The ins and outs of microglial cells in brain health and disease.

Microglia are the brain's resident macrophages that play pivotal roles in immune surveillance and maintaining homeostasis of the Central Nervous System (CNS). Microglia are functionally implicated in various cerebrovascular diseases, including stroke, aneurysm, and tumorigenesis as they regulate neuroinflammatory responses and tissue repair processes. Here, we review the manifold functions of microglia in the brain under physiological and pathological conditions, primarily focusing on the implication of microglia in glioma propagation and progression. We further review the current status of therapies targeting microglial cells, including their re-education, depletion, and re-population approaches as therapeutic options to improve patient outcomes for various neurological and neuroinflammatory disorders, including cancer.

An autophagy program that promotes T cell egress from the lymph node controls responses to immune checkpoint blockade.

Lymphatic endothelial cells (LECs) of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady-state and tumor-bearing conditions is lacking. Intravital imaging combined with immune phenotyping shows that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs by increasing the availability of the main egress signal sphingosine-1-phosphate. Single-cell RNA sequencing of tumor-draining LNs shows that loss of ATG5 remodels niche-specific LEC phenotypes involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC autophagy prevents recruitment of tumor-infiltrating T and natural killer cells and abrogates response to immunotherapy. Thus, an LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics.

Tumor endothelial cell autophagy is a key vascular-immune checkpoint in melanoma.

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.

Multiomics and spatial mapping characterizes human CD8+ T cell states in cancer.

Clinically relevant immunological biomarkers that discriminate between diverse hypofunctional states of tumor-associated CD8+ T cells remain disputed. Using multiomics analysis of CD8+ T cell features across multiple patient cohorts and tumor types, we identified tumor niche-dependent exhausted and other types of hypofunctional CD8+ T cell states. CD8+ T cells in "supportive" niches, like melanoma or lung cancer, exhibited features of tumor reactivity-driven exhaustion (CD8+ TEX). These included a proficient effector memory phenotype, an expanded T cell receptor (TCR) repertoire linked to effector exhaustion signaling, and a cancer-relevant T cell-activating immunopeptidome composed of largely shared cancer antigens or neoantigens. In contrast, "nonsupportive" niches, like glioblastoma, were enriched for features of hypofunctionality distinct from canonical exhaustion. This included immature or insufficiently activated T cell states, high wound healing signatures, nonexpanded TCR repertoires linked to anti-inflammatory signaling, high T cell-recognizable self-epitopes, and an antiproliferative state linked to stress or prodeath responses. In situ spatial mapping of glioblastoma highlighted the prevalence of dysfunctional CD4+:CD8+ T cell interactions, whereas ex vivo single-cell secretome mapping of glioblastoma CD8+ T cells confirmed negligible effector functionality and a promyeloid, wound healing-like chemokine profile. Within immuno-oncology clinical trials, anti-programmed cell death protein 1 (PD-1) immunotherapy facilitated glioblastoma's tolerogenic disparities, whereas dendritic cell (DC) vaccines partly corrected them. Accordingly, recipients of a DC vaccine for glioblastoma had high effector memory CD8+ T cells and evidence of antigen-specific immunity. Collectively, we provide an atlas for assessing different CD8+ T cell hypofunctional states in immunogenic versus nonimmunogenic cancers.

High endothelial venules in cancer: Regulation, function, and therapeutic implication.

The lack of sufficient intratumoral CD8+ T lymphocytes is a significant obstacle to effective immunotherapy in cancer. High endothelial venules (HEVs) are organ-specific and specialized postcapillary venules uniquely poised to facilitate the transmigration of lymphocytes to lymph nodes (LNs) and other secondary lymphoid organs (SLOs). HEVs can also form in human and murine cancer (tumor HEVs [TU-HEVs]) and contribute to the generation of diffuse T cell-enriched aggregates or tertiary lymphoid structures (TLSs), which are commonly associated with a good prognosis. Thus, therapeutic induction of TU-HEVs may provide attractive avenues to induce and sustain the efficacy of immunotherapies by overcoming the major restriction of T cell exclusion from the tumor microenvironment. In this review, we provide current insight into the commonalities and discrepancies of HEV formation and regulation in LNs and tumors and discuss the specific function and significance of TU-HEVs in eliciting, predicting, and aiding anti-tumoral immunity.

GBM tumors are heterogeneous in their fatty acid metabolism and modulating fatty acid metabolism sensitizes cancer cells derived from recurring GBM tumors to temozolomide.

Glioblastoma is a highly lethal grade of astrocytoma with very low median survival. Despite extensive efforts, there is still a lack of alternatives that might improve these prospects. We uncovered that the chemotherapeutic agent temozolomide impinges on fatty acid synthesis and desaturation in newly diagnosed glioblastoma. This response is, however, blunted in recurring glioblastoma from the same patient. Further, we describe that disrupting cellular fatty acid homeostasis in favor of accumulation of saturated fatty acids such as palmitate synergizes with temozolomide treatment. Pharmacological inhibition of SCD and/or FADS2 allows palmitate accumulation and thus greatly augments temozolomide efficacy. This effect was independent of common GBM prognostic factors and was effective against cancer cells from recurring glioblastoma. In summary, we provide evidence that intracellular accumulation of saturated fatty acids in conjunction with temozolomide based chemotherapy induces death in glioblastoma cells derived from patients.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of T cell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTßR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 T cell progenitors that differentiate into GrzB+PD1+ CD8 T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

A cellular hierarchy in melanoma uncouples growth and metastasis.

Although melanoma is notorious for its high degree of heterogeneity and plasticity1,2, the origin and magnitude of cell-state diversity remains poorly understood. Equally, it is unclear whether growth and metastatic dissemination are supported by overlapping or distinct melanoma subpopulations. Here, by combining mouse genetics, single-cell and spatial transcriptomics, lineage tracing and quantitative modelling, we provide evidence of a hierarchical model of tumour growth that mirrors the cellular and molecular logic underlying the cell-fate specification and differentiation of the embryonic neural crest. We show that tumorigenic competence is associated with a spatially localized perivascular niche, a phenotype acquired through an intercellular communication pathway established by endothelial cells. Consistent with a model in which only a fraction of cells are fated to fuel growth, temporal single-cell tracing of a population of melanoma cells with a mesenchymal-like state revealed that these cells do not contribute to primary tumour growth but, instead, constitute a pool of metastatic initiating cells that switch cell identity while disseminating to secondary organs. Our data provide a spatially and temporally resolved map of the diversity and trajectories of melanoma cell states and suggest that the ability to support growth and metastasis are limited to distinct pools of cells. The observation that these phenotypic competencies can be dynamically acquired after exposure to specific niche signals warrant the development of therapeutic strategies that interfere with the cancer cell reprogramming activity of such microenvironmental cues.

Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis.

Autophagy has vasculoprotective roles, but whether and how it regulates lymphatic endothelial cells (LEC) homeostasis and lymphangiogenesis is unknown. Here, we show that genetic deficiency of autophagy in LEC impairs responses to VEGF-C and injury-driven corneal lymphangiogenesis. Autophagy loss in LEC compromises the expression of main effectors of LEC identity, like VEGFR3, affects mitochondrial dynamics and causes an accumulation of lipid droplets (LDs) in vitro and in vivo. When lipophagy is impaired, mitochondrial ATP production, fatty acid oxidation, acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes are dwindled. Enforcing mitochondria fusion by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC fails to restore LDs turnover and lymphatic gene expression, whereas supplementing the fatty acid precursor acetate rescues VEGFR3 levels and signaling, and lymphangiogenesis in LEC-Atg5-/- mice. Our findings reveal that lipophagy in LEC by supporting FAO, preserves a mitochondrial-PROX1 gene expression circuit that safeguards LEC responsiveness to lymphangiogenic mediators and lymphangiogenesis.

LGL1 binds to Integrin ß1 and inhibits downstream signaling to promote epithelial branching in the mammary gland.

Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin ß1 and inhibits its downstream signaling, and Integrin ß1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin ß1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.

High Endothelial Venules: A Vascular Perspective on Tertiary Lymphoid Structures in Cancer.

High endothelial venules (HEVs) are specialized postcapillary venules composed of cuboidal blood endothelial cells that express high levels of sulfated sialomucins to bind L-Selectin/CD62L on lymphocytes, thereby facilitating their transmigration from the blood into the lymph nodes (LN) and other secondary lymphoid organs (SLO). HEVs have also been identified in human and murine tumors in predominantly CD3+T cell-enriched areas with fewer CD20+B-cell aggregates that are reminiscent of tertiary lymphoid-like structures (TLS). While HEV/TLS areas in human tumors are predominantly associated with increased survival, tumoral HEVs (TU-HEV) in mice have shown to foster lymphocyte-enriched immune centers and boost an immune response combined with different immunotherapies. Here, we discuss the current insight into TU-HEV formation, function, and regulation in tumors and elaborate on the functional implication, opportunities, and challenges of TU-HEV formation for cancer immunotherapy.

The metabolism of cancer cells during metastasis.

Metastasis formation is the major cause of death in most patients with cancer. Despite extensive research, targeting metastatic seeding and colonization is still an unresolved challenge. Only recently, attention has been drawn to the fact that metastasizing cancer cells selectively and dynamically adapt their metabolism at every step during the metastatic cascade. Moreover, many metastases display different metabolic traits compared with the tumours from which they originate, enabling survival and growth in the new environment. Consequently, the stage-dependent metabolic traits may provide therapeutic windows for preventing or reducing metastasis, and targeting the new metabolic traits arising in established metastases may allow their eradication.

Tumors vs. Chronic Wounds: An Immune Cell's Perspective.

The wound repair program is tightly regulated and coordinated among different cell constituents including epithelial cells, fibroblasts, immune cells and endothelial cells following consecutive steps to ensure timely, and proper wound closure. Specifically, innate and adaptive immune cells are pivotal participants that also closely interact with the vasculature. Tumors are portrayed as wounds that do not heal because they undergo continuous stromal remodeling and vascular growth with immunosuppressive features to ensure tumor propagation; a stage that is reminiscent of the proliferative resolution phase in wound repair. There is increasing evidence from mouse model systems and clinical trials that targeting both the immune and vascular compartments is an attractive therapeutic approach to reawaken the inflammatory status in the "tumor wound" with the final goal to abrogate tumor cells and invigorate tissue homeostasis. In this review, we compare the implication of immune cells and the vasculature in chronic wounds and tumor wounds to underscore the conceptual idea of transitioning tumors into an inflammatory wound-like state with antiangiogenic immunotherapies to improve beneficial effects in cancer patients.

Peptide-guided nanoparticles for glioblastoma targeting.

Tumor-selective drug conjugates can potentially improve the prognosis for patients affected by glioblastoma (GBM) - the most common and malignant type of brain cancer with no effective cure. Here we evaluated a novel tumor penetrating peptide that targets cell surface p32, LinTT1 (AKRGARSTA), as a GBM targeting ligand for systemically-administered nanoparticles. LinTT1-functionalization increased tumor homing of iron oxide nanoworms (NWs) across a panel of five GBM models ranging from infiltratively-disseminating to angiogenic phenotypes. LinTT1-NWs homed to CD31-positive tumor blood vessels, including to transdifferentiated endothelial cells, and showed co-localization with tumor macrophages and lymphatic vessels. LinTT1 functionalization also resulted in increased GBM delivery of other types of systemically-administered nanoparticles: silver nanoparticles and albumin-paclitaxel nanoparticles. Finally, LinTT1-guided proapoptotic NWs exerted strong anti-glioma activity in two models of GBM, including doubling the lifespan of the mice in an aggressive orthotopic stem cell-like GBM that recapitulates the histological hallmarks of human GBM. Our study suggests that LinTT1 targeting strategy can be used to increase GBM uptake of systemic nanoparticles for improved imaging and therapy.

Regulation of Blood and Lymphatic Vessels by Immune Cells in Tumors and Metastasis.

Research over the last decades has provided strong evidence for the pivotal role of the tumor-associated blood and lymphatic vasculature in supporting immunoevasion and in subverting T cell-mediated immunosurveillance. Conversely, tumor blood and lymphatic vessel growth is in part regulated by the immune system, with infiltrating innate as well as adaptive immune cells providing both immunosuppressive and various angiogenic signals. Thus, tumor angiogenesis and escape of immunosurveillance are two cancer hallmarks that are tightly linked and interregulated by cell constituents from compartments secreting both chemokines and cytokines. In this review, we discuss the implication and regulation of innate and adaptive immune cells in regulating blood and lymphatic angiogenesis in tumor progression and metastases. Moreover, we also highlight novel therapeutic approaches that target the tumor vasculature as well as the immune compartment to sustain and improve therapeutic efficacy in cancer.

Where Have All the T Cells Gone?

Glioblastoma are highly immunosuppressive brain tumors that are known for their T cell paucity. In a recent issue of Nature Medicine, Chongsathidkiet et al. (2018) discovered a brain-specific mechanism of tumors to escape immunosurveillance by trapping T cells in the bone marrow through the loss of sphingosine-1-phosphate (S1P) receptor on the T cell surface.

A tension-mediated glycocalyx-integrin feedback loop promotes mesenchymal-like glioblastoma.

Glioblastoma multiforme (GBMs) are recurrent lethal brain tumours. Recurrent GBMs often exhibit mesenchymal, stem-like phenotypes that could explain their resistance to therapy. Analyses revealed that recurrent GBMs have increased tension and express high levels of glycoproteins that increase the bulkiness of the glycocalyx. Studies showed that a bulky glycocalyx potentiates integrin mechanosignalling and tissue tension and promotes a mesenchymal, stem-like phenotype in GBMs. Gain- and loss-of-function studies implicated integrin mechanosignalling as an inducer of GBM growth, survival, invasion and treatment resistance, and a mesenchymal, stem-like phenotype. Mesenchymal-like GBMs were highly contractile and expressed elevated levels of glycoproteins that expanded their glycocalyx, and they were surrounded by a stiff extracellular matrix that potentiated integrin mechanosignalling. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignalling and a bulky glycocalyx, implying a causal link towards a mesenchymal, stem-like phenotype in GBMs. Strategies to ameliorate GBM tissue tension offer a therapeutic approach to reduce mortality due to GBM.