Interrogation of endothelial and mural cells in brain metastasis reveals key immune-regulatory mechanisms.

Brain metastasis (BrM) is a common malignancy, predominantly originating from lung, melanoma, and breast cancers. The vasculature is a key component of the BrM tumor microenvironment with critical roles in regulating metastatic seeding and progression. However, the heterogeneity of the major BrM vascular components, namely endothelial and mural cells, is still poorly understood. We perform single-cell and bulk RNA-sequencing of sorted vascular cell types and detect multiple subtypes enriched specifically in BrM compared to non-tumor brain, including previously unrecognized immune regulatory subtypes. We integrate the human data with mouse models, creating a platform to interrogate vascular targets for the treatment of BrM. We find that the CD276 immune checkpoint molecule is significantly upregulated in the BrM vasculature, and anti-CD276 blocking antibodies prolonged survival in preclinical trials. This study provides important insights into the complex interactions between the vasculature, immune cells, and cancer cells, with translational relevance for designing therapeutic interventions.

Compromised Blood-Brain Barrier Junctions Enhance Melanoma Cell Intercalation and Extravasation.

Melanoma frequently metastasises to the brain, and a detailed understanding of the molecular and cellular mechanisms underlying melanoma cell extravasation across the blood-brain barrier (BBB) is important for preventing brain metastasis formation. Making use of primary mouse brain microvascular endothelial cells (pMBMECs) as an in vitro BBB model, we imaged the interaction of melanoma cells into pMBMEC monolayers. We observed exclusive junctional intercalation of melanoma cells and confirmed that melanoma-induced pMBMEC barrier disruption can be rescued by protease inhibition. Interleukin (IL)-1ß stimulated pMBMECs or PECAM-1-knockout (-ko) pMBMECs were employed to model compromised BBB barrier properties in vitro and to determine increased melanoma cell intercalation compared to pMBMECs with intact junctions. The newly generated brain-homing melanoma cell line YUMM1.1-BrM4 was used to reveal increased in vivo extravasation of melanoma cells across the BBB of barrier-compromised PECAM-1-deficient mice compared to controls. Taken together, our data indicate that preserving BBB integrity is an important measure to limit the formation of melanoma-brain metastasis.

The local microenvironment drives activation of neutrophils in human brain tumors.

Neutrophils are abundant immune cells in the circulation and frequently infiltrate tumors in substantial numbers. However, their precise functions in different cancer types remain incompletely understood, including in the brain microenvironment. We therefore investigated neutrophils in tumor tissue of glioma and brain metastasis patients, with matched peripheral blood, and herein describe the first in-depth analysis of neutrophil phenotypes and functions in these tissues. Orthogonal profiling strategies in humans and mice revealed that brain tumor-associated neutrophils (TANs) differ significantly from blood neutrophils and have a prolonged lifespan and immune-suppressive and pro-angiogenic capacity. TANs exhibit a distinct inflammatory signature, driven by a combination of soluble inflammatory mediators including tumor necrosis factor alpha (TNF-ɑ) and Ceruloplasmin, which is more pronounced in TANs from brain metastasis versus glioma. Myeloid cells, including tumor-associated macrophages, emerge at the core of this network of pro-inflammatory mediators, supporting the concept of a critical myeloid niche regulating overall immune suppression in human brain tumors.

Phenotypic diversity of T cells in human primary and metastatic brain tumors revealed by multiomic interrogation.

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.

Immunogenomic analysis of human brain metastases reveals diverse immune landscapes across genetically distinct tumors.

Brain metastases (BrMs) are the most common form of brain tumors in adults and frequently originate from lung and breast primary cancers. BrMs are associated with high mortality, emphasizing the need for more effective therapies. Genetic profiling of primary tumors is increasingly used as part of the effort to guide targeted therapies against BrMs, and immune-based strategies for the treatment of metastatic cancer are gaining momentum. However, the tumor immune microenvironment (TIME) of BrM is extremely heterogeneous, and whether specific genetic profiles are associated with distinct immune states remains unknown. Here, we perform an extensive characterization of the immunogenomic landscape of human BrMs by combining whole-exome/whole-genome sequencing, RNA sequencing of immune cell populations, flow cytometry, immunofluorescence staining, and tissue imaging analyses. This revealed unique TIME phenotypes in genetically distinct lung- and breast-BrMs, thereby enabling the development of personalized immunotherapies tailored by the genetic makeup of the tumors.

Multispectral fluorine-19 MRI enables longitudinal and noninvasive monitoring of tumor-associated macrophages.

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 (19F 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 19F MRI in preclinical models of gliomagenesis, breast-to-brain metastasis, and breast cancer and showed that the major cellular source of 19F signal consists of TAMs. Moreover, multispectral 19F 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.

PI3K Signaling in Dendritic Cells Aggravates DSS-Induced Colitis.

Aberrant innate immune responses to the gut microbiota are causally involved in the pathogenesis of inflammatory bowel diseases (IBD). The exact triggers and main signaling pathways activating innate immune cells and how they modulate adaptive immunity in IBD is still not completely understood. Here, we report that the PI3K/PTEN signaling pathway in dendritic cells enhances IL-6 production in a model of DSS-induced colitis. This results in exacerbated Th1 cell responses and increased mortality in DC-specific PTEN knockout (PTENΔDC) animals. Depletion of the gut microbiota using antibiotics as well as blocking IL-6R signaling rescued mortality in PTENΔDC mice, whereas adoptive transfer of Flt3L-derived PTEN-/- DCs into WT recipients exacerbated DSS-induced colitis and increased mortality. Taken together, we show that the PI3K signaling pathway in dendritic cells contributes to disease pathology by promoting IL-6 mediated Th1 responses.

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory T cell induction.

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+ T cell expansion, less regulatory T cells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ T cells into Tregs in vitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

An integrated pipeline for comprehensive analysis of immune cells in human brain tumor clinical samples.

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.

Interrogation of the Microenvironmental Landscape in Brain Tumors Reveals Disease-Specific Alterations of Immune Cells.

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.

Gliomasphere marker combinatorics: multidimensional flow cytometry detects CD44+/CD133+/ITGA6+/CD36+ signature.

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.

A Long-Distance Relay-tionship between Tumor and Bone.

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.

CD28 Blockade Ex Vivo Induces Alloantigen-Specific Immune Tolerance but Preserves T-Cell Pathogen Reactivity.

Donor T-cells contribute to reconstitution of protective immunity after allogeneic hematopoietic stem cell transplantation (HSCT) but must acquire specific tolerance against recipient alloantigens to avoid life-threatening graft-versus-host disease (GvHD). Systemic immunosuppressive drugs may abrogate severe GvHD, but this also impedes memory responses to invading pathogens. Here, we tested whether ex vivo blockade of CD28 co-stimulation can enable selective T-cell tolerization to alloantigens by facilitating CD80/86-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) signaling. Treatment of human allogeneic dendritic cell/T-cell co-cultures with a human CD28 blocking antibody fragment (α-huCD28) significantly abrogated subsequent allospecific immune responses, seen by decreased T-cell proliferation and of type 1 cytokine (IFN-γ and IL-2) expression. Allo-tolerization persisted after discontinuation of CD28 blockade and secondary alloantigen stimulation, as confirmed by enhanced CTLA-4 and PD-1 immune checkpoint signaling. However, T-cells retained reactivity to pathogens, supported by clonotyping of neo-primed and cross-reactive T-cells specific for Candida albicans or third-party antigens using deep sequencing analysis. In an MHC-mismatched murine model, we tolerized C57BL/6 T-cells by ex vivo exposure to a murine single chain Fv specific for CD28 (α-muCD28). Infusion of these cells, after α-muCD28 washout, into bone marrow-transplanted BALB/c mice caused allo-tolerance and did not induce GvHD-associated hepatic pathology. We conclude that selective CD28 blockade ex vivo can allow the generation of stably allo-tolerized T-cells that in turn do not induce graft-versus-host reactions while maintaining pathogen reactivity. Hence, CD28 co-stimulation blockade of donor T-cells may be a useful therapeutic approach to support the immune system after HSCT.

A novel chemoradiation targeting stem and nonstem pancreatic cancer cells by repurposing disulfiram.

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.

Loss of MAPK-activated protein kinase 2 enables potent dendritic cell-driven anti-tumour T cell response.

Maintaining dendritic cells (DC) in a state of dysfunction represents a key mechanism by which tumour cells evade recognition and elimination by the immune system. Limited knowledge about the intracellular mediators of DC dysfunction restricts success of therapies aimed at reactivating a DC-driven anti-tumour immune response. Using a cell type-specific murine knock-out model, we have identified MAPK-activated protein kinase 2 (MK2) as a major guardian of a suppressive DC phenotype in the melanoma tumour microenvironment. MK2 deletion in CD11c+ cells led to an expansion of stimulatory CD103+ DCs, mounting a potent CD8+ T cell response that resulted in elimination of highly aggressive B16-F10 tumours upon toll-like receptor (TLR) activation in the presence of tumour antigen. Moreover, tumour infiltration by suppressive myeloid cells was strongly diminished. These insights into the regulation of DC functionality reveal MK2 as a targetable pathway for DC-centred immunomodulatory cancer therapies.

Loss of Phosphatase and Tensin Homolog in APCs Impedes Th17-Mediated Autoimmune Encephalomyelitis.

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-Activated Kinase MK2 Attenuates Dendritic Cell-Mediated Th1 Differentiation and Autoimmune Encephalomyelitis.

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/ß 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.

Radiation meets immunotherapy - a perfect match in the era of combination therapy?

PURPOSE: This review focuses on recent advances in the field of combining radiation with immunotherapy for the treatment of malignant diseases, since various combinatorial cancer therapy approaches have lately proven highly successful. RESULTS: With initial case reports and anecdotes progressively converting into solid clinical data, interest in cancer immunotherapy (CIT) has risen steeply. Especially immune checkpoint blockade therapies have recently celebrated tremendous successes in the treatment of severe malignancies resistant to conventional treatment strategies. Nevertheless, the high variability of patient responses to CIT remains a major hurdle, clearly indicating an urgent need for improvement. It has been suggested that successful cancer therapy most probably involves combinatorial treatment approaches. Radiotherapy (RT) has been proposed as a powerful partner for CIT due to its broad spectrum of immune modulatory characteristics. Several preclinical studies, supported by an increasing number of clinical observations, have demonstrated synergistic interactions between RT and CIT resulting in significantly improved therapy outcomes. CONCLUSIONS: Numerous reports have shown that radiation is capable of tipping the scales from tumor immune evasion to elimination in different tumor types. The next puzzle to be solved is the question of logistics - including types, schedule and dosage of combinatorial RT and CIT strategies.