bernadette Blauensteiner, Bastien Huber, Mario Kuttke, Gernot Schabbauer, Lydia Zopf, Jelena Zinnanti, Alexander Michael Dohnal. MAPK-activated protein kinase MK2 exerts immune regulatory functions in the microenvironment of orthotopic tumors. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A43.

In the current study we are investigating the effects of PTEN-deficient myeloid cells on tumor immune surveillance. We could previously show that hyper-activation of the PI3K signaling cascade by genetic knock-out of the counteracting phosphatase PTEN induced an anti-inflammatory phenotype in myeloid cells. This resulted in protection of conditional knock-out mice in models of acute infection and inflammation. A reduction in pro-inflammatory responses could however increase tumor burden. To address this question we induced colitis associated colon cancer in conditional PTEN-KO mice and found an increase in tumor burden and a reduction in survival in male KO mice. This was accompanied by increased numbers of splenic antigen-presenting cells (APC) expressing the immune checkpoint regulators PD-L1 and PD-L2. Furthermore, transcriptome analysis in these cells revealed a shift towards gene expression profiles found in professional APCs capable of cross-presentation. As expected, ex-vivo stimulated T-cells from KO-mice showed a reduction in proliferative capacity. These findings were further substantiated by findings in a second tumor model using implanted B16 melanoma cells. In this model myeloid PTEN-deficient mice showed a decrease in T-cell activation and a reduction in melanoma cell killing capacity. Taken together, our findings show that genetic deletion of PTEN in cells of myeloid origin increases splenic APCs expressing immune checkpoint regulators resulting in a decrease in tumor immune surveillance. Our study shows that PI3K-inhibitors which are currently tested as anti-cancer drugs might have additional beneficial effects on immune cells by shifting their inflammatory phenotype. Citation Format: Mario Kuttke, Emine Sahin, Julia Pisoni, Sophie Percig, Andrea Vogel, Daniel Kraemmer, Leslie Hanzl, Julia Stefanie Brunner, Hannah Paar, Klara Soukup, Angela Halfmann, Alexander Dohnal, Carl-Walter Steiner, Stephan Blüml, Jose Basilio, Bernhard Hochreiter, Manuel Salzmann, Bastian Hoesel, Günther Lametschwandtner, Robert Eferl, Johannes Schmid, Gernot Schabbauer. Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 527.

Dendritic cells (DCs) represent important mediators at the interface of innate and adaptive immunity. Our research focuses on the immune regulatory phenotype of DCs, which can be induced following stimulation via toll-like receptor ligands such as lipopolysaccharide (LPS). We identified the p38 substrate MAPK-activated protein kinase 2 (MK2) as a potential candidate molecule involved in directing DCs into the regulatory phenotype. As we are interested in enhancing effector T cell functions in the context of DC-based cancer immunotherapy, we investigated the role of MK2 in inflammation and immune regulation mediated by DCs using both conditional MK2 knock-out mice (CD11cCre-MK2fl/fl) and the MK2-specific small molecule kinase inhibitor MK2-I3. Bone marrow- and tissue-derived murine DCs lacking MK2 activity exhibit an enhanced potential to differentiate TH1 effector cells upon LPS stimulation. MK2-deficient DCs show an altered cytokine profile leading to significantly increased IL-12 along with reduced IL-10 secretion. These observations correlate with higher levels of phosphorylated p38 and a down-modulation of JAK/STAT3 signaling. Accordingly, numbers of IFN-γ producing CD4+ T cells, as well as proliferation and cytotoxicity of CD8+ T cells are elevated as demonstrated in vitro and in vivo using ovalbumin as a model antigen. In a murine experimental autoimmune encephalomyelitis (EAE) model, we saw severely enhanced disease progression in CD11cCre-MK2fl/fl mice compared to wildtype littermate controls. We found clinical EAE manifestation to be mediated via IFN-γ producing cells, as symptoms are abrogated upon administration of an IFN-γ blocking antibody. Finally, we studied effects of MK2 deficiency in DCs on tumor growth in murine transplantable B16 and MC-38 models, showing significantly reduced tumor growth in CD11cCre-MK2fl/fl mice upon DC activation by administration of LPS together with whole tumor cell lysate as compared to wildtype littermate controls. Furthermore, in a model of DC-based immunotherapy, LPS-activated MK2-I3-treated DCs exhibit enhanced capacity to reduce tumor burden after injection into wildtype tumor bearing mice. Our data contrast the previously described role of MK2 in pro-inflammatory mechanisms of the p38 signaling route and suggest an additional anti-inflammatory feedback function, which might be important in preventing DCs from prolonging excessive inflammation. Taken together, these findings provide novel strategies for the improvement of current DC-based tumor immunotherapy approaches by enhancing TH1-priming capacities of DCs. Citation Format: Klara Soukup, Angela Halfmann, Fiona Poyer, Thomas Felzmann, Alexander Michael Dohnal. MAPK-activated protein kinase MK2 attenuates TH1 differentiation mediated by dendritic cells – implications for autoimmunity and cancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B46.