Macrophage-mediated myelin recycling fuels brain cancer malignancy.

Tumors growing in metabolically challenged environments, such as glioblastoma in the brain, are particularly reliant on crosstalk with their tumor microenvironment (TME) to satisfy their high energetic needs. To study the intricacies of this metabolic interplay, we interrogated the heterogeneity of the glioblastoma TME using single-cell and multi-omics analyses and identified metabolically rewired tumor-associated macrophage (TAM) subpopulations with pro-tumorigenic properties. These TAM subsets, termed lipid-laden macrophages (LLMs) to reflect their cholesterol accumulation, are epigenetically rewired, display immunosuppressive features, and are enriched in the aggressive mesenchymal glioblastoma subtype. Engulfment of cholesterol-rich myelin debris endows subsets of TAMs to acquire an LLM phenotype. Subsequently, LLMs directly transfer myelin-derived lipids to cancer cells in an LXR/Abca1-dependent manner, thereby fueling the heightened metabolic demands of mesenchymal glioblastoma. Our work provides an in-depth understanding of the immune-metabolic interplay during glioblastoma progression, thereby laying a framework to unveil targetable metabolic vulnerabilities in glioblastoma.

Rapid Autopsy to Define Dendritic Cell Spatial Distribution and T Cell Association in Lung Adenocarcinoma.

Immunotherapy response is associated with the presence of conventional dendritic cells (cDCs). cDC type 1 (cDC1) is critically important for CD8+ T cell activation, cDC type 2 (cDC2) regulates CD4+ T cell responses, and mature regulatory cDCs may dampen T cell responses in the tumor microenvironment (TME). However, we lack a clear understanding of cDC distribution in the human TME, cDC prevalence in metastatic sites, and cDC differences in early- versus late-stage disease. Rapid autopsy specimens of 10 patients with lung adenocarcinoma were evaluated to detect cDCs and immune cells via multiplex immunofluorescence using 18 markers and 42 tumors. First, we found that T cells, cDC1, and cDC2 were confined to stroma, whereas mature regulatory DCs were enriched in tumor, suggesting unique localization-specific functions. Second, lung and lymph node tumors were more enriched in T cells and cDCs than liver tumors, underscoring differences in the TME of metastatic sites. Third, although the proportion of T cells and cDC1 did not differ in different stages, an increase in the proportion of cDC2 and macrophages in late stage suggests potential differences in regulation of T cell responses in different stages. Collectively, these findings provide new, to our knowledge, insights into cDC biology in human cancer that may have important therapeutic implications.

Grading of lung adenocarcinomas with simultaneous segmentation by artificial intelligence (GLASS-AI).

Preclinical genetically engineered mouse models (GEMMs) of lung adenocarcinoma are invaluable for investigating molecular drivers of tumor formation, progression, and therapeutic resistance. However, histological analysis of these GEMMs requires significant time and training to ensure accuracy and consistency. To achieve a more objective and standardized analysis, we used machine learning to create GLASS-AI, a histological image analysis tool that the broader cancer research community can utilize to grade, segment, and analyze tumors in preclinical models of lung adenocarcinoma. GLASS-AI demonstrates strong agreement with expert human raters while uncovering a significant degree of unreported intratumor heterogeneity. Integrating immunohistochemical staining with high-resolution grade analysis by GLASS-AI identified dysregulation of Mapk/Erk signaling in high-grade lung adenocarcinomas and locally advanced tumor regions. Our work demonstrates the benefit of employing GLASS-AI in preclinical lung adenocarcinoma models and the power of integrating machine learning and molecular biology techniques for studying the molecular pathways that underlie cancer progression.

Combination IFNß and Membrane-Stable CD40L Maximize Tumor Dendritic Cell Activation and Lymph Node Trafficking to Elicit Systemic T-cell Immunity.

Oncolytic virus therapies induce the direct killing of tumor cells and activation of conventional dendritic cells (cDC); however, cDC activation has not been optimized with current therapies. We evaluated the adenoviral delivery of engineered membrane-stable CD40L (MEM40) and IFNß to locally activate cDCs in mouse tumor models. Combined tumor MEM40 and IFNß expression induced the highest cDC activation coupled with increased lymph node migration, increased systemic antitumor CD8+ T-cell responses, and regression of established tumors in a cDC1-dependent manner. MEM40 + IFNß combined with checkpoint inhibitors led to effective control of distant tumors and lung metastases. An oncolytic adenovirus (MEM-288) expressing MEM40 + IFNß  in phase I clinical testing induced cancer cell loss concomitant with enhanced T-cell infiltration and increased systemic presence of tumor T-cell clonotypes in non-small cell lung cancer (NSCLC) patients. This approach to simultaneously target two major DC-activating pathways has the potential to significantly affect the solid tumor immunotherapy landscape.

Cell death, therapeutics, and the immune response in cancer.

Induction of cell death is inexorably linked with cancer therapy, but this can also initiate wound-healing processes that have been linked to cancer progression and therapeutic resistance. Here we describe the contribution of apoptosis and the lytic cell death pathways in the response to therapy (including chemotherapy and immunotherapy). We also discuss how necroptosis, pyroptosis, and ferroptosis function to promote tumor immunogenicity, along with emerging findings that these same forms of death can paradoxically contribute to immune suppression and tumor progression. Understanding the duality of cell death in cancer may allow for the development of therapeutics that shift the balance towards regression.

Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunotherapy efficacy.

Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.

Cytokines drive prostate cancer lineage plasticity.

Lineage plasticity is a critical mechanism of therapeutic resistance in cancer. In a recent issue of Science, Chan and colleagues demonstrate that early lineage plasticity in prostate cancer is driven by JAK-STAT inflammatory cytokine signaling.

Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and type I interferon to promote anti-tumor T cell responses.

Activation of unfolded protein responses (UPRs) in cancer cells undergoing endoplasmic reticulum (ER) stress promotes survival. However, how UPR in tumor cells impacts anti-tumor immune responses remains poorly described. Here, we investigate the role of the UPR mediator pancreatic ER kinase (PKR)-like ER kinase (PERK) in cancer cells in the modulation of anti-tumor immunity. Deletion of PERK in cancer cells or pharmacological inhibition of PERK in melanoma-bearing mice incites robust activation of anti-tumor T cell immunity and attenuates tumor growth. PERK elimination in ER-stressed malignant cells triggers SEC61ß-induced paraptosis, thereby promoting immunogenic cell death (ICD) and systemic anti-tumor responses. ICD induction in PERK-ablated tumors stimulates type I interferon production in dendritic cells (DCs), which primes CCR2-dependent tumor trafficking of common-monocytic precursors and their intra-tumor commitment into monocytic-lineage inflammatory Ly6C+CD103+ DCs. These findings identify how tumor cell-derived PERK promotes immune evasion and highlight the potential of PERK-targeting therapies in cancer immunotherapy.

LAG3 ectodomain structure reveals functional interfaces for ligand and antibody recognition.

The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible 'loop 2' region in LAG3 domain 1. We also defined the LAG3-FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.

Detection of exogenous DNA uptake by murine dendritic cells.

This protocol has been developed to measure exogenous DNA uptake by murine dendritic cells (DCs) using supernatant containing cellular debris, which allows for DNA uptake in the absence of transfection reagents. Inhibitors or antibodies that alter the process can be added, and either flow cytometry or fluorescent microscopy can be used to measure DNA uptake. This is intended to mimic the exposure of DCs to dying cells in the tumor microenvironment or other pathological conditions of high cellular death. For complete details on the use and execution of this protocol, please refer to de Mingo Pulido et al. (2021).

Metabolism in tumor-associated macrophages.

Macrophages functionally adapt to a diverse set of signals, a process that is critical for their role in maintaining or restoring tissue homeostasis. This process extends to cancer, where macrophages respond to a series of inflammatory and metabolic cues that direct a maladaptive healing response. Tumor-associated macrophages (TAMs) have altered glucose, amino acid, and lipid metabolic profiles, and interfering with this metabolic shift can blunt the ability of macrophages to promote tumor growth, metastasis, and the creation of an immunosuppressive microenvironment. Here we will review changes in metabolites and metabolic pathways in TAMs and link these with the phenotypic and functional properties of the cells. We will also discuss current strategies targeting TAM metabolism as a therapeutic intervention in cancer.

TIM-3 blockade enhances IL-12-dependent antitumor immunity by promoting CD8+ T cell and XCR1+ dendritic cell spatial co-localization.

BACKGROUND: T cell immunoglobulin and mucin domain containing-3 (TIM-3) blocking antibodies are currently being evaluated in clinical trials for solid and hematological malignancies. Despite its identification on T cells, TIM-3 is predominantly expressed by myeloid cells, including XCR1+ type I conventional dendritic cells (cDC1s). We have recently shown that TIM-3 blockade promotes expression of CXCR3 chemokine ligands by tumor cDCs, but how this drives a CD8+ T cell-dependent response to therapy is unclear. METHODS: T cell infiltration, effector function, and spatial localization in relation to XCR1+ cDC1s were evaluated in a murine orthotopic mammary carcinoma model during response to TIM-3 blockade and paclitaxel chemotherapy. Mixed bone marrow chimeras and diphtheria toxin depletion were used to determine the role of specific genes in cDC1s during therapeutic responses. RESULTS: TIM-3 blockade increased interferon-γ expression by CD8+ T cells without altering immune infiltration. cDC1 expression of CXCL9, but not CXCL10, was required for response to TIM-3 blockade. CXCL9 was also necessary for the increased proximity observed between CD8+ T cells and XCR1+ cDC1s during therapy. Tumor responses were dependent on cDC1 expression of interleukin-12, but not MHCI. CONCLUSIONS: TIM-3 blockade increases exposure of intratumoral CD8+ T cells to cDC1-derived cytokines, with implications for the design of therapeutic strategies using antibodies against TIM-3.

Cavity macrophages stop anti-tumor T cells.

Precisely how macrophages regulate response to immunotherapy remains unclear. In this issue of Cancer Cell, Chow et al. demonstrate a tumor-promoting role of TIM-4+ cavity macrophages which affects the response of metastatic malignancies to immunotherapy. Specifically, TIM-4+ macrophages engage with phosphatidylserine-expressing cytotoxic T lymphocytes, inhibiting their activity even during PD-1 blockade.

Macrophage-Derived Cholesterol Contributes to Therapeutic Resistance in Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is a lethal stage of disease in which androgen receptor (AR) signaling is persistent despite androgen deprivation therapy (ADT). Most studies have focused on investigating cell-autonomous alterations in CRPC, while the contributions of the tumor microenvironment are less well understood. Here we sought to determine the role of tumor-associated macrophages in CRPC, based upon their role in cancer progression and therapeutic resistance. In a syngeneic model that reflected the mutational landscape of CRPC, macrophage depletion resulted in a reduced transcriptional signature for steroid and bile acid synthesis, indicating potential perturbation of cholesterol metabolism. As cholesterol is the precursor of the five major types of steroid hormones, we hypothesized that macrophages were regulating androgen biosynthesis within the prostate tumor microenvironment. Macrophage depletion reduced androgen levels within prostate tumors and restricted AR nuclear localization in vitro and in vivo. Macrophages were also cholesterol-rich and were able to transfer cholesterol to tumor cells in vitro. AR nuclear translocation was inhibited by activation of liver X receptor (LXR)-ß, the master regulator of cholesterol homeostasis. Consistent with these data, macrophage depletion extended survival during ADT and the presence of macrophages correlated with therapeutic resistance in patient-derived explants. Taken together, these findings support the therapeutic targeting of macrophages in CRPC. SIGNIFICANCE: These results suggest that macrophage-targeted therapies can be combined with androgen deprivation therapy to treat patients with prostate cancer by limiting cholesterol bioavailability and the production of intratumoral androgens.See related commentary by Al-Janabi and Lewis, p. 5399.

The inhibitory receptor TIM-3 limits activation of the cGAS-STING pathway in intra-tumoral dendritic cells by suppressing extracellular DNA uptake.

Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1+ classical dendritic cells (cDC1), thereby limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.

Tumour-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

Dendritic Cells and Their Role in Immunotherapy.

Despite significant advances in the field of cancer immunotherapy, the majority of patients still do not benefit from treatment and must rely on traditional therapies. Dendritic cells have long been a focus of cancer immunotherapy due to their role in inducing protective adaptive immunity, but cancer vaccines have shown limited efficacy in the past. With the advent of immune checkpoint blockade and the ability to identify patient-specific neoantigens, new vaccines, and combinatorial therapies are being evaluated in the clinic. Dendritic cells are also emerging as critical regulators of the immune response within tumors. Understanding how to augment the function of these intratumoral dendritic cells could offer new approaches to enhance immunotherapy, in addition to improving the cytotoxic and targeted therapies that are partially dependent upon a robust immune response for their efficacy. Here we will discuss the role of specific dendritic cell subsets in regulating the anti-tumor immune response, as well as the current status of dendritic cell-based immunotherapies, in order to provide an overview for future lines of research and clinical trials.

The Unfolded Protein Response Mediator PERK Governs Myeloid Cell-Driven Immunosuppression in Tumors through Inhibition of STING Signaling.

The primary mechanisms supporting immunoregulatory polarization of myeloid cells upon infiltration into tumors remain largely unexplored. Elucidation of these signals could enable better strategies to restore protective anti-tumor immunity. Here, we investigated the role of the intrinsic activation of the PKR-like endoplasmic reticulum (ER) kinase (PERK) in the immunoinhibitory actions of tumor-associated myeloid-derived suppressor cells (tumor-MDSCs). PERK signaling increased in tumor-MDSCs, and its deletion transformed MDSCs into myeloid cells that activated CD8+ T cell-mediated immunity against cancer. Tumor-MDSCs lacking PERK exhibited disrupted NRF2-driven antioxidant capacity and impaired mitochondrial respiratory homeostasis. Moreover, reduced NRF2 signaling in PERK-deficient MDSCs elicited cytosolic mitochondrial DNA elevation and, consequently, STING-dependent expression of anti-tumor type I interferon. Reactivation of NRF2 signaling, conditional deletion of STING, or blockade of type I interferon receptor I restored the immunoinhibitory potential of PERK-ablated MDSCs. Our findings demonstrate the pivotal role of PERK in tumor-MDSC functionality and unveil strategies to reprogram immunosuppressive myelopoiesis in tumors to boost cancer immunotherapy.

Correction: Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Immune Microenvironment and Cancer Metastasis.

The dynamic interplay between neoplastic cells and the immune microenvironment regulates every step of the metastatic process. Immune cells contribute to invasion by secreting a cornucopia of inflammatory factors that promote epithelial-to-mesenchymal transition and remodeling of the stroma. Cancer cells then intravasate to the circulatory system assisted by macrophages and use several pathways to avoid recognition by cytotoxtic lymphocytes and phagocytes. Circulating tumor cells that manage to adhere to the vasculature and encounter premetastic niches are able to use the associated myeloid cells to extravasate into ectopic organs and establish a dormant microscopic colony. If successful at avoiding repetitive immune attack, dormant cells can subsequently grow into overt, clinically detectable metastatic lesions, which ultimately account to most cancer-related deaths. Understanding how disseminated tumor cells evade and corrupt the immune system during the final stages of metastasis will be pivotal in developing new therapeutic modalities that combat metastasis.